Chicken Embryo As A Diabetic Animal Model

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Editor-in-Chief
Dr. S.G. Ramachandra
Associate Editors
Dr. Suresh Poosala Dr. Nagendra R. Hegde
Editorial Board Members
Dr. S.S.Y.H. Qadri Dr. Vyas M. Shingatgeri
Dr. T.V. Anilkumar Dr. K.B.Patel
Dr. Kalidas Kohale Dr. C.G. Raut
Dr. Yogananda Moolemath Dr. Sheshagiri Gaonkar
Dr. Deepak Sharma Dr. S.P. Muthukumar
Dr. V.S. Harikrishnan Dr. M.J . Mahesh Kumar
Editorial Ofce
Secretary
Laboratory Animal Scientists Association
H.No. 12-13-483/A/1
Lane Opp. Mahalakshmi Super Market
Nagarjuna Nagar
St. No. 14, Tarnaka
Secunderabad-560017, Andhra Pradesh
Phone : 040-27008921
Mobile phone: 09849518510
email : editor.jlas@gmail.com
Cover page image: Courtesy - National Human Genome Research Institute, Bethesda, MD 20892-2152, USA
Printed and Published by
Laboratory Animal Scientists Association, India
Contents
Editorial
-Ramachandra S.G. 1
Short communication
Laboratory animal science and experimentation in India : Review of the last
decade
-Qadri S.S.Y.H.
2 - 3
Special feature
The AAALAC International accreditation program in India
-Kathryn B.
4 - 8
Review
The saga of the humanized mouse: a giant leap into the galaxy or a small step for
humankind?
-Nagendra R. H.
9 - 23
Research
Effect of graded doses of nimbolide on biochemical and sperm functional
parameters in male albino rats
-Ravindranath H. A, Sukesh B, Umadevi C. J and Murigendra B. H.
24- 30
Dietary supplementation of curcumin ameliorate Afatoxin B
1
induced oxidative
stress in mice
-Prakash N, Sunilchandra U, Vijaykumar M, Pavitra B.H, Nagabhushana V and
Ramachandra S.G.
31 - 33
Evaluation of certain bioactive compounds using in ovo diabetic model
-Srinivasan K, Venkateswaran G and Muthukumar S.P.
34 - 38
Effect of graded levels of Jojoba meal in the complete feed of rabbits on voluntary
intake and nutrient utilization
-Kiran L, Nagabhushana V, Prakash N, Jadhav N.V, Madhavaprasad C.B, Ashok
Pawar and Shettar V.B.
39 - 42
Effect of varying doses of cisplatin on rat intestinal apoptosis
-Uday Kumar P, Vijayalakshmi B, Kalyanasundaram S, Raghunath M and Sesikeran B.
43 - 48
Sub chronic toxicity study of Fusarium xylarioides culture fltrate from ground
nut hay in rats
-Vinay T, Shridhar N.B, Narayanaswamy H.D, Shrikrishna I and Jayakumar K.
49 - 53
Protective effect of caffeine against cyclophosphamide induced in vivo genotoxicity
-Prakash G, Ingle A.D and Hosetti B.B.
54 - 58
Clinical
Hematological values of mice and rat strains : ACTREC laboratory animal facility
-Ingle A.D, Shinde A.D and Ahire S.D.
59 - 63
Eradication of Pinworms (Syphacia spp.) in rodent colony
-Harikrishnan V.S, Ranaraj V.R, Sreeja. K.R and Fernandez A.C.
64 - 67
Care and management
Sentinel monitoring program: Quality assurance in laboratory animal facility
-Shakthi Devan R.K, Parthiban N, Samarendra N and Suresh P.
68 - 76
An overview of the key concepts required for managing a laboratory animal
(rodent) facility
-Sai T.
77 - 83
Production facility of purpose-bred beagle dogs
-Keen J and Jasmin B.
84 - 87
Environmental enrichment for laboratory animals
-Satish P.
88 - 91
Vol -1, Issue - 1, Jan - Apr 2011 ii
Meet our Editor
Dr. Ramachandra S.G.

Dr. S.G. Ramachandra graduated from the Bangalore Veterinary College in 1987 and
completed post graduation in Veterinary Pharmacology and Toxicology in 1990.
Subsequently, he received his PhD in Veterinary Pharmacology and Toxicology from
the Bangalore Veterinary College. He completed one year certifcate course on Animal
Welfare from the Madras Veterinary College, Tamilnadu Veterinary and Animal Sciences
University, Chennai in 2001 and has undergone an advanced training on primate medicine,
surgery and management at German Primate Centre, Gottingen, Germany in 2003.
He has been working in the feld of Laboratory Animal Science for the last two decades
and is currently working as Principal Research Scientist at the Central Animal Facility,
Indian Institute of Science, Bangalore. He has been nominated as Fellow by the National
Academy of Veterinary Science, New Delhi for his signifcant contribution in the area of
Laboratory Animal Science in India. He is presently serving as the President of Laboratory
Animal Scientists Association and also as a Member of the Council in Asian Federation
of Laboratory Animal Science Associations, Japan. He has been appointed as Adhoc
Specialist by the Association for Assessment and Accreditation of Laboratory Animal
Care International (AAALAC International), USA. Recently, he has been nominated as
GLP Inspector by the National GLP Compliance Monitoring Authority, Dept. of Science
and Technology, Govt. of India.
Currently, he is serving as a nominee of the Committee for the Purpose of Control
and Supervision of Experiments on Animals (CPCSEA) in the Institutional Animal
Ethics Committee (IAEC) of several organisations. He is also a member of IAEC and
Institutional Bio Safety Committee (IBSC) at several research institutes in Karnataka.
He is serving as a member of Animal House Inspection Committee, CPCSEA, Ministry
of Environment and Forests, New Delhi and has inspected several animal facilities in
Karnataka, Andhra Pradesh and Maharastra.
To promote the laboratory animal science and to build a strong base of trained personnel,
he is regularly offering a continuing education programme on Laboratory Animal
Management through the Centre for Continuing Education, Indian Institute of Science
and conducting workshops, basic and advanced training courses on Laboratory Animal
Management for laboratory animal house managers/care takers. His unstinted technical
guidance in establishing several animal facilities in and around Bangalore is worth
mentioning. In his endeavor to create awareness and disseminate knowledge on recent
developments in laboratory animal science, several workshops and conferences were
successfully organized. It is noteworthy to mention that under his guidance, as President
of LASA, three international conferences on laboratory animal science were conducted in
the last 5 years. He has delivered several lectures at national and international conferences
and chaired several scientifc sessions on laboratory animal science. He was invited by the
South African Association for Laboratory Animal Science as the Chief Guest during their
SAALAS Congress (2009) and was an invited speaker at the III and IV Asian Federation
of Laboratory Animal Science Association Congress which were held at Beijing in 2008
and Taiwan in 2010 respectively.
During his tenure at the Primate Research Laboratory, Indian Institute of Science, he was
involved in various research projects on primates, which were focused mainly on fertility
regulation. This includes physiology of hypothalamo-pituitary-gonadal axis, hormonal
regulation of fertility, development of contraceptive drugs and vaccines for males.
He was actively involved in testing several compounds/drugs for their contraceptive
effcacy, determining the need for estrogen during implantation and fertility regulation
by using immuno-contraception approach in bonnet monkeys by active immunization
with phage-expressed peptides of the extra cellular domain of FSH receptor and Eppin,
a testis/epididymis-specifc protein. Published several research articles in national and
international journals.
Vol -1, Issue - 1, Jan - Apr 2011 iii
Vol -1, Issue - 1, Jan - Apr 2011 iv
Journal of Laboratory Animal Science
The use of laboratory animals in scientifc and medical
research is essential for the improvement of quality of life.
Hence, humane and responsible care of laboratory animals is
vital to quality research. In India, experimentation on animals
is regulated by the Committee for the Purpose of Control
and Supervision of Experiments on Animals (CPCSEA), a
statutory body under the Prevention of Cruelty to Animals
Act (1960), in the Ministry of Environment and Forests. The
persons/institutes engaged in scientifc experiments on animals
must act in conformity with the provisions of the Prevention
of Cruelty to Animals Act (1960) and the Breeding of and
Experiments on Animals (Control and Supervision) Rules
(1998). These provisions are enforced by the independent
committee, CPCSEA. The CPCSEA has formulated
guidelines for experimentation on animals with animal
welfare and well-being as its utmost concern. The main aim
of these guidelines is to ensure humane and ethical treatment
of animals, while carrying out legitimate scientifc research
involving experiments on animals. The goal is to promote
the humane care of animals used in biomedical research and
to provide information that will enhance animal well-being,
the quality of biomedical research, and the advancement of
biologic/scientifc knowledge that is relevant to humans or
animals. To oversee the effective implementation of these
guidelines at the institutional level and also to approve the
project proposals on animal experimentation, the CPCSEA
nominates persons to every registered institute/organization.
The nominee appointed by the Member Secretary, CPCSEA
will serve as a link between the institute/organization and the
CPCSEA. The primary responsibility of the nominee is to
supervise the well-being and welfare of the animals housed or
used for experiments/breeding.
The recent economic boom that is being experienced in India is
largely attributed to the globalization and liberalization of the
Indian economy. This has resulted in unprecedented growth
in information and biotechnology sectors. The availability
of a vast pool of English speaking science graduates; good
regulatory processes and the cost advantage have positioned
India as a favorable investment destination. The large pool of
scientifc talent, world-class information technology industry
and vibrant pharmaceutical sector has helped India to emerge
as a signifcant player in the global biotech arena. Several
overseas pharmaceutical giants and drug discovery frms
are outsourcing their work to India on a large scale. Hence,
several state-of-the-art facilities have been established in
India in the last decade.
The recent galvanization of CPCSEA and implementation
of stringent rules have made this sector organized and
improved animal facilities as well as the practice of using
animals for experimentation. At present, more than 1400
institutes/organizations that are using animals in biomedical
research are registered with the CPCSEA. Even though, many
researchers and other personnel are working in the feld of
laboratory animal science, there is no common platform to
discuss the issues relating to comparative medicine and
laboratory animal science in India. Hence, Laboratory Animal
Scientists Association (LASA) was established with an
intention to create a forum for all those who are working in
the feld of laboratory animal science. The LASA is making
attempts to bring all the institutes and the people working
in this feld under one platform. The LASA was established
with objectives to provide and disseminate education and
training in laboratory animal science and to promote and
encourage the highest level of ethics within the profession
of laboratory animal science. The LASA is making efforts
to disseminate leading edge laboratory animal science and
welfare information through seminars, workshops and
discussions among the members. The motto of LASA is to
promote and coordinate the development of laboratory animal
science throughout our country, to collect and disseminate
information on laboratory animals and to promote nationwide
harmonization in the care and use of laboratory animals. As
a prelude to this, LASA is making an attempt to disseminate
the information through the journal. This dedicated journal on
laboratory animal science will be an ideal platform to reach a
broad and multidisciplinary audience in the feld of laboratory
animal science and biomedical research. The contents would
include original peer-reviewed contributions in the form of
invited reviews, research articles, rapid/short communications
and clinical articles, covering all topics in research relating
to laboratory animals. The launch of this journal is timely,
given the recent growth in the feld of laboratory animal
science in India. I am sure that the strong track record and
well experienced team of LASA would strive hard to serve
the communities involved in laboratory animal science and
biomedical research.
Ramachandra S.G.
Editorial
Vol -1, Issue - 1, Jan - Apr 2011 1
Editorial
Ever since the revival of the Prevention of Cruelty to Animals
Act (1960)in 1998, the law was amended and implemented
with renewed vigor, the animal welfare and laboratory animal
experimentation has seen a major change. The strict imple-
mentation of the law during the early part of the decade led
to the spilling of skeletons from cupboards of some very well
known and established institutions in India. The implemen-
tation of the law not only revealed the lack of basic facilities
and poor and unhygienic conditions in animal facilities, it also
revealed the laid back attitude of those who were responsible.
The increase in awareness that was achieved through the
animal welfare movement in India was enormous. It has
led to the improvement of laboratory animal facilities in
several institutions and the commissioning and constitution
of Institutional Animal Ethics Committees (IAEC) in all
the CPCSEA (Committee for the Purpose of Control and
Supervision of Experiments on Animals), Animal Welfare
Division, Ministry of Environment and Forests, Government
of India, registered organizations. The IAEC is in turn playing
a major role in improving the welfare of the animals used
for experiments. Nevertheless, the improvement of animal
facilities in the Pharmaceutical industry and the Contract
Research Organizations (CRO) to this day are driven more
by the need to attract and capture business than a true change
in the heart for the welfare of animals. The race for acquiring
Good Laboratory Practices and Association for Assessment
and Accreditation of Laboratory Animal Care International
(AAALAC International) accreditation in the private industry,
and the contrasting laid back attitude in the Government sector
as was evident before 1998 is a clear indication of this fact.
It may also be considered here that the threat of the law and
negative public opinion has played a signifcant effect on the
Government Institutions and several of them did release funds
and have improved the animal facilities in their respective
organizations.
Till date, approximately 1400 organizations have registered
with the CPCSEA. This includes a large number of organi-
zations from the pharmaceutical industry, CROs, medical,
pharmacy and university colleges in the private sector
and biomedical research institutions, medical, veterinary,
pharmacy and university colleges in the Government sector.
Majority of these facilities are very primitive and consist
of two to few rooms. Some have installed air-conditioners
whereas others have only fans or air coolers. Several medical
and pharmacy colleges have 2-3 room animal facilities with
concrete or stone shelves on the walls that are used for placing
the cages. This design was in vogue during the 1950s and
60s. The authorities probably had no idea about the concept
of clean and dirty corridors. Most facilities do not have proper
electricity and water. The staff is not trained and in many of
these institutions, the animals to this day are fed some bread,
soaked horse gram in case of rats and mice and cabbage and
carrots to rabbits.
About half a dozen organizations in the private sector
obtained AAALAC accreditation, while some of these
have also achieved GLP accreditation from Netherlands or
Germany or India. Unfortunately, most of these accreditation
bodies concentrate more on the facilities and the protocols
that pertain to animal welfare giving very little importance to
the quality (health and genetics) of the animals. This has led to
a situation where a majority of registered organizations prefer
to procure cheaper animals and feeds of inferior quality for
their research. The regulatory authorities most often are either
not aware of these issues or are not much concerned about it.
He completed post graduation in Veterinary Pathology in 1987 from A.P. Agricultural University.
He is recipient of gold medal in Veterinary Pathology (1987), a Young Scientist award (1995)
and Mahaveera Vishwa Sadhbhavna Puraskar for animal welfare (1999). Currently he is working
as Senior Scientist (Veterinary Pathologist) in the Pathology Division at the National Institute
of Nutrition (ICMR), Ministry of Health and Family Welfare, Government of India. He worked as
an Advisor (Expert Consultant) to the CPCSEA for three years on deputation. He is a Certifcate
holder in Lab Animal Science from Utrecht University, Netherlands and FELASA level C from
Europe. He is a Board Certifed Veterinary Pathologist in India (Diplomate ICVP). He is the founder
of SPCA and PFA at Hyderabad and Secunderabad. He is also the founder and present Secretary
General of the Laboratory Animal Scientists Association of India. He has vast experience in
animal experimentation especially dogs, primates and other smaller laboratory rodents and has
completed over a hundred preclinical toxicology projects and reviewed histopathology slides
from toxicology studies in more than 40 projects..
Qadri S.S.Y.H.
Laboratory animal science and experimentation in India
: Review of the last decade
Qadri S.S.Y.H.
National Institute of Nutrition
Hyderabad 500007, India
Key words : laboratory animals, experimentation, India
Corresponding author :
S.S.Y.H. Qadri, Dept. of Pathology
National Institute of Nutrition, Hyderabad 500 007
Phone : 09849518510, email : ssyhq@yahoo.com
Short communication
Laboratory animal science in India, Qadri S.S.Y.H.
The availability of human resources is another important
factor that is a major impediment in the proper growth of
this sector. There is a huge gap in the availability of qualifed
and trained technical staff to work in the laboratory animal
facilities compared to the number of animal facilities
mushrooming in this country. The problem is compounded
by the unavailability of veterinarians. A majority of animal
facilities in the Government sector are headed by either the
heads of the pharmacology departments or by the zoology
departments in the colleges which is more often an additional
charge of the facility. The Act of the Parliament of India, the
Veterinary Council of India Act 1984 and its amendment of
1992 authorizes only qualifed and registered veterinarians to
breed and experiment on animals. The CPCSEA rules under
the PCA Act 1960, Chapter IV, have also laid the rules for
laboratory animal breeding and experimentation, and the
qualifcations and expertise of those who plan to conduct the
experiments. However, there is lot of ambiguity in the law
and needs clarifcation. The ambiguity in the law has also
led to a situation where any body, is permitted to do animal
experiments whether he/she is qualifed or not. This has led
to a situation where the intention and purpose for which the
CPCSEA has been formed is at stake.
Majority of the organizations have obtained CPCSEA
permission to breed as well as experiment on animals.
Unfortunately, most of the facilities do not have the capacity
nor have the trained staff to breed quality animals for research.
A majority of these organizations are breeding animals
randomly. Invariably, most of them do not have the means
to phenotypically differentiate between the strains nor are
these facilities conducting any genetic monitoring to confrm
the strain. This in turn refects the present situation where
the animals that are available from the suppliers do not have
either the basic genotype data or the suppliers aware of the
microbial, viral load or even the latent infections prevalent in
these animals. Background pathology of most of such animals
is very poor with a majority of animals showing respiratory
infections, abscesses in lungs, kidneys, livers, parasitic loads,
degeneration and necrosis in the liver, interstitial nephritis
etc., besides spontaneously developed tumors. Literature
surveys reveal that most of the pathogens causing diseases in
animals are known to interfere with the research results and
in most cases invalidate the data. Therefore, the quality of the
animals that are available for research gives a clear indication
of the quality of the research carried out in most institutions.
These issues do not come either under the purview of the
CPCSEA nor under the AAALAC accreditation system. This
is compounded by the fact that most scientists and adminis-
trators alike would like to see the other way when such issues
are raised as quality animals and feed costs more.
Off late, it has become a fashion to say that one is following the
3 Rs principle of Russel and Bursch. It is unfortunate to note
that many of the experimenters have not realized the scientifc
basis of these principles. No honest attempts are being made
to fnd alternative to replace their animal experiments. It is
quite common to observe in proceeding of the national confer-
ences and symposia especially related to pharmacological
sciences and toxicology that hundreds of experiments are
being conducted by students for their dissertation work or for
PhD thesis. Majority of these experiments are on testing some
herbal drugs. The number of animal experiments conducted,
are doubling every year and a majority of these experiments
look redundant and could have been done using alternate
methods. Again the regulatory authorities have been unsuc-
cessful in controlling such experiments. There is an urgent
need for educating the concerned authorities to encourage and
educate the students to use alternatives and avoid duplication
or redundant research using poor quality animals.
The reduction in number of animals in each experiment is
only possible when the quality of the animal facilities and also
the animals are improved thus reducing the noise (variability)
in the experiments, in turn reducing the number of animals
required in each experiment as well as the number of experi-
ments required to obtain the desired information. There are
no minimum requirements/stipulations for organizations that
breed and supply animals. The improvement in the quality
of animals can be brought about only by permitting only
those breeders and suppliers who breed healthy and geneti-
cally defned animals. This would necessitate the breeders
and suppliers to regularly screen their animals for bacteria,
viruses, parasites, mycoplasma, and the feed and water for
their quality and content of toxins, contaminants such as
heavy metals and pesticide residues. This would be the only
way to even weed out profteering and unscrupulous uncon-
trolled breeding of animals.
The refnement of techniques based on the 3 Rs principles
pertains to the design of experiments and the methodology
adopted in conducting the experiments. This would require
a comprehensive training of the experimenters on handling,
restraint, dosing, sample collection, anesthesia, euthanasia
etc. as well as the IAEC members who scrutinize the research
proposals. Animals are being transported from north India
to south India vice versa are under tremendous stress due
to changes in climate conditions hence uniform handling
techniques, environmental condition, feed and water is highly
essential. All these factors have a tremendous impact on the
stress status of the animal. These effects become confounding
variables in an experiment. This can be avoided only when
similar techniques and standards are followed all over the
country.
With India joining the OECD and the opening of free trade
between India and the European Union, would eventually
lead to the outsourcing of regulatory toxicity data generation
especially for REACH compliance. This would in turn lead
to further mushrooming of animal facilities and breeding of
laboratory animals on a large scale. The number of animal
experiments would also increase in leaps and bounds. With
the existing infrastructure and the quality standards, the future
as far as the quality research and regulatory data generation
is concerned it doesnt look very encouraging, unless drastic
steps are taken by the authorities and scientists themselves in
improving the situation.
Use of large animals especially beagles and non-human
primates in India has already been curtailed to a large extent
both by the inability of the scientifc community to foresee the
future and develop breeding facilities and by the unwritten
laws of land where sentiments overrule scientifc judgment.
This is leading to a situation where, in the pharmaceutical
sector a large number of studies are getting delayed as the
necessary permission to conduct large animal experiments is
not forthcoming. In the long run, this may become a major
impediment for the growth of the pharmaceutical sector/drug
development in India. The other effect of this that has been
observed is the fast mushrooming of CROs in neighboring
countries especially the northern neighbor and the diversion
of CRO business to these countries.
Hence, it is high time to all those who are concerned to
highlight these issues in all the forum to bring about a change
and an improvement in this sector.
Introduction
The globalization of the biomedical research enterprise is
occurring at an accelerating pace (Bayne and Miller, 2000).
Increasingly, scientifc collaborations and contracts cross
national borders. The need for assurance that the caliber of
animal research and animal welfare are consistent and that
such animal use is done in a humane and conscientious manner
is of concern to the scientifc community, the general public,
and other stakeholders. Bridging these international interac-
tions is a clear scientifc imperative for reproducibility of
results and statistical validity of data (Bayne, 2008). One way
to mitigate the potential confounding effects the quality of the
animals may have on the research data is to harmonize animal
care practices and procedures, thereby ensuring a similar level
Dr. Kathryn Bayne, MS, PhD, DVM, DACLAM, CAAB, is Global Director for the
Association for Assessment and Accreditation of Laboratory Animal Care
International (AAALAC International). In this role, she directs the global
accreditation program and travels globally to advance AAALACs accreditation
program and laboratory animal welfare. She has published over forty articles
on the subject and is internationally renowned for her work in laboratory animal
behavior. Dr. Bayne is a past President of the American College of Laboratory
Animal Medicine, the Association of Primate Veterinarians, as well as the District
of Columbia Veterinary Medical Association. She is past Chair of the American
Veterinary Medical Associations Animal Welfare Committee. Dr. Bayne is the 2009
recipient of the American Veterinary Medical Associations Animal Welfare Award
and Washington Sate Universitys 2009 Excellence in Research and Teaching Award.
She is also a recipient of AALASs prestigious Garvey award. Kathryn Bayne
Abstract
Science is a global enterprise, as illustrated by the number and scope of international research collaborations
and scientifc meetings, as well as the number of journals publishing articles from the international scientifc
community. As institutions seek to compete on the global market it is critical to ensure an internationally
accepted level of animal care and welfare is maintained by institutions using animals for research, testing and
education. AAALAC Internationals accreditation program utilizes the fexibility of performance standards; is
sensitive to differing legal and cultural issues; promotes the cross fertilization of information among institu-
tions through the site visit process; highlights best practices; and serves as a benchmark of quality. Through
this process, uniform, high standards of animal care and use may be achieved under a broad range of varying
national requirements because of the overarching quality assurance derived from AAALAC Internationals
accreditation program. Accreditation of animal care and use programs in India is a fairly new proposition, but
is rapidly gaining attraction. As the biomedical research enterprise in India continues to gain momentum, so
too have the merits of an international quality assurance validation system become increasingly recognized.
To that end, the AAALAC International accreditation program is herewith described, including observations
of fndings identifed during on-site assessments.
Key words: accreditation, India, AAALAC
Corresponding author:
Kathryn Bayne, MS, PhD, DVM, DACLAM, CAAB, 5283 Corporate Dr., Suite 203, Frederick, MD 21703 USA.
Phone: 301.696.9626, Fax: 301.696.9627, email: kbayne@aaalac.org
Kathryn B
AAALAC International, Frederick, MD, USA
The AAALAC International Accreditation Program in India
Special feature
Accreditation in India, Kathryn B.
of animal health and welfare, and thus making the data more
reproducible. By putting into place standards that promote
harmonization, some assurance of quality animal care and use
is achieved.
The Association for Assessment and Accreditation
of Laboratory Animal Care International (AAALAC
International) has been accrediting institutions in countries
outside the United States since 1981. Today, more than 860
animal care and use programs in 34 countries have achieved
AAALAC International accreditation. AAALAC is in a
unique position to harmonize animal care and use programs
as, collectively, the expert teams that conduct the on-site
evaluations have visited an average of 212 institutions each
year for the last six years. Thus, the AAALAC site visitors
have a profound depth of experience in reviewing a wide range
of animal care and use programs. The frst institution in India
to become accredited by AAALAC occurred in 2001. That
institution, then known as the Rallis Research Center, today is
Advinus Therapeutics. Eight years passed before other Indian
institutions applied for and achieved AAALAC International
accreditation. At the time of this writing, six institutions in
India, all in the private or not-for-proft sectors have achieved
accreditation, and an additional four more institutions have
recently hosted their initial on-site assessment in the process
towards accreditation.
The AAALAC International is a non-governmental, not-for-
proft organization that has been accrediting laboratory
animal care and use programs since 1965. It was the frst
organization in the world to provide this service and is the
only organization today that provides a global accreditation
service for animal research, testing and teaching programs.
AAALAC is a voluntary accrediting organization that
enhances the quality of research, teaching and testing by
promoting humane, responsible animal care and use. It
provides advice and independent assessments to participating
institutions and accredits those that meet or exceed applicable
standards. The AAALAC International accreditation program
is science-based and sensitive to cultural and legal differ-
ences, and thus it is a logical approach for the international
scientifc community to adopt to facilitate the harmonization
of animal care and use standards (Bayne and Miller, 2000).
The AAALAC has global regional offces to better serve the
institutions currently participating in the accreditation program
or desirous of becoming accredited. The headquarters offce
is located in Maryland, United States; a European offce is
located in Pamplona, Spain; and a Southeast Asia offce is
located just outside of Bangkok, Thailand. As a not-for-proft
organization, AAALAC is guided by a Board of Trustees.
This Board is comprised of scientifc organizations, veter-
inary medical organizations, and research advocacy groups.
It currently has 67 member organizations, Approximately
37 organizations represent research disciplines, 18 represent
veterinary medicine or animal sciences specialty groups, and
the balance represent patient or science advocacy groups
and industry/academic interest groups. Of these, several are
international associations, including the Asian Federation of
Laboratory Animal Science, and which provide guidance to
AAALAC in its global mission.
The AAALAC International
Accreditation Program-
Standards of Accreditation
Harmonizing an animal care and use program to an interna-
tional level requires applying the provisions and principles
of the Guide for the Care and Use of Laboratory Animals
(Guide) (NRC, 1996 et seq.) and several other standards.
Specifcally, the country-specifc legal and regulatory
requirements applicable to the institution being assessed
by AAALAC constitute the baseline for accreditation. No
program can become AAALAC accredited if it is in violation
of local legal and regulatory requirements. Once AAALAC
is satisfed that these local baseline requirements are met, the
Guide recommendations are overlayed on the local require-
ments to meet the AAALAC International standard. It is
important to note that when local requirements are more
stringent than Guide recommendations, the former must be
met in order to achieve accreditation. In some instances,
the Guide includes provisions not addressed in national or
supranational animal welfare legislation or regulations, for
example in the area of occupational health and safety. In
the absence of national standards, the Guide recommenda-
tions are used as the basis for evaluating program elements
in these areas. When necessary and appropriate, specifc
Reference Resources (e.g., the American Veterinary Medical
Associations Guidelines on Euthanasia, 2007) are used to
evaluate program components where the Guide is silent or not
specifc (see http://www.aaalac.org/accreditation/resources.
cfm for a list of the Reference Resources used by AAALAC).
Critically important is that all principles of the Guide must be
met. Finally, the expert professional judgment of AAALACs
Council on Accreditation is applied through the peer review
process before a fnal accreditation status is granted.
Various editions of the Guide have been the basis for the
assessment process and for the application of performance
standards. The Guide has recently been revised (NRC, 2010),
and the updated version will be used by AAALAC International
beginning in the Fall 2011. Recently, AAALACs Board
of Trustees approved the use of three primary standards by
AAALACs Council on Accreditation: the Guide, the Guide
for the Care and Use of Agricultural Animals in Research and
Testing (FASS, 2010) and European Treaty Series (ETS) 123
(1986). Application of these standards will depend on the
geographic location of the institution and the type of animal
use. Because of its reliance on these standards, AAALAC
does not formulate animal care and use polices or regulations
of its own as a separate standard to achieve accreditation.
Each of the three primary standards is grounded in a
performance-based approach to animal care and use, which
is typically based on the scientifc literature. A performance
approach requires professional input, sound judgment, and a
team approach to achieve specifc goals; thus, there is some
fexibility in how the desired outcome (e.g., sanitized cages)
or goal is reached (NRC, 2010).
The Application Process
Any public or private institution, organization or agency
maintaining, using, importing or producing animals for
purposes of scientifc research, teaching or testing may be
accredited. At the time of this writing, 83% of the institu-
tions accredited in India are commercial organizations, either
pharmaceutical companies or contract research organizations.
AAALAC Internationals Rules of Accreditation stipulate
that an active animal care and use program must be in place,
specifcally, that for a program to be accreditable it must have
a reasonable activity level relative to the space available for
animal holding and use. For new applicants, the active animal
care and use program must be in place and operational prior
to application for accreditation. AAALAC International will
not conduct a site visit to new applicants that do not meet the
criteria of an accreditable organization and that do not have
an active animal care and use program. Accredited institu-
tions must have all components of an active animal care and
use program at the time of a site revisit, to include: animals;
facilities; equipment; professional, technical, and adminis-
trative support; and policies and programs for institutional
responsibilities, animal husbandry and veterinary care. An
institution interested in applying for accreditation submits a
two-page form which provides the AAALAC Executive Offce
with contact information of key personnel and designates the
Attending Veterinarian and Institutional Offcial, as well as
the Chair of the Institutional Anima Ethics Committee (IAEC)
and what AAALAC refers to as the Unit Contact (that person
identifed by the institution to serve as the offcial liaison
with AAALAC). This form is accompanied by the Program
Description, which must be completed using the templates
available on the AAALAC website.
The application for accreditation initially undergoes an
administrative review by AAALAC Executive Offce staff.
The application is accepted if it is complete, specifcally
that it contains all the required appendices and the two page
application form. If an application is found to be incomplete,
the institution is contacted to request the missing information
before the application can be accepted and processed. Once
the missing information has been provided, the institution
can be scheduled for its initial visit. The actual quality of
the programwhich may or may not be well described in the
written materials submitted in the applicationis reviewed
before and during the on-site assessment using AAALACs
standards as the benchmark for assessment. This process has
led to some confusion among Indian colleagues who misun-
derstood the difference between accepting the application
and conferring accreditation. Acceptance of the application
packet merely makes the institution eligible to proceed to the
next phase, hosting the accreditation site visit. Acceptance of
the application in no way implies that accreditation will be
achieved.
Peer Review
The AAALAC International uses an interactive, peer review
system that relies on expert professional judgment both in the
application of performance standards and in assessing perfor-
mance outcomes (Bayne and Martin, 1998). Specifcally, the
composition of the site visit team is tailored to the institutions
animal-based research program, and includes team members
familiar with the species used at the institution and the types
of research done there. The team members are colleagues who
share similar experiences and knowledge base to the institu-
tions they visit. For example, a site visitor who works at a
diverse academic program would be well-qualifed to conduct
a site visit to another large, complex academic institution.
As an important measure of ensuring that high quality
animal care and use programs are maintained at institutions
accredited by the AAALAC International, formal site visits
are conducted at three year intervals, and initially for institu-
tions seeking to gain accreditation. Information obtained from
these on-site assessments, together with Annual Reports and
direct correspondence, constitute the basis for ongoing evalu-
ation and assessment by the Council on Accreditation. The
site visit and resultant report provide the Council with factual
information and serve as a basis for subsequent Council action
regarding the institutions accreditation status. The fndings
noted in the site visit report are deliberated upon by members
of the Council on Accreditation, and thus the site visitors
observations undergo another level of peer review.
Concerns identifed during the site visit are categorized as
either Suggestions for Improvement or Mandatory Items for
correction. Suggestions for Improvement are items which, if
implemented, would enhance an already acceptable or even
commendable program and they do not impact the accreditation
status of an institution. Mandatory items are more serious
defciencies which must be corrected for Full Accreditation
to be awarded or continued. The Council review classifes
the fndings observed during the on-site assessment into one
of these categories. Ultimately, a formal vote is taken by the
Council regarding the question of the institutions proposed
accreditation status.
Initially the report of the site visit undergoes a thorough review
by several other members of the Council on Accreditation
before the convened meeting. Electronic discussions occur
which provide the opportunity for clarifcations and modifca-
tions to the site visit report before the Council convenes. The
review that occurs before the convened meeting involves very
senior members on the Council who serve as elected Offcers of
the Council. This review may then be extended into additional
discussions during the convened meeting of Council. The
letter that will be sent to the institution indicating the institu-
tions accreditation status is carefully crafted, based on the
review of the site visit report by the Council. The letter then
also undergoes further detailed review by the Council Offcers
and senior staff within AAALAC International. In this way,
these multiple layers of peer review help to ensure an accurate
assessment of the animal care and use program and a product
(i.e., the accreditation letter) that is designed to be meaningful
and resourceful to the institution.
Fundamental to the accreditation process is the fact that
AAALAC International evaluates the entire animal care and
use program. This program is framed in the Guide (NRC,
1996; NRC, 2010) and each institution seeking accreditation
must describe the details of the animal care and use program
using a Program Description outline, provided by AAALAC,
which generally follows the content of the Guide. Thus, the
Council on Accreditation reviews the system of oversight and
monitoring of the institutions animal care and use program
(e.g., by an Institutional Animal Care and Use Committee
(IACUC), IAEC, or similar ethical review committee); the
provision of veterinary medical care; personnel qualifcations
and training; the occupational health and safety program;
animal housing; behavioral management; husbandry proce-
dures and population management; animal procurement and
transportation; quarantine, stabilization, and separation of
species; sentinel programs; oversight of the surgery program;
anesthesia and analgesia guidelines; euthanasia guidelines;
structural soundness of the facility, facility maintenance,
ability to prevent harborage of vermin in the facility; and
proper functioning of the heating, ventilation, and air condi-
tioning system (HVAC).
The Annual Report and Prompt Reporting
In accordance with AAALACs Rules of Accreditation, the
accredited institution must submit an Annual Report which
describes elements of the animal care and use program as
specifed by AAALAC International. The Annual Report
form is typically distributed to accredited institutions in
mid-December. The report is designed to be completed and
submitted in an on-line format. Questions range from updates
on the contact information for key personnel, actions taken
to address any Suggestions for Improvement offered by the
Council on Accreditation, to declarations of issues that arose
in the program during the year (such as suspended protocols).
Although the Annual Report forms are distributed at the end
of the calendar year, each institution may establish its own
reporting period. Many Indian institutions initially chose to
use the month in which they were accredited (based on the
date of the letter from the Council on Accreditation), but some
have moved to the calendar year for reporting. Globally, insti-
tutions also use the fscal year, academic year or government
fscal cycle, depending on what is most convenient.
In addition, each accredited institution is expected to
promptly notify AAALAC International (e.g., through written
correspondence or e-mail) of adverse events relating to the
animal care and use program. Examples include investiga-
tions by the government oversight bodies, as well as other
serious incidents or concerns that negatively impact animal
well-being. This information is included in the accredited
programs fle and the Council on Accreditation typically
reviews this correspondence to determine how the institution
addressed the problem and what systems were put in place to
prevent a similar occurrence.
Accreditation Site Visit Findings
Commendations to Institutions
Although AAALACs experience in India is limited, with
accredited Indian institutions comprising less than one
percent of the total number of accredited animal care and
use programs, some patterns are clear in both the positive
aspects of the animal research programs and in areas that
have proven challenging. Across the site visits conducted
to-date in India, the Council has commended the strong
institutional commitment to and support for a quality
animal care and use program. In addition, the Council has
consistently complimented the dedication of the staff, both
professional and technical, as well as their enthusiasm,
qualifcations and genuine compassion and concern for the
animals. Record keeping practices (e.g., standard operating
procedures, training records, protocol documentation) have
amply ensured thorough and detailed information. In recent
years, the Council has determined that the institutions it has
visited have become suffciently familiar with AAALACs
expectations for the occupational health and safety program
that it is often mentioned as a strong element of the animal
care and use program.
Findings Corrected by Post Site Visit
Communication
At the conclusion of each site visit, the AAALAC International
representatives conduct an exit briefng for personnel from
the institution. The purpose of this briefng is to provide the
institution with a preliminary evaluation based on fndings
and impressions of the site visitors. Comments and views
expressed by site visitors are independent, preliminary
opinions and may not necessarily refect the fnal peer reviewed
judgment of the AAALAC International Council. The exit
briefng offers the institution the opportunity to clarify any
possible misunderstandings about the program and to provide
additional information through post site visit communication
(PSVC). In general, issues corrected by PSVC should not be
of such signifcance that they entail signifcant expense until
such time that the formal recommendations from the Council
are sent by written correspondence to the institution. Rather,
corrections described in the PSVC should focus on those
concerns that can be corrected with the stroke of a pen or
require immediate attention to ensure the health and safety of
staff or the animals. The PSVC is considered by the Council
during their review of the site visit report and determination
of the institutions accreditation status.
Similar to other countries around the world, the function
of the IACUC and/or IAEC, with reference to the activities
stipulated by the Guide, were commonly identifed during
the exit briefng as incomplete and in need of amplifcation.
Specifcally, the conduct of semiannual program reviews
and facility inspections had not been fully implemented in
some cases. Less frequently, but still noted, the IACUC
and/or IAEC had not reviewed and approved standard
operating procedures and in a few cases had not established
clear, appropriate endpoints for studies impacting animal
welfare. These issues were fully addressed by PSVC. Also
corrected after the site visit were items such as ensuring
that animals were housed in cages in accordance with the
space recommendations in the Guide and Committee for
the Purpose of Control and Supervision on Experiments on
Animals (CPCSEA) Guidelines, ensuring daily observations
of all animals were performed and documented, ensuring
euthanasia procedures conformed with the recommendations
of the American Veterinary Medical Association (AVMA)
guidelines (AVMA, 2007), and ensuring that environmental
enrichment was consistently applied across the animal
program. In few cases some refnements to the occupational
health and safety program were recommended in the exit
Accreditation in India, Kathryn B.
briefng. For example, AAALAC site visitors occasionally
noted that waste anesthetic gas was not scavenged properly, or
that appropriate personal protective equipment was either not
available in certain areas of the facility or not used.
Concerns Expressed to Institutions in AAALAC
Correspondence
Of these numerous aspects of an animal care and use program,
the three most commonly identifed mandatory items requiring
correction worldwide are: 1) the occupational health and
safety program; 2) the function of the IACUC/IAEC; and 3)
the HVAC system performance. In the Pacifc Rim region the
ranking is different, so that the three most commonly identifed
program areas requiring correction are: 1) the function of the
IACUC; 2) the animal environment; and 3) the veterinary
care program. In India over the last decade, issues deemed
mandatory items for correction by AAALACs Council on
Accreditation were a blend of those observed globally and
more specifcally in the Pacifc Rim. They included physical
plant and equipment maintenance, cage space, sanitation
practices, HVAC function, IACUC/IAEC function, daily
animal observation, and a comprehensive and coordinated
program of veterinary care.
AAALACs site visit teams also offered several Suggestions
for Improvement over the many years of conducting accredi-
tation site visits in India. These included: discontinuing the
use of ether as an anesthetic; ensuring fuorescent light fxtures
had protective covers; discontinuing the use of picric acid
to identify rodents; ensuring the provision of raised resting
surfaces for dogs exposed to wet foors after cleaning proce-
dures; ensuring the provision of enrichment to singly housed
animals or correcting an inconsistent provision of enrichment
to animals across the program; discontinuing inappropriate
feed storage practices; implementing a health monitoring
program for animals housed long-term; and ensuring a safety
evaluation and categorization of test substances, as well
as associated procedures for the use and disposal of these
substances, to protect personnel. These suggestions typify
those offered to institutions throughout the Pacifc Rim region.
Conclusions
There are risks to not benchmarking the animal care and use
program against those of other institutions, and thereby not
working towards harmonizing animal care practices. Public
relations problems, increased costs, loss of innovation, and
possibly erosion of public and community trust may result
when an animal care and use program becomes stagnant. In
addition, potential collaborators or clients may look to place
their work elsewhere if they determine that the institution
does not meet an international level of quality. Thus, institu-
tions should determine the risks of not looking outward with
the value gained by learning from others. The AAALAC
International accreditation program is a valuable and integral
component of any quality assessment benchmarking program
because it stimulates an extensive internal review and
provides a comprehensive external review. Throughout the
process, areas of excellence are highlighted so that these may
be perpetuated and shared with others. Further, AAALAC
offers for consideration by the institution a path for continuous
improvement within the animal care and use program, with
the expectation that both animal welfare and science will
beneft. Institutional achievement of AAALAC International
accreditation denotes not only that a high standard of animal
care and use has been attained and validated, but also that its
program is comparable to hundreds of others around the world
that have also gained accreditation. In this way, AAALAC has
a key role in ensuring high standards and the global harmoni-
zation of animal care and use programs and in facilitating the
international exchange of reliable research data.
References
American Veterinary Medical Association (2007). AVMA
Guidelines on Euthanasia. http://www.avma.org/issues/
animal_welfare/euthanasia.pdf
Bayne K (2008). Animal Care and Use Programs: Global
Harmonization Through Alternatives. Special Issue,
March 31, 2008. AATEX .14:749-752.
Bayne K, Martin D (1998). AAALAC International: Using
performance standards to evaluate an animal care and
use program. Lab. Anim.. 27(4):32-35.
Bayne K, Miller J (2000). Assessing animal care and use
programs internationally. Lab. Anim.29(6):27-29.
ETS 123 (1986). European Convention for the Protection of
Vertebrate Animals used for Experimental and Other
Scientifc Purposes, Strasbourg, 18.III.1986. Text
amended according to the provisions of the Protocol
(ETS No. 170) as of its entry into force on 2 December
2005. http://conventions.coe.int/Treaty/en/Treaties/
html/123.htm
Federation of Animal Science Societies (2010). Guide for
the Care and Use of Agricultural Animals. http://www.
fass.org/page.asp?pageID=216
Klein H, Bayne K (2007). Establishing a culture of care,
conscience, and responsibility: Addressing the
improvement of scientifc discovery and animal welfare
through science-based performance standards. ILAR.J.
48(1):3-11.
Miller J (1998). International harmonization of animal
care and use: The proof is in the practice. Lab. Anim.
27(5):28-31.
National Research Council (1996). Guide for the Care and
Use of Laboratory Animals, Seventh Edition. National
Academy Press, Washington, D.C.
National Research Council (2010). Guide for the Care and
Use of Laboratory Animals, Eighth Edition. National
Academy Press, Washington, D.C.
Overview
Social, ethical and economical considerations as well as
technical limitations have constrained the study of physiology
and pathology directly in humans and higher primates.
Understanding biology from molecular to organismal level
has included both expansionist and reductionist approaches.
The problem has been in sythesizing them together because
the reductionist methods do not appropriately refect the
complex interactions between molecules, tissues, organs, and
organ systems in vivo. On the other hand, in silico modeling
or in vitro reproduction of complex phenotypes is almost
impossible. While gross pathology and exploratory studies
have taught us a lot about normal and abnormal processes,
microscopic and molecular studies have yielded the fner
details of the underlying mechanisms as well as how to
tackle diseases. Experimental animals have been used as
surrogates to create human-like systems. Unlike humans,
mice and rats can be manipulated by experimentally inducing
disease, and the diseased tissue can be sampled at various
stages when alive or post mortem. In addition, genetically
modifed or transgenic mice can be used for functional
analysis of gene function(s) in vivo. Such animal models
have provided important fundamental insights into biological
processes. Indeed, the slogan laboratory animals save more
lives than the emergency telephone number may not be an
overstatement (Baenziger et al. 2008).
Dr. Nagendra Hegde completed BVSc and MVSc from Veterinary College, Bangalore,
and PhD from University of Nebraska, USA. He then obtained post-doctoral training
from Oregon Health and Science University, USA, and worked as Research Assistant
Professor at the same place before returning to India. He has worked on immune
responses to and immune evasion by viruses aficting both man and animals, and
on virus-host interactions. His current interests are host-pathogen interactions,
and vaccines and diagnostics. He is bestowed with International Scientist of the
Year by International Biographical Centre, Cambridge, U.K. in 2002. He has pub-
lished several papers in national and international journals and holds two patents.
Currently, he is working as Group Leader (Virology) at Ella Foundation, Hyderabad.
Nagendra R. Hegde
The saga of the humanized mouse: a giant leap into
the galaxy or a small step for humankind?
Nagendra R. H.
Ella Foundation, Genome Valley, Turkapally, Shameerpet Mandal,
Hyderabad , India
Abstract
Laboratory animals have contributed greatly in understanding human physiology and pathology. However,
differences in biology do not allow us to defnitively interpret the results of experiments in animals.
Transplanting human cells and tissues into mice has therefore been one of the milestones in humanizing
laboratory animals. However, this necessitates complete replacement of the mouse system by the human
system, the most explored of which is the immune system. But this has not been easy as each phenotypic
deletion in mice has unraveled the complexity of host (mouse) defense mechanisms and at the same time shed
more light on the development of the human immune system. Because humanized immune mice lack the
armory to reject xenotransplants, they are being extensively used to implant and study other human systems,
including infectious diseases. This review summarizes the history of humanizing the mouse, the technical
advancements, the knowledge gained in the process, their use as well as the outlook in this fascinating and
rapidly advancing feld.
Keywords: humanized mice, transgenic, immunology
Nagendra R. Hegde, Ella Foundation, Genome Valley, Turkapally, Shameerpet Mandal, Hyderabad 500078,
Phone: 040-23480570, Fax: 040-2348-0571, email: hegden@ellafoundation.org
Saga of humanized mouse, Nagendra R. H.
Review
However, direct translation of the results from rodents to
humans is intricate. Although similarities in genome, protein
structure and function, and physiology are very high, humans
and mice differ substantially. Thus one of the greatest reasons
for major triumphs in mice often being lost in translational
research is that mice are not men (Mestas and Hughes
2004, Steinman and Mellman 2004; Lin, 2008). Moreover,
the phenotype of each inbred strain differs (Diwan et al.
1986; Zumbach et al. 2001), leading to potentially differing
interpretations of results. Further, the mouse immune system
rejects any transplant which is used study the biology of the
implanted cell, tissue or organ. This has led to the discovery
and engineering of several strains of mice which now serve as
the basis to reconstitute human biological systems. However,
the progress has been slow because ablation of a specifc arm
of the immune system leaves another arm at freedom to attack
the transplant. Based on the knowledge accumulated over the
years, humanized mice have been generated to investigate
human hematopoiesis, innate and adaptive immunity, autoim-
munity, infectious diseases, cancer biology, stem cell biology
and regenerative medicine. However, further improvements
are needed to achieve completely humanized mice.
A crash course in immunology
In order to understand the history of humanized mouse, it is
essential to understand the biology of the immune system. The
lymphoid organs can be divided into primary and peripheral.
The primary lymphoid organs bone marrow (BM) and
thymus generate and educate lymphocytes. While all
lymphocytes originate in the BM, T and B cells mature in the
thymus and the BM, respectively. The peripheral lymphoid
organs mainly spleen and lymph nodes (LN) initiate and
sustain specifc responses. The cells of the immune system
include granulocytes (neutrophils, basophils, eosinophils),
lymphocytes, monocyte-macrophages, dendritic cells (DCs),
natural killer (NK) cells and mast cells.
Immunity can be classifed into innate and adaptive. Innate
immunity is inborn and not specifc to any pathogen. It is
initiated by the activation of a group of receptors, including
toll-like receptors (TLRs), by molecular patterns frequently
encountered on pathogens. A variety of soluble factors,
including lysozyme, interferons (IFNs), complements,
collectins and acute phase proteins as well as phagocytes and
NK cells contribute to innate immunity. Complements, which
are activated either by certain pathogen molecules or via
antibodies bound to pathogens, function by killing pathogens
directly or by recruiting and/or aiding in the function of
phagocytes that then clear the pathogens. IFN-, or block
viral replication and also activate cells of both innate and
adaptive immunity. NK cells act by recognizing aberrant cells
and responding rapidly with the release of cytolytic granules
and cytokines.
Adaptive immunity is initiated only upon exposure to antigen,
and is characterized by specifcity, diversity, memory, and
self/non-self recognition. The specifcity of each T and B cell
is determined during maturation in the thymus or BM even
before its contact with antigen. By a combination of multiple
gene rearrangement and mutations, adaptive immunity
generates an enormous diversity to recognize an infnite
variety of antigens. This generation of diversity in B and T
cell receptors (TCR) is mediated by recombination activating
gene 1 and 2 (enciphered by Rag1 and Rag2 genes) as well
as other proteins. Exposure to antigen activates T and B cells,
and expands the population of cells with a given antigenic
specifcity, and produces effector (immediately functional)
and memory (long-lasting) cells. Unlike B cells which
recognize antigen directly, T cells can only recognize antigen
bound to major histocompatibility complex (MHC) molecules
(human leukocyte antigen or HLA in humans), and presented
by antigen presenting cells (APC). There are two classical
MHC molecules: class I, expressed by nearly all nucleated
cells of vertebrate species, and class II, expressed only by
specialized APC (B cells, monocytes, macrophages, DCs).
Both are heterodimeric, class I containing a heavy chain and
2
-microglobulin (
2
m), class II made of and chains. T
cells are educated to recognize non-self antigens presented by
self MHC molecules.
Antibodies, or immunoglobulins (Igs), of various types and
subtypes, are the effector molecules of B cells. IgM is the
frst Ig produced upon exposure to an antigen (a primary
response), as well as in neonates. IgG is the most abundant Ig
in the serum, appears after IgM, is predominant in secondary
or booster responses, and is produced by a phenomenon called
class switching. Antibodies carry out their functions by inhib-
iting the invasion of pathogens, or by activating complements,
or by aiding macrophages and NK cells to engulf or destroy
pathogens.
There are two major subpopulations of T cells: helper (Th) and
cytotoxic (Tc), which carry the surface molecules CD4 and
CD8, and are hence typically referred to as CD4+and CD8+
T cells, respectively. Stimulated Th cells secrete cytokines
which activate various cells, including B cells, Tc cells,
macrophages, and other immune cells. Th cells are further
divided into Th1 and Th2, which originate from the common
precursor Th0, depending on the initial signals and cytokine
environment. Typically, Th1 and Th2 cells polarize immunity
towards cell-mediated and antibody responses, respectively.
The Tc cells exhibit cytotoxic activity mediated through the
exocytosis of granules containing granzyme and perforin, and
inhibit intracellular pathogens via the secretion of IFN-, thus
playing a vital role in monitoring and eliminating aberrant
cells, such as virus-infected cells, tumor cells, and cells of a
foreign tissue graft. The other important T cell subpopulation
is the regulatory T cell (Treg), which acts in controlling
immune responses by regulating various pathways. NK cells
function like Tc cells but are a part of innate immunity. NK
cells express two distinct families of receptors: the activating
receptors and the inhibitory receptors, belonging to one of
the CD94/NKG2, Ly49, killer-cell Ig-like receptor (KIR), or
leukocyte immunoglobulin-like receptor (LIR) families. In
general, the most important inhibitory signal for NK cells is
the presence of MHC class I molecules. Lack or absence of
MHC class I molecules is the most important reason for NK
cell activation, and mice lacking 2m have NK cell defciency
as they lack education during NK cell development.
All the cells of the immune system derive from the same
Vol -1, Issue - 1, Jan - Apr 2011 10
precursor cells, the hematopoietic stem cells (HSC), in the
BM. HSCs are a heterogeneous population of cells with
long-term and short-term regeneration capacities and differ-
ential repopulation kinetcs. In vivo, they are mostly found in
the BM, but can be isolated from several fetal tissues (liver,
spleen, aorta, and gonads), umbilical cord blood, placenta,
peripheral blood as well as adult tissues like skin. The HSCs
can be defned by the cell surface expression of specifc
markers, including CD34, and the absence of other markers,
including CD38. Contact with stromal cells and soluble
mediators secreted by them are necessary for HSCs to differ-
entiate. Two major progenitor lines, myeloid and lymphoid,
generate the hematopoietic cells. The myeloid progenitor
undergoes thrombopoiesis (platelets), erythropoiesis (eryth-
rocytes), granulopoiesis (granulocytes), monocytopoiesis
(monocyte-macrophages, DCs) and differentiation into mast
cells. Granulocytes are produced in increased numbers and
migrate from blood to sites of infection or infammation.
Macrophages and DCs act as the major initiators of specifc
immunity.
The lymphoid progenitor produces T, B, and NK cells. T
lymphopoiesis occurs in the thymus and absolutely requires
contact with thymic stromal cells and the subsequent signal
transduction events. The T cells then pass through a series
of steps involving no, overlapping or exclusive expression
of CD4 and CD8, during which, TCRs undergo gene
rearrangement. The T cells, each being clonal but collectively
bearing a vast array of specifcities, come in contact with self
MHC molecules and undergo selection and retention to react
to non-self antigen (i.e., self-reactive T cells are deleted).
The T cells can belong to one of the (conventional) or
type based on the kind of TCR that they express. Other types
include NKT and regulatory T cells.
B lymphopoiesis occurs in the BM, whose niche of stromal
cells, extracellular matrix and cytokines is essential for the
process. B cell development also goes through a series of
well defned steps which can be recognized by the surface
expression of particular markers. Further maturation of B
cells occurs in spleen and lymph nodes. Similar to T cells,
B cells rearrange their Ig genes to generate antibodies with a
vast variety of specifcities. These are then selected specif-
cally on contacting the antigen in secondary lymphoid organs.
The identity of human NK cell precursors, their location, and
factors involved in development are not as well defned. Fetal
NK cell precursors arise in the liver, thymus, BM, and LN,
and the BM is the major location of NK cell development in
adults. Different HSC cell populations can generate mature
NK cells in the presence of stromal cells and cytokines, and
transcription factors play a critical role in the development of
NK cells.
The generation, maturation, differentiation and effector
functions of various immune cells are initiated or regulated
by a variety of soluble mediators. The principle ones are the
colony stimulating factors (CSF), interleukins (IL), IFNs,
and tumor necrosis factor (TNF). CSFs stimulate HSCs to
differentiate into various blood cell lineages, and include
macrophage, granulocyte and granulocyte-macrophage
CSFs as well as erythropoietin and thrombopoietin. Among
the IFNs, only IFN- is secreted exclusively by cells of the
immune system. It protects cells by initiating molecular
antiviral pathways, and along with TNF-, an infammatory
cytokine, it is involved in activation and regulation of innate
and adaptive immune cells.
Interleukins are a group of >30 cytokines which regulate
the generation, differentiation and function of immune cells.
IL-1, produced by many cells, particularly macrophages and
epithelial cells, performs multiple immune and non-immune
functions, including the initiation and regulation of acute
infammation. IL-2 is a T cell growth factor, and in synergy
with IL-15, it induces and regulates T and NK cell activation,
proliferation and function. IL-3 and IL-7 are mostly produced
by stromal cells and promote commitment and differentiation
of myeloid and lymphoid lineages, respespectively. IL-4
and IL-13 induce Th0 cells to differentiate into Th2 cells,
and their action is further supported by IL-10, a pleiotrophic
immune regulator. IL-6 has multiple effects, the principal of
which is end stage B cell differentiation. IL-8 is a neutrophil
chemotactic factor. IL-9 supports IL-2- and IL-4-independent
growth of Th cells. IL-11 supports megakaryopoiesis and
osteoclast activation. IL-12, produced by APCs upon antigen
stimulation, polarizes Th0 cells to differentiate into Th1 cells,
which in turn secrete IFN-. IL-17, along with IL-1 and
TNF-, mediates delayed type and infammatory reactions,
is produced by Th type 17 (Th17) cells. IL-17, IL-21, IL-22
and IL-23 control the differentiation and function of Th17
cells. IL-10 and TNF- are the major effectors of immune
regulation mediated by Treg cells.
The interleukins function by binding mostly unique, and
sometimes shared, cell surface receptors, many of which have
oligomeric components. The IL-2 receptor (IL2R) common
-chain (IL2Rc), is a common receptor subunit used by
IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, and is indispensable
for high-affnity ligand binding and signaling of all these
cytokines. Knocking out IL2Rc therefore cause profound
immunodefciency. The defciencies in the immune system
can be generated by knocking out genes encoding immune
effector molecules like granzyme and perforin B or specifc
cytokines or CSFs. These immunodefcient mice have played
an important role in the study of in vivo function of human
cells and tissues.
Humanization of mice: the genesis (or
transgenesis) and refnement
The humanized mouse is one in which human proteins
are transgenically expressed or human tissue is transplanted
(Nomura et al. 2008; Pearson et al. 2008). Transgenesis can
be used to (a) study immune responses, e.g., identifcation of
antigenic epitopes presented by HLA molecules, (b) study
infectious diseases specifc to humans through the expression
of virus-specifc receptors, or by knocking out genes encoding
IFNs or their receptors, or TLRs, and (c) validate and further
evaluate autoimmune and infammatory diseases. Study of
several diseases, including those caused by hepatitis C, human
corona, dengue and other viruses, as well as rheumatoid
Vol -1, Issue - 1, Jan - Apr 2011 11
arthritis, spondylitis, diabetes, encephalomyelitis etc. has
been facilitated by these mice. Alternatively, engraftment of
immunodefcient mice with human cells and tissues permits
investigation of the development and function of these tissues
in vivo, and the study of diseases.
Implantation of human cells and tissues (humanization) started
with athymic nude mice, which lack both T and B cell functions
(Segre et al. 1995), and other immunodefcient mice, either
individually or in combination. However, initial attempts to
engraft human hematopoietic cells were not successful. The
breakthrough was the discovery of CB17-scid mice (Bosma
et al. 1983), which contain a spontaneous mutation in the
DNA-dependent protein kinase gene Prkdc (protein kinase,
DNA activated, catalytic polypeptide), which is involved in
the DNA double-strand repair and in the rearrangements of
TCR and Ig gene segments (Blunt et al. 1995, Kirchgessner
et al. 1995; Miller et al. 1995). Consequently, the mice lack
T and B cell progenitors, and are unable to reject grafted
cells or tissues. Rag1 and Rag2 mutations also result in a
similar phenotype (Mombaerts et al. 1992; Shinkai et al.
1992). Later, it was demonstrated that human fetal tissues and
peripheral blood mononuclear cells (PBMC) could engraft to
produce human hematopoietic cells in these mice (McCune et
al. 1988; Mosier et al. 1988).
The Scid, Rag1 or Rag2 mice, however, retain innate
immunity, posing a signifcant barrier to acceptance of the
graft, especially of hematopoietic origin (Christianson et al.
1996). The major reason is the high level of host NK cell
activity, to deplete which, antibodies to asialoGM1 were used
unsuccessfully as this also targets human NK and activated
CD8
+
T cells and macrophages present in the graft inoculum
(Habu et al. 1981; Yoshino et al. 2000). On the other hand,
IL-2 receptor -specifc antibodies, which target murine, and
not human, NK cells, are a better option (Tanaka et al. 1993).
Other approaches have targeted neutrophils with anti-Gr1
antibodies, and macrophages with liposome-encapsulated
clodronate (Pearson et al. 2008). Genetic attempts to reduce
innate immunity have included combining scid mutation with
macrophage abnormalities, reduction in complement activity
or defects in NK cell function, e.g., generation of NOD
(non-obese diabetic)-scid mice (Lowry et al. 1996; Pfumio
et al. 1996; Shultz et al. 1995; van der Loo et al. 1998).
Efforts to further decrease innate immunity have included
targeted mutation in
2
m (Christianson et al. 1997; Zijlstra
et al. 1989), perforin (Kagi et al. 1994; Shultz et al. 2003) or
granzyme B (Shresta et al. 1995) genes, leading to defciency
or reduced toxicity of NK cells.
An obstacle to the development of human hematopoietic
cell functions is the lack of cross-reactivity of many mouse
hormones, growth factors, and cytokines for the development,
survival, and function of these cells (Auffray et al. 1994;
Uze et al. 1990). In addition, many required human-specifc
factors are produced by nonhematopoietic stromal cells not
present in the inoculum. Exogenous factors important for the
engraftment, homing, and differentiation of human HSC as
well as the function or regulation of the differentiated cells
have been used in an attempt to overcome these obstacles
(Pearson et al. 2008; Chen et al. 2009). Alternate approaches
have included transgenic expression (Bock et al. 1995;
Nicolini et al. 2004) or manipulating the human HSC inoculum
ex vivo, especially with cocktails containing multiple human
cytokines (Pearson et al. 2008). The latter approach has been
hampered by the diffculty in achieving ex vivo expansion
of human CD34+HSC without loss of their capacity to
repopulate stem and progenitor cells in vivo (Sorrentino,
2004). Another approach is to use lineage-specifc differen-
tiation cytokines to drive human stem and progenitor cells ex
vivo (Ishikawa et al. 2005; van Hensbergen et al. 2006). One
more method is to co-inject HSC with human mesenchymal
stem cells (MSC), somatic stem cells, or cytokine-transduced
stromal cells to provide a stromal environment (Ramasamy et
al. 2007; Ringden et al. 2006; Pearson et al. 2008).
Other obstacles in the frst generation immunodefcient mice
were the the development of murine T and B cells on aging
(referred to as leakiness), high occurrence of thymomas and
hence short life-span, and the inability to generate human T
cells (Bosma 1992; Custer et al. 1985; Ito et al. 2008a; Pearson
et al. 2008; Shultz et al. 2005). The most recent breakthrough
has been the development of mice carrying a mutation in the
IL2R chain (Il2rg or Il2rgc) gene, leading to severe impair-
ments in innate and adaptive immunity, including comple-
ments, and differentiation and function of APCs (Cao et al.
1995; DiSanto et al. 1995; Ohbo et al. 1996). Combining
these with NOD/Shi-scid or NOD/LtSz-scid phenotype has
resulted in NOG and NOD/LtSz-scid IL2rg mice, respectively,
leading to extreme immunodefciency derived from the three
parental strains of mice: (a) reduced innate immunity derived
from the NOD strain, (b) lack of functional T and B cells due
to the scid mutation, and (c) NK cell, DC, and other unknown
defciencies resulting from inactivation of the IL-2R gene.
Lymphoid organs, including thymus and LNs, in both NOG
and NOD/LtSz-scid IL2rg null mice are immature, atrophic
and rudimentary. NOG mice can live as long as conventional
inbred mice under strict specifc pathogen-free conditions
(Pearson et al. 2008) whereas half of the NOD/LtSz-scid
IL2rg null mice die within one year (Shultz et al. 2003).
The incidence of thymoma in NOG mice is much lower than
that in NOD/LtSz-scid IL2rg null mice (Ito et al. 2008a;
Prochazka et al. 1992). Similarly, little leakiness is observed
in NOD/Shi-scid mice, with no leakiness at all in NOG mice
(Ito et al. 2008a). These mice show fairly complete human
immune systems, including thymocytes and peripheral mature
T and B cells, myeloid cells, myeloid and plasmacytoid DCs,
platelets, and erythrocytes (Ito et al. 2002; Shultz et al. 2005;
Traggiai et al. 2004). T cells that develop in these mice have
a diverse TCR repertoire and direct antigen-specifc IgM and
IgG responses after immunization with T-dependent antigens
(Shultz et al. 2007) in newborn and adult recipients, and by
different routes of injection (Ito et al. 2008a). Therefore, these
mice are closest to being humanized.
Applications of humanized mice
Hematopoiesis
Transplantations of immunodefcient mice are the most
reliable methods to evaluate stages of human hematopoiesis
Vol -1, Issue - 1, Jan - Apr 2011 12
and the function of human HSCs (Shultz et al. 1995;
Larochelle et al. 1996). However, despite their ability to
produce all mature lineages (Galy et al. 1995; Yahata et al.
2006), HSCs have limited self-renewal capacity in this setting
(Ema et al. 2005), possibly due to the lack of a special niche
(Ando et al. 2008). Stem cell niches and key molecules which
regulate niche function have been identifed in mice (Arai
et al. 2004; Calvi et al. 2003; Kiel et al. 2005; Sugiyama et
al. 2005; Zhang et al. 2003), but this may not translate to
humans. In vitro, MSCs can give rise to cells that produce
a number of cytokines and extracellular matrix proteins, and
express cell adhesion molecules, all of which contribute to
the hematopoietic microenvironment (HME) (Conget and
Minguell, 1999; Majumdar et al. 1998; Pittenger et al. 1999).
However, little is known about their in vivo phenotypic and
functional characteristics. Although MSCs accelerate the
hematopoietic recovery of cotransplanted human HSCs (Koc
et al. 2000; Noort et al. 2002), and donor MSCs may exist in
recipient BM (Bensidhoum et al. 2004) and reconstitute HME
(Muguruma et al. 2006), there is no concrete evidence that the
transplanted MSCs indeed engraft and function directly
Self-renewal, multilineage differentiation, frequency, surface
marker expression, homing, hierarchy, and dynamic behavior
have been demonstrated in scid mouse repopulation assays
(Bhatia et al. 1997, Guenechea et al. 2001; McKenzie et al.
2006; Peled et al. 1999). However, in NOD-scid mice, CD34
+
cells appear to reconstitute only B-lymphoid and myeloid cells
and not T cells, although it can be enhanced by exogenous
addition of human IL-7 (Hiramatsu et al. 2003; Shultz et
al. 2005). On the other hand, NOD-scid/2m
/
or RAG2

/
c
/
mice demonstrate human T cell development and
functionality (Hiramatsu et al. 2003; Ishikawa et al. 2005;
Shultz et al. 2005; Traggiai et al. 2004; Yahata et al. 2002).
A lymphoid-like structure develops in the spleen and contains
human macrophages and DCs (Watanabe et al. 2007), but
follicular DCs are not observed (Ito et al. 2008a). T cell areas
and B cell follicles are observed in the spleen, and B cells
differentiate frst, followed by development of T cells (Ito et
al. 2008a). The mice produce antigen-specifc IgM, but not
much IgG, even with multiple immunizations (Traggiai et al.
2004; Legrand et al. 2006), possibly due to the overwhelming
repopulation of B1 type B cells (Matsumura et al. 2003),
which are known to produce mainly IgM (Kipps, 1989).
Human T cells generated in these transplanted mice include a
fairly normal ratio of CD4
+
and CD8
+
cells with broad TCR
diversity, regulatory and cells (Ito et al. 2008a). Mature T
cells exit the thymus and home to secondary lymphoid organs,
and stages of T cells are consistent with T cell development in
humans (Hiramatsu et al. 2003, Matsumura et al. 2003, Yahata
et al. 2002). However, human T cells are educated by mouse
thymic stroma in these mice. In addition, myeloid lineage
cells are not well generated: erythrocytes, neutrophils and
platelets are too few in number, although some fully matured
erythrocytes can be observed in newborn NOD/LtSz-scid
IL2rg null mice (Ishikawa et al. 2005; Nakamura et al. 2006).
As far as NK cell development is concerned, even though
differentiated human NK cells arising from the graft express
most of the cell surface antigens and activation functions
found with fresh human NK cells (Huntington and Di Santo
,2008), inhibitory receptors and their MHC ligands (Anfossi
et al. 2006; Yokoyama and Kim, 2006) are absent. Another
factor to consider is the unavailability of human IL-7 and
IL-15, which are required for NK cell generation and survival
(Kennedy et al. 2000; Huntington and Di Santo, 2008;
Vossehenrich et al. 2006).
Adaptive immunity
Initial studies to induce human antibody production in
immunodefcient mice implanted with human PBMC (Abedi
et al. 1992; Mosier et al. 1988; Sandhu et al. 1994) were
unsuccessful because of graft versus-host disease (GVHD)
(Ito et al. 2008b). GVHD was reducely greatly by decreasing
the number of engrafted PBL, but antigen-specifc antibodies
were almost undetectable (Sandhu et al. 1994). In NOD-scid
or NOD-scid/2m
/
or NOG mice, the developed human
CD4
+
or CD8
+
T cells express mature T cell markers and
produce cytokines when stimulated with ionophores or
superantigens, or via TCR ligation (Saito et al. 2002; Yeoman
et al. 1993). However, spenic T cells from immunized mice
lack the ability to produce IL-2 or proliferate. Further,
although serum IgM and IgG levels are high, the level of
antigen-specifc IgG is extremely low (Ishikawa et al. 2005;
Ito et al. 2008b). This rudimentary adaptive immunity is
presumably because human T cells are positively selected
by the murine MHC in the thymus of these mice (Ishikawa
et al. 2008). To overcome the impediment, one can express
human MHC transgenically (Faulkner et al. 1998; Friese et
al. 2006) or engraft human lymphoid tissues (e.g., embryonic
thymus) to direct the selection of human T cells. Indeed, when
adult LNs or embryonic liver, thymus, or skin are engrafted
into NOG mice which are then immunized with antigen,
human immune cells proliferate and are activated effciently
with given antigens (Carballido et al. 2000; Ishikawa et al.
2008). In addition, in NOD-scid-IL2R null mice, signifcant
amounts of IgG and IgM specifc to T-dependent antigen
are detectable, suggesting functionality and effective class
switching (Ishikawa et al. 2008). Recent studies also show
the generation of functional T cell subsets specifc to a viral
pathogen (J aiswal et al. 2009; Shultz et al. 2010). Of great
clinical signifcance is the ability to easily generate completely
human monoclonal antibodies for therapeutic use (Becker et
al. 2010).
Infectious Disease
Humanized mice are very useful in the investigation of
human-specifc infectious diseases, including viral infec-
tions caused by human immunodefciency, herpes (human
cytomegalovirus, Epstein-Barr virus), hanta, Chikungunya,
hepatitis C, dengue and other viruses, as well as parasites
such as Plasmodium. Scid mice coimplanted with human fetal
thymus and liver tissues show high susceptibility to human
immunodefciency virus type 1 (HIV-1) infection (Aldrovandi
et al. 1993; Namikawa et al. 1988), and can be used to assess
effcacy of anti-HIV compounds (McCune et al. 1990; Rabin
et al. 1996). However, direct injection of virus into the implant
is required because of the low numbers of circulating CD4
+
T cells, and no antiviral immune response is observed. Scid
mice injected with PBMC from healthy adults can also be
Vol -1, Issue - 1, Jan - Apr 2011 13
used (Mosier et al. 1991; Nakata et al. 2005), but these mice
develop GVHD (Sandhu et al. 1995), and both T cell phenotype
and coreceptor usage by HIV-1 do not refect that in humans
(Mosier et al. 1993; Nakata et al. 2005). Nevertheless, some
immune responses are induced in these mice (Gorantla et al.
2005). Further, MHC-restricted anti-HIV-1 T cell responses
can be elicited by using autologous skin that contains tissue
DC (Delhem et al. 1998). Similarly, inactivated HIV-1-
pulsed, human monocyte-derived DC (MDDC) can elicit
partially protective anti-HIV-1 antibody production (Santini
et al. 2000). The lack of a suitable HME in these mice can
be overcome by transferring PBMC together with inactivated
HIV-1-pulsed autologous MDDC directly into the mouse
spleen (Yoshida et al. 2003). This reduces excessive GVHD,
produces larger yields of human T cells, elicits a protective T
cell immunity, and the sera from the immunized mice contain
a soluble HIV-1 suppressive factor produced by human
antigen-specifc CD4
+
T cells (Yoshida et al. 2005).
The HIV-1 replication occurs at high levels and the virus
persists for a long time without any GVHD in the human
hematopoietic cell reconstituted NOD/Shi-scid mice
(Koyanagi et al. 1997a; 1997b). NOG (Nakata et al.
2005; Watanabe et al. 2007) and Rag2 null Il2rg scid mice
(Baenziger et al. 2006; Gorantla et al. 2007; Ince et al. 2010;
Sun et al. 2007) have long-lasting infection of human cells
by HIV-1 as well as HIV-specifc human immune responses.
Plasma viral RNA and antigen load, stabilization of viremia,
cell tropism, quasispecies generation, CD4
+
T cell depletion
and sometimes syncytium formation all recapitulate what
happens in humans, including late-stage disease. However, no
robust IgG or T cell responses are detected, dampening the
excitement about testing vaccines and antivirals (Koyanagi et
al. 2008).
The NOG mice are susceptible to infection with Epstein-Barr
virus (EBV), a causal agent of epithelial carcinomas and B
cell lymphomas, and human T-lymphotropic virus type I
(HTLV-I), which causes adult T-cell leukemia/lymphoma
(ATL). EBV DNA can be detected in the peripheral blood and
spleen cells, EBV
+
B lymphoblastoid cells can be isolated
from NOG mice, and specifc immunity can be elicited
(Melkus et al. 2006, Mosier et al. 1989; Shultz et al. 2010).
The NOG mice develop clinical signs following inoculation
of ATL cells, with massive infltration of cells in various
organs (Dewan et al. 2006; Imada et al. 1995), providing a
model for testing targets for ATL therapy (Dewan et al. 2003;
Dewan et al. 2006; Mori et al. 1999).
Immunodefcient mice can also be used to reproduce the
pathogenic process and clinical manifestations of disease,
especially in the case of agents which do not cause any
disease in laboratory animals. For example, immunodefcient
mice expressing HLA molecules and lacking endogenous
MHC class I molecules, are able to produce symptoms of
Lassa fever. Further, the exacerbation of disease following
depletion of human T cells revealed the hitherto unknown
possibility of immunopathology in this disease (Flatz et al.
2010). Work with dengue virus has also shown recapitulation
of human-like disease and genotype-dependent severity
(Bente et al. 2005; Mota and Rico-Hesse, 2009) as well as
specifc antibody and T cell responses (Jaiswal et al. 2009).
Other examples include symptoms of paralysis induced by
poliovirus infection (Horie et al. 1994; Ito et al. 2002) and
induction of streptococcal impetigo (Scaramuzzino et al.
2000).
Cancer
Immunodefcient mice have been used to study cancer
biology for over three decades (Giovanella and Fogh, 1985;
Shultz et al. 2007), but the early strains (nude and scid) could
only support a limited number of tumors (Friese et al. 2006;
Hudson et al. 1998). The development of NOD-scid or NOD/
Ltz-scid IL2rg mice has permitted the study of many primary
human lymphomas and leukemias as well as liver metastatic
models (Dewan et al. 2004; Dewan et al. 2005; Ishikawa et al.
2007; Suemizu et al. 2007).
Hematogenous metastasis models, which mimic events that
occur after cells enter the blood, are useful to understand
mechanisms of human gastrointestinal, hepatic and pulmonary
cancers (Nakamura and Suemizu, 2008). Several models of
liver metastasis have been established in nude mice (Bresalier
et al. 1987; Nomura et al. 2002), but it is unlikely that such
a large number (> 106) of cells would enter the liver simul-
taneously and form metastatic foci in patients (Nakamura
and Suemizu, 2008). The incidences of liver metastases in
NOG mice are very high and reproducible, and the lesions are
dose dependent over a wide range of inoculum, but are better
produced by the elimination of NK cells (Nakamura and
Suemizu, 2008). Lymphatic metastasis models are diffcult
to develop as the cancers need to frst invade the lymphatic
vessels and metastasize to regional LNs, whose structures are
not developed in immunodefeicnt mice. On the other hand,
dissemination in body cavities, a late event of malignancy,
is much easier to develop, but it gives limited information
for targeting therapy for early-stage cancer (Nakamura and
Suemizu, 2008). Several human melanoma cell lines are
reported to metastasize in nude mice (Cornil et al. 1989;
Iliopoulos et al. 1989), albeit at low rates. On the other hand,
metastasis is high in NOG and NOD/Shi-scid mice (Nakamura
and Suemizu, 2008).
The concept of tumor stem cells, small number of cells with the
capacity to self-renew and differentiate to give rise to tumor
(Shultz et al. 2007), has also been validated with humanized
mice (Passague et al. 2003; Reya et al. 2001; Warner et al.
2004) for myeloma, and breast, brain and pancreatic cancers
(Al-Hajj et al. 2003, Li et al. 2007, Pilarski and Belch, 2002;
Singh et al. 2004). This concept could be critical in treating
early stage cancers, rather than targeting reduction of tumor
mass (Nakamura and Suemizu, 2008). Molecular mecha-
nisms of carcinogenesis have also been explored with these
and transgenic mice (Mitsumori et al. 1997; Nakamura and
Suemizu, 2008).
Stem Cells and Regenerative Medicine
The scid mice have been used extensively as models for
human stem cell transplant (Greiner et al. 1998). BM cells
possess the capacity to generate hepatocytes, hematopoietic
Vol -1, Issue - 1, Jan - Apr 2011 14
cells, epithelial cells, cardiomyocytes as well as functional
pancreatic beta cells (Camargo et al. 2004; Fujino et al. 2007;
Harris et al. 2004; Hess et al. 2003; Ianus et al. 2003; Ishikawa
et al. 2006; Ishikawa et al. 2008; Krause et al. 2001; Ma et
al. 2006; Petersen et al. 1999; Wang et al. 2005). Several
investigators have also reported the differentiative capacity of
human MSCs in vivo (Ishikawa et al. 2008; Sato et al. 1999;
Toma et al. 2002). Other studies include reconstitution of
human endometrium, resulting in induction of human sexual
hormone-dependent menstrual cycle (Masuda et al. 2007;
Matsuura-Sawada et al. 2005), and therapeutic evaluation of
thrombopoietics (Nakamura et al. 2006). Recognition that
transdifferentiation and/or cell fusion of transplanted HSC
also occurs in damaged tissues has led to intense investigation
in this area (Ishikawa et al. 2008; Prockop et al. 2003; Shultz
et al. 2007).
Autoimmunity
The study of autoimmune diseases has benefted extensively
by animal models, where, the genetic basis for the disease can
be identifed, the genome can be altered, and therapies can
readily be tested without ethical concerns (Pearson et al. 2008).
Humanized mice have been used for the study of autoimmune
type 1 diabetes, thyroiditis, and rheumatoid arthritis (Shultz
et al. 2007). In diabetes, PBMC from diabetic individuals
adoptively transferred to CB17-scid mice led to the detection
of autoantibodies to islet components, but no infltration or
beta cell destruction (Petersen et al. 1993). In studies of the
adoptive transfer of human T cell clones with specifcities to
islet autoantigens into NOD-scid mice, infltration, but not
islet cell destruction was detected (van Halteren et al. 2005).
The use of newer models based on the IL2rg null mutation
and transgenic expression of human HLA molecules should
permit the direct study of human autoreactive T cells in vivo.
Drug metabolism and pharmacology
Metabolism of pharmacological compounds is mainly
mediated by the liver, whose enzymes, including cytochrome
P450, display tremendous heterogeneity as well as species-
specifcity. Transgenic immunodefcient mice in which
endogenous hepatocytes are depleted through genetic altera-
tions causing hepatocyte damage and human hepatocytes are
transplanted to repopulate the liver (Katoh and Yokoi, 2007;
Meuleman et al. 2005; Strom et al. 2010) serve as excellent
models to study receptor specifcity, metabolism, toxicity
and excretion (Cheung and Gonzalez, 2008; Kamimura et al.
2010; Yoshizato and Tateno, 2009).
The road ahead
There are three broad limitations in humanized mice: (a)
many human-specifc molecules are absent, (b) remaining
innate immune mechanisms present obstacles to engraftment,
and (c) the architecture of the lymphoid system remains
undeveloped (Ito et al. 2008a; Pearson et al. 2008). These
can be forfended by (a) the introduction of human genes that
code for growth or differentiation factors, (b) the depletion
of macrophages, DCs, mast cells, neutrophils, etc., or their
differentiation factors, and (c) the introduction of HLA genes
(Ito et al. 2008a).
The expression of human-specifc genes, especially the HLA
genes (Camacho et al. 2004), is of great importance for
thymic selection. However, the expteme polymorphism of
HLA means that multiple HLA genes may need to be intro-
duced, by using, for example, artifcial chromosomes (Chen
et al. 2009; Kuriowa et al. 2002). Additional molecules such
as cytokines and growth factors should facilitate the devel-
opment, differentiation, and survival of a functional human
immune system. Genes related to infammatory responses
and allergic reactions, e.g., IL-4 and IL-5, may be useful in
developing disease models (Wills-Karp, 1999).
Both genetic approaches and exogenous administration of
reagents can be used to further depress innate immunity.
Better engraftment has already been achieved through the
ablation of remaining cells, either partially or completely, by
various techniques (Heyman et al. 1989; J ung et al. 2002;
Kamogawa et al. 1993; Saito et al. 2001; Santini et al. 1998).
For example, transgenic expression of simian diphtheria
toxic receptor can selectively deplete DCs, macrophages, and
other cells in combination with exogenous administration of
diphtheria toxin (Bennett et al. 2005; Duffeld et al. 2005;
J ung et al. 2002; Matsumura et al. 2004), to which mouse
cells are relatively insensitive (Cha et al. 2003).
Poor secondary lymphoid organ development is another
concern in humanized mice. Appropriate lymphoid archi-
tecture requires interaction of follicular DCs with lympho-
cytes and other nonlymphoid cells for full development (Fu
et al. 1997a; 1997b). Engraftment of human stromal cells,
artifcial thymic stroma or biocompatible scaffolding may
facilitate restoration of the lymphoid structure (Poznansky et
al. 2000; Suematsu and Watanabe, 2004). In addition, trans-
duction of BM or thymic stromal cells with factors that are
needed for human hematopoiesis or lymphocyte development
may prove useful (Brenner et al. 2004). Additional molecules,
such as adhesion and homing molecules expressed on tissues
in the host, may overcome some of the issues associated with
homing of human immune cells. The genes for lymphotoxin-
related molecules and chemokines that are necessary for
reconstruction of human secondary lymphoid organs may
also be targets for introduction (Fu et al. 1997b; Suemetsu
and Watanabe, 2004).
Distant metastasis of malignant neoplasms consists of
entrapment in vessels, invasion of the basement membrane,
destruction of the vasculature, and entry into blood or lymph
(Nakamura and Suemezu, 2008). Metastatic models only
refect late events, i.e., after adhesion or entrapment in organs.
Models for initial steps or local invasion mechanisms at the
primary lesion of the cancer must be developed (Nakamura
and Suemizu, 2008). Further, developments in in vivo
imaging techniques could help in the study of events as they
happen (Masuda et al. 2008).
Recent advances have allowed us to identify genes responsible
for self-renewal of stem cells (Seita et al. 2007; Yamazaki
et al. 2007), and their introduction into mice may make it
possible to maintain human stem cells for a long time. As
far as stem cells and regenerative medicine are concerned,
there is a clear need for targeting research into replacement
Vol -1, Issue - 1, Jan - Apr 2011 15
of tissues constituting organs or areas of extensive loss. In
addition, infammatory and autoimmune disorders, which
involve immunological disturbances, need to be addressed
separately as both regenerative approaches and normalization
of immune system may be required.
One other issue that needs to be addressed is the husbandry
of immunodefcient mice. Infections with microbes including
mouse hepatitis virus and Staphylococcus aureus in nude
mice and Pneumocystis carinii (P. carinii) in scid mice are
a constant problem (Nomura et al. 2008). Ordinary rearing
environment can cause mortality in NOG mice. For example,
contamination of transplated human tumors with Pseudomonas
aeruginosa and spread to the rearing environment via drinking
water, researchers, or caretakers has been observed (Nomura
et al. 2008). Indeed, nude and scid mice show resistance to
P. aeruginosa and P. pneumotropica but NOD-scid and NOG
mice die (Nomura et al. 2008). In addition, the intestinal
fora, e.g., E. coli, of NOG mice has been found to change
because of stress caused by transport or changes in rearing
temperature (Nomura et al. 2008). These incidents confrm
that higher-level control, e.g., germ-free breeding techniques
and rearing facilities, is indispensable for the generation and
maintenance of extremely immunodefcient mice (Nomura et
al. 2008).
Conclusions
The ability to experimentally manipulate in small animal
models each incremental step in complex human biological
processes offers great promise for rapid advances. Humanized
mice having human cells, tissues, or organs, should facilitate
the study of human disease pathogenesis and help develop
therapeutics and prophylactics. While there is no alternative
to the accuracy of information obtained from human clinical
trials, these mice should allow derivation of valuable
preclinical information, and substantially reduce the cost
and time associated with extensive clinical trials. Humanized
mice also provide a platform to attempt novel interventions
in regenerative medicine and cancer treatment before their
application to the clinic.
Acknowledgements
I thank Springer-Verlag, Heidelberg, Germany, for
permitting me to use information contained in Humanized
mice, Volume 324, Current Topics in Microbiology and
Immunology. I apologize to those whose work could not be
cited because of space limitations.
References
Abedi MR, Christensson B, Islan KB, Hammarstrom L,
Smith CI (1992). Immunoglobulin production in severe
combined immunodefcient (SCID) mice reconstituted
with human peripheral blood mononuclear cells. Eur. J.
Immunol. 22:823-828.
Aldrovandi GM, Feuer G, Gao L, et al. (1993). The
SCID-hu mouse as a model for HIV-1 infection. Nature.
363:732-736.
Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison
SJ, Clarke MF (2003). Prospective identifcation of
tumorigenic breast cancer cells. Proc. Natl. Acad. Sci.
USA. 100:3983-3988.
Ando K, Muguruma Y, Yahata T (2008). Humanizing
bone marrow in immune-defcient mice. Curr. Topics.
Microbiol. Immunol. 324:78-86.
Anfossi N, Andre P, Guia S, et al. (2006). Human NK cell
education by inhibitory receptors for MHC class I.
Immunity. 25:331-342.
Arai F, Hirao A, Ohmura M et al. (2004). Tie2/
angiopoietin-1 signaling regulates hematopoietic
stem cell quiescence in the bone marrow niche. Cell.
118:149-161.
Auffray I, Dubart A, Izac B, Vainchenker W, Coulombel
L (1994). A murine stromal cell line promotes the
proliferation of the human factor-dependent leukemic
cell line UT-7. Exp. Hematol. 22:417-424.
Baenziger S, Tussiwand R, Schlaepfer E, et al. (2006).
Disseminated and sustained HIV infection in CD34+
cord blood cell-transplanted Rag2
/
c
/
mice. Proc.
Natl. Acad. Sci. USA. 103:15951-15956.
Baenziger S, Ziegler P, Mazzucchelli L, Bronz L, Speck RF,
Manz MG (2008). Human T cell development and HIV
infection in human hemato-lymphoid system mice.
Curr. Topics. Microbiol. Immunol. 324:125-131.
Becker PD, Legrand N, van Geelen CM, et al. (2010).
Generation of human antigen-specifc monoclonal IgM
antibodies using vaccinated human immune system
mice. PLoS One. 5:e13137.
Bennett CL, van Rijn E, Jung S, et al. (2005). Inducible
ablation of mouse Langerhans cells diminishes but
fails to abrogate contact hypersensitivity. J. Cell. Biol.
169:569-576.
Bensidhoum M, Chapel A, Francois S. et al. (2004). Homing
of in vitro expanded Stro-1
or Stro-1
+
human mesen-
chymal stem cells into the NOD/SCID mouse and their
role in supporting human CD34 cell engraftment. Blood.
103:3313-3319.
Bente DA, Melkus MW, Garcia JV, Rico-Hesse R (2005).
Dengue fever in humanized NOD-SCID mice. J . Virol.
79:13797-13799.
Bhatia M, Wang JC, Kapp U, Bonnet D, Dick JE (1997).
Purifcation of primitive human hematopoietic cells
capable of repopulating immune-defcient mice. Proc.
Natl. Acad. Sci. USA. 94:5320-5325.
Blunt T, Finnie NJ , Taccioli GE, et al. (1995). Defective
DNA-dependent protein kinase activity is linked to VDJ
recombination and DNA repair defects associated with
the murine scid mutation. Cell. 80:813-823.
Vol -1, Issue - 1, Jan - Apr 2011 16
Bock TA, Orlic D, Dunbar CE, Broxmeyer HE, Bodine DM
(1995). Improved engraftment of human hematopoietic
cells in severe combined immunodefcient (SCID)
mice carrying human cytokine transgenes. J. Exp. Med.
182:2037-43.
Bosma GC, Custer RP and Bosma MJ (1983). A severe
combined immunodefciency mutation in the mouse.
Nature. 301:527-530.
Bosma MJ (1992). B and T cell leakiness in the scid mouse
mutant. Immunodefciency Rev. 3:261-276.
Brenner S, Whiting-Theobald N, Kawai T, et al. (2004).
CXCR4-transgene expression signifcantly improves
marrow engraftment of cultured hematopoietic stem
cells. Stem Cells. 22:1128-1133.
Bresalier RS, Raper SE, Hujanen ES, Kim YS (1987). A new
animal model for human colon cancer metastasis. Int. J.
Cancer. 39:625-630.
Calvi LM, Adams GB, Weibrecht KW, et al. (2003).
Osteoblastic cells regulate the haematopoietic stem cell
niche. Nature. 425:841-846.
Camacho RE, Wnek R, Shah K, et al. (2004). Intra-thymic/
splenic engraftment of human T cells in HLADR1
transgenic NOD/scid mice. Cell Immunol. 232:86-95.
Camargo FD, Finegold M, Goodell MA (2004).
Hematopoietic myelomonocytic cells are the major
source of hepatocyte fusion partners. J. Clin. Invest.
113, 1266-70.
Cao X, Shores EW, Hu-Li J, et al. (1995). Defective
lymphoid development in mice lacking expression of
the common cytokine receptor gamma chain. Immunity.
2:223-238.
Carballido J M, Namikawa R, Carballido-Perrig N,
Antonenko S, Roncarolo MG, de Vries JE (2000).
Generation of primary antigen-specifc human T- and
B-cell responses in immunocompetent SCID-hu mice.
Nat. Med. 6:103-106.
Cha JH, Chang MY, Richardson JA, Eidels L (2003).
Transgenic mice expressing the diphtheria toxin
receptor are sensitive to the toxin. Mol. Microbiol.
49:235-240.
Chen Q, Khoury M, Chen J (2009). Expression of human
cytokines dramatically improves reconstitution of
specifc human-blood lineage cells in humanized mice.
Proc. Natl. Acad. Sci. USA. 106:21783-21788.
Cheung C, Gonzalez FJ (2008). Humanized mouse lines
and their application for prediction of human drug
metabolism. J. Pharmacol. Exp. Ther. 327:288-299.
Christianson SW, Greiner DL, Hesselton RA, et al. (1997).
Enhanced human CD4+T cell engraftment in beta2-
microglobulin-defcient NOD-scid mice. J. Immunol.
158:3578-3586.
Christianson SW, Greiner DL, Schweitzer IB, et al.
(1996). Role of natural killer cells on engraftment of
human lymphoid cells and on metastasis of human
T-lymphoblastoid leukemia cells in C57BL/6J-scid
mice and in C57BL/6J-scid bg mice. Cell Immunol.
171:186-199.
Conget PA, Minguell JJ (1999). Phenotypical and functional
properties of human bone marrow mesenchymal
progenitor cells. J. Cell Physiol. 181:67-73.
Cornil I, Man S, Fernandez B, Kerbel RS (1989). Enhanced
tumorigenicity, melanogenesis, and metastases of a
human malignant melanoma after subdermal implan-
tation in nude mice. J. Natl. Cancer Inst. 81:938-944.
Custer RP, Bosma GC, Bosma MJ (1985). Severe combined
immunodefciency (SCID) in the mouse. Pathology,
reconstitution, neoplasms. Am. J. Pathol. 120:464-477.
Delhem N, Hadida F, Gorochov G, et al. (1998). Primary
Th1 cell immunization against HIVgp160 in SCID-hu
mice coengrafted with peripheral blood lymphocytes
and skin. J. Immunol. 161:2060-2069.
Dewan MZ, Terashima K, Taruishi M, et al. (2003). Rapid
tumor formation of human T-cell leukemia virus type
1-infected cell lines in novel NODSCID/gc null mice:
suppression by an inhibitor against NF-kappaB. J. Virol.
77:5286-5294.
Dewan MZ, Uchihara J N, Terashima K, et al. (2006).
Effcient intervention of growth and infltration of
primary adult T-cell leukemia cells by an HIV protease
inhibitor, ritonavir. Blood. 107:716-724.
Dewan MZ, Watanabe M, Ahmed S, et al. (2005). Hodgkins
lymphoma cells are effciently engrafted and tumor
marker CD30 is expressed with constitutive nuclear
factor-kappaB activity in unconditioned NOD/SCID/gc
mice. Cancer Sci. 96:466-473.
Dewan MZ, Watanabe M, Terashima K, et al. (2004). Prompt
tumor formation and maintenance of constitutive
NF-kappaB activity of multiple myeloma cells in NOD/
SCID/gc null mice. Cancer Sci. 95:564-568.
Di Santo JP, Muller W, Guy-Grand D, Fischer A, Rajewsky
K (1995). Lymphoid development in mice with a
targeted deletion of the interleukin 2 receptor gamma
chain. Proc. Natl. Acad. Sci. USA. 92:377-381.
Diwan BA, Rice JM, Ohshima M, Ward JM (1986).
Interstrain differences in susceptibility to liver
carcinogenesis initiated by N-nitrosodiethylamine and
its promotion by phenobarbital in C57BL/6NCr, C3H/
HeNCrMTV- and DBA/2NCr mice. Carcinogenesis.
7:215-220.
Duffeld JS, Forbes SJ, Constandinou CM, et al. (2005).
Selective depletion of macrophages reveals distinct,
opposing roles during liver injury and repair. J. Clin.
Invest. 115:56-65.
Vol -1, Issue - 1, Jan - Apr 2011 17
Ema H, Sudo K, Seita J , et al. (2005). Quantifcation of
self-renewal capacity in single hematopoietic stem
cells from normal and lnk-defcient mice. Dev. Cell.
8:907-914.
Faulkner L, Borysiewicz LK, Man S (1998). The use of
human leucocyte antigen class I transgenic mice to
investigate human immune function. J. Immunol.
Methods. 221:1-16.
Flatz L, Rieger T, Merkler D, et al. (2010). T cell-
dependence of Lassa fever pathogenesis. PLoS Pathog.
6:e1000836.
Friese MA, Jensen LT, Willcox N, Fugger L (2006).
Humanized mouse models for organ-specifc
autoimmune diseases. Curr. Opin. Immunol.
18:704-709.
Fu XY, Huang G, Matsumoto M, Molina H, Chaplin DD
(1997a). Independent signals regulate development of
primary and secondary follicle structure in spleen and
mesenteric lymphnode. Proc. Natl. Acad. Sci. USA.
94:5739-5743.
Fu YX, Molina H, Matsumoto M, Huang G, Min J , Chaplin
DD (1997b). Lymphotoxin-alpha (LTa) supports devel-
opment of splenic follicular structure that is required for
IgG responses. J. Exp. Med. 185:2111-2120.
Fujino H, Hiramatsu J, Tsuchiya A, et al. (2007). Human
cord blood CD34
+
cells develop into hepatocytes in the
livers of NOD/SCID/gcnull mice through cell fusion.
FASEB J. 21:3499-3510.
Galy A, Travis M, Cen D, Chen B (1995). Human T, B,
natural killer, and dendritic cells arise from a common
bone marrow progenitor cell subset. Immunity.
3:459-473.
Giovanella BC, Fogh J (1985). The nude mouse in cancer
research. Adv. Cancer Res. 44:69-120.
Gorantla S, Santos K, Meyer V, et al. (2005). Human
dendritic cells transduced with herpes simplex virus
amplicons encoding human immunodefciency virus
type 1 (HIV-1) gp120 elicit adaptive immune responses
from human cells engrafted into NOD/SCID mice and
confer partial protection against HIV-1 challenge. J.
Virol. 79:2124-2132.
Gorantla S, Sneller H, Walters L, et al. (2007). Human
immunodefciency virus type 1 pathobiology studied
in humanized BALB/c-Rag2
/
c
/
mice. J. Virol.
81:2700-2712.
Greiner DL, Hesselton RA. Shultz LD (1998). SCID mouse
models of human stem cell engraftment. Stem Cells.
16:166-177.
Guenechea G, Gan OI, Dorrell C, Dick JE (2001). Distinct
classes of human stem cells that differ in proliferative
and self-renewal potential. Nat. Immunol. 2:75-82.
Habu S, Fukui H, Shimamura K, et al. (1981). In vivo effects
of anti-asialo GM1. I. Reduction of NK activity and
enhancement of transplanted tumor growth in nude
mice. J. Immunol. 127:34-38.
Harris RG, Herzog EL, Bruscia EM, Grove JE, Van Arnam
JS, Krause DS (2004). Lack of fusion requirement for
bone marrow-derived epithelia. Science. 305:90-93.
Hess D, Li L, Martin M, et al. (2003). Bone marrow-
derived stem cells initiate stem cell regeneration. Nat.
Biotechnol. 21:763-770.
Heyman RA, Borrelli E, Lesley J, et al. (1989). Thymidine
kinase obliteration: creation of transgenic mice with
controlled immune defciency. Proc. Natl. Acad. Sci.
USA. 86:2698-2702.
Hiramatsu H, Nishikomori R, Heike T, et al. (2003).
Complete reconstitution of human lymphocytes from
cord blood CD34
+
cells using the NOD/SCID/gc null
mice model. Blood. 102:873-880.
Horie H, Koike S, Kurata T, et al. (1994). Transgenic mice
carrying the human poliovirus receptor: new animal
models for study of poliovirus neurovirulence. J. Virol.
68:681-688.
Hudson WA, Li Q, Le C, Kersey JH (1998).
Xenotransplantation of human lymphoid malignancies
is optimized in mice with multiple immunologic defects.
Leukemia. 12:2029-2033.
Huntington ND, Di Santo JP (2008). Humanized immune
system (HIS) mice as a tool to study human NK
cell development. Curr. Topics Microbiol. Immunol.
324:109-124.
Ianus A, Holz GG, Theise ND, Hussain MA (2003). In vivo
derivation of glucose-competent pancreatic endocrine
cells from bone marrow without evidence of cell fusion.
J. Clin. Invest. 111:843-850.
Iliopoulos D, Ernst C, Steplewski Z, et al. (1989). Inhibition
of metastases of a human melanoma xenograft by
monoclonal antibody to the GD2/GD3 gangliosides. J.
Natl. Cancer Inst. 81:440-444.
Imada K, Takaori-Kondo A, Akagi T, et al. (1995).
Tumorigenicity of human T-cell leukemia virus type
I-infected cell lines in severe combined immunodef-
cient mice and characterization of the cells proliferating
in vivo. Blood. 86:2350-2357.
Ince WL, Zhang L, Jiang Q, Arrildt K, Su L, Swanstrom R
(2010). Evolution of the HIV-1 env gene in the Rag2
-/-
C
-/-
humanized mouse model. J. Virol. 84:2740-2752.
Ishikawa F, Saito Y, Yoshida S, Harada M, Shultz LD (2008).
The differentiative and regenerative properties of human
hematopoietic stem/ progenitor cells in NOD-SCID/
IL2rnull mice. Curr. Topics Microbiol. Immunol.
324:87-94.
Vol -1, Issue - 1, Jan - Apr 2011 18
Ishikawa F, Shimazu H, Shultz LD, et al. (2006). Purifed
human hematopoietic stem cells contribute to the
generation of cardiomyocytes through cell fusion.
FASEB J. 20:950-952.
Ishikawa F, Yasukawa M, Lyons B, et al. (2005).
Development of functional human blood and immune
systems in NOD/SCID/IL2 receptor chain null mice.
Blood. 106:1565-1573.
Ishikawa F, Yoshida S, Saito Y, et al. (2007). Chemotherapy-
resistant human AML stem cells home to and engraft
within the bone-marrow endosteal region. Nat.
Biotechnol. 25:1315-1321.
Ito M, Hiramatsu H, Kobayashi K, et al. (2002). NOD/SCID/
c null mouse: an excellent recipient mouse model for
engraftment of human cells. Blood. 100:3175-3182.
Ito M, Kobayashi K, Nakahata T (2008a). NOD/Shi-scid
IL2rg null (NOG) mice more appropriate for humanized
mouse models. Curr. Topics Microbiol. Immunol.
324:54-76.
Ito R, Shiina M, Saito Y, Tokuda Y, Kametani Y, Habu S
(2008b). Antigen-specifc antibody production of human
B cells in NOG mice reconstituted with the human
immune system. Curr. Topics Microbiol. Immunol.
324:95-107.
J aiswal S, Pearson T, Friberg H, et al. (2009). Dengue virus
infection and virus-specifc HLA-A2 restricted immune
responses in humanized NOD-scid IL2Rnull mice.
PLoS One 4:e7251.
J ung S, Unutmaz D, Wong P, et al. (2002). In vivo depletion
of CD11c+dendritic cells abrogates priming of CD8
+
T
cells by exogenous cell-associated antigens. Immunity.
17:211-220.
Kagi D, Ledermann B, Burki K, et al. (1994). Cytotoxicity
mediated by T cells and natural killer cells is greatly
impaired in perforin-defcient mice. Nature. 369:31-37.
Kamimura H, Nakada N, Suzuki K, et al. (2010). Assessment
of chimeric mice with humanized liver as a tool for
predicting circulating human metabolites. Drug Metab.
Pharmacokinet. 25:223-235.
Kamogawa Y, Minasi LA, Carding SR, Bottomly K,
Flavell RA (1993). The relationship of IL-4- and
IFN-producing T cells studied by lineage ablation of
IL-4-producing cells. Cell. 75:985-995.
Katoh M, Yokoi T (2007). Application of chimeric mice with
humanized liver for predictive ADME. Drug Metab.
Rev. 39:145-157.
Kennedy MK, Glaccum M, Brown SN, et al. (2000).
Reversible defects in natural killer and memory CD8
T cell lineages in interleukin 15-defcient mice. J. Exp.
Med.191:771-780.
Kiel M, Ylimaz OH, Iwashita T, Ylimaz OH, Terhorst D,
Morrison SJ (2005). SLAM family receptors distinguish
hematopoietic stem and progenitor cells and reveal
endothelial niches for stem cells. Cell. 121:1109-1121.
Kipps TJ (1989). The CD5 B cell. Adv. Immunol.
47:117-185.
Kirchgessner CU, Patil CK, Evans J W, et al. (1995).
DNA-dependent kinase (p350) as a candidate gene for
the murine SCID defect. Science. 267:1178-1183.
Koc ON, Gerson SL, Cooper BW, et al. (2000). Rapid
hematopoietic recovery after coinfusion of autologous-
blood stem cells and culture-expanded marrow
mesenchymal stem cells in advanced breast cancer
patients receiving high-dose chemotherapy. J. Clin.
Oncol. 18:307-316.
Koyanagi Y, Takana Y, Ito M, Yamamoto N (2008).
Humanized mice for human retrovirus infection. Curr.
Topics Microbiol. Immunol. 324:133-148.
Koyanagi Y, Tanaka Y, Kira J , et al. (1997a). Primary
human immunodefciency virus type 1 viremia and
central nervous system invasion in a novel hu-PBL-
immunodefcient mouse strain. J. Virol. 71:2417-2424.
Koyanagi Y, Tanaka Y, Tanaka R, et al. (1997b). High levels
of viremia in hu-PBL-NOD-scid with HIV-1 infection.
Leukemia. 11 Suppl 3:109-112.
Krause DS, Theise ND, Collector MI, et al. (2001). Multi-
organ, multi-lineage engraftment by a single bone
marrow-derived stem cell. Cell. 105:369-377.
Kuroiwa Y, Yoshida H, Ohshima T, et al. (2002). The use
of chromosome-based vectors for animal transgenesis.
Gene Therapy. 9:708-712.
Larochelle A, Vormoor J, Hanenberg H, et al. (1996).
Identifcation of primitive human hematopoietic cells
capable of repopulating NOD/SCID mouse bone
marrow: implications for gene therapy. Nat. Med.
2:1329-1337.
Legrand N, Cupedo T, van Lent AU, et al. (2006).
Transient accumulation of human mature thymocytes
and regulatory T cells with CD28 superagonist in
human immune system Rag2
/
c
/
mice. Blood.
108:238-245.
Li C, Heidt DG, Dalerba P, et al. (2007). Identifcation of
pancreatic cancer stem cells. Cancer Res. 67:1030-1037.
Lin JH (2008). Applications and limitations of genetically
modifed mouse models in drug discovery and devel-
opment. Curr. Drug Metab. 9:419-438.
Lowry PA, Shultz LD, Greiner DL, et al. (1996). Improved
engraftment of human cord blood stem cells in NOD/
LtSz-scid/scid mice after irradiation or multiple-day
injections into unirradiated recipients. Biol. Blood
Marrow Transplant. 2:15-23.
Vol -1, Issue - 1, Jan - Apr 2011 19
Ma N, Ladilov Y, Kaminski A, et al. (2006). Umbilical cord
blood cell transplantation for myocardial regeneration.
Transplant Proc. 38:771-773.
Majumdar MK, Thiede MA, Mosca JD, Moorman
M, Gerson SL (1998). Phenotypic and functional
comparison of cultures of marrow-derived mesenchymal
stem cells (MSCs) and stromal cells. J. Cell Physiol.
176:57-66.
Masuda H, Maruyama T, Hiratsu E, et al. (2007).
Noninvasive and real-time assessment of reconstructed
functional human endometrium in NOD/SCID/c null
immunodefcient mice. Proc. Natl. Acad. Sci. USA.
104:1925-1930.
Masuda H, Okano HJ, Maruyama T, Yoshimura Y (2008).
In vivo imaging in humanized mice. Curr. Topics
Matsumura H, Hasuwa H, Inoue N, Ikawa M, Okabe M
(2004). Lineage-specifc cell disruption in living mice
by Cre-mediated expression of diphtheria toxin A chain.
Biochem. Biophys. Res. Commun. 321:275-279.
Matsumura T, Kametani Y, Ando K, et al. (2003). Functional
CD5+B cells develop predominantly in the spleen of
NOD/SCID/c null (NOG) mice transplanted either
with human umbilical cord blood, bone marrow, or
mobilized peripheral blood CD34+cells. Exp. Hematol.
31:789-797
Matsuura-Sawada R, Murakami T, Ozawa Y, et al. (2005).
Reproduction of menstrual changes in transplanted
human endometrial tissue in immunodefcient mice.
Hum. Reprod. 20:1477-1484.
McCune JM, Namikawa R, Kaneshima H, Shultz LD,
Lieberman M, Weissman IL (1988). The SCID-hu
mouse: murine model for the analysis of human
hematolymphoid differentiation and function. Science.
241:1632-1639.
McCune JM, Namikawa R, Shih CC, Rabin L, Kaneshima
H (1990). Suppression of HIV infection in AZT-treated
SCID/hu mice. Science. 247:564-566.
Mckenzie J I, Gan OI, Doedens M, Wang J CY, Dick J E
(2006). Individual stem cells with highly variable
proliferation and self-renewal properties comprise the
human hematopoietic stem cell compartment. Nat.
Immunol. 11:1225-1233.
Melkus MW, Estes J D, Padgett-Thomas A, et al. (2006).
Humanized mice mount specifc adaptive and innate
immune responses to EBV and TSST-1. Nat. Med.
12:1316-1322.
Mestas J, Hughes CC (2004). Of mice and not men:
differences between mouse and human immunology. J.
Immunol. 172:2731-2738.
Meuleman P, Libbrecht L, De Vos R, et al. (2005).
Morphological and biochemical characterization of a
human liver in a uPA-SCID mouse chimera. Hepatology.
41:847-856.
Miller RD, Hogg J , Ozaki J H, Gell D, J ackson SP, Riblet
R (1995). Gene for the catalytic subunit of mouse
DNA-dependent protein kinase maps to the scid locus.
Mitsumori K, Wakana S, Yamamoto S, Kodama Y, Yasuhara
K, Nomura T, Hayashi Y, Maronpot RR (1997).
Susceptibility of transgenic mice carrying human
prototype c-Ha-ras gene in a short-term carcinogenicity
study of vinyl carbamate and ras gene analyses of the
induced tumors. Mol. Carcinog. 20(3):298-307.
Mombaerts P, Iacomini J , J ohnson RS, Herrup K, Tonegawa
S, Papaioannou VE (1992). RAG-1-defcient mice have
no mature B and T lymphocytes. Cell. 68:869-877.
Mori N, Fujii M, Ikeda S, et al. (1999). Constitutive
activation of NF-kappaB in primary adult T-cell
leukemia cells. Blood. 93:2360-2368.
Mosier DE, Gulizia RJ, Baird SM, Wilson DB (1988).
Transfer of a functional human immune system to
mice with severe combined immunodefciency. Nature.
335:256-259.
Mosier DE, Gulizia RJ , Baird SM, et al. (1989). Studies of
HIV infection and the development of Epstein-Barr
virus-related B cell lymphomas following transfer of
human lymphocytes to mice with severe combined
immunodefciency. Curr. Top. Microbiol. Immunol.
152:195-199.
Mosier DE, Gulizia RJ , Baird SM, Wilson DB, Spector
DH, Spector SA (1991). Human immunodefciency
virus infection of human-PBL-SCID mice. Science.
251:791-794.
Mosier DE, Gulizia RJ, MacIsaac PD, Torbett BE, Levy
JA (1993). Rapid loss of CD4+ T cells in human-PBL-
SCID mice by noncytopathic HIV isolates. Science.
260:689-692.
Mota J, Rico-Hesse R (2009). Humanized mice show clinical
signs of dengue fever according to infecting virus
genotype. J. Virol. 83:8638-8645.
Muguruma Y, Yahata T, Miyatake H, et al. (2006).
Reconstitution of the functional human hematopoietic
microenvironment derived from human mesenchymal
stem cells in the murine bone marrow compartment.
Blood. 107:1878-1887.
Nakamura M, Suemizu H (2008). Novel metastasis models
of human cancer in NOG mice. Curr. Topics Microbiol.
Immunol. 324:167-177.
Vol -1, Issue - 1, Jan - Apr 2011 20
Nakamura T, Miyakawa Y, Miyamura A, et al. (2006). A
novel nonpeptidyl human c-Mpl activator stimulates
human megakaryopoiesis and thrombopoiesis. Blood.
107:4300-4307.
Nakata H, Maeda K, Miyakawa T, et al. (2005). Potent
anti-R5 human immunodefciency virus type 1 effects
of a CCR5 antagonist, AK602/ONO4128/GW873140,
in a novel human peripheral blood mononuclear
cell nonobese diabetic-SCID, interleukin-2 receptor
-chain knocked-out AIDS mouse model. J. Virol.
79:2087-2096.
Namikawa R, Kaneshima H, Lieberman M, Weissman IL,
McCune JM (1988). Infection of the SCID-hu mouse by
HIV-1. Science. 242:1684-1686.
Nicolini FE, Cashman J D, Hogge DE, Humphries RK, Eaves
CJ (2004). NOD/SCID mice engineered to express
human IL-3, GM-CSF and Steel factor constitutively
mobilize engrafted human progenitors and compromise
human stem cell regeneration. Leukemia. 18:341-347.
Nomura T, Tamaoki N, Takakura A, Suemizu H (2008).
Basic concepts of development and practical application
of animal models for human diseases. Curr. Topics
Noort WA, Kruisselbrink AB, Anker PS, et al. (2002).
Mesenchymal stem cells promote engraftment of human
umbilical cord blood-derived CD34+cells in NOD/
SCID mice. Exp. Hematol. 30:870-878.
Ohbo K, Suda T, Hashiyama M, et al. (1996). Modulation
of hematopoiesis in mice with a truncated mutant of the
interleukin-2 receptor gamma chain. Blood. 87:956-967.
Passegue E, Jamieson CH, Ailles LE, Weissman IL (2003).
Normal and leukemic hematopoiesis: are leukemias a
stem cell disorder or a reacquisition of stem cell charac-
teristics? Proc. Natl. Acad. Sci. USA. 100:11842-11849.
Pearson T, Greiner DL, Shultz LD (2008). Humanized SCID
mouse models for biomedical research. Curr. Topics
Peled A, Petit I, Kollet O, Magid M, et al. (1999).
Dependence of human stem cell engraftment and
repopulation of NOD/SCID mice on CXCR4. Science.
283:845-848.
Petersen BE, Bowen WC, Patrene KD, et al. (1999). Bone
marrow as potential source of hepatic oval cells.
Science. 284:1168-1170.
Petersen JS, Marshall MO, Baekkeskov S, Hejnaes KR,
Hoier-Madsen M, Dyrberg T (1993). Transfer of type
1 (insulin-dependent) diabetes mellitus associated
autoimmunity to mice with severe combined immuno-
defciency (SCID). Diabetologia. 36:510-515.
Pfumio F, Izac B, Katz A, Shultz LD, Vainchenker W
and Coulombel L (1996). Phenotype and function of
human hematopoietic cells engrafting immune-defcient
CB17-severe combined immunodefciency mice and
nonobese diabetic-severe combined immunodefciency
mice after transplantation of human cord blood
mononuclear cells. Blood. 88:3731-3740.
Pilarski LM, Belch AR (2002). Clonotypic myeloma cells
able to xenograft myeloma to nonobese diabetic
severe combined immunodefcient mice copurify with
CD34+hematopoietic progenitors. Clin. Cancer Res.
8:3198-3204.
Pittenger MF, Mackay AM, Beck SC, et al. (1999).
Multilineage potential of adult human mesenchymal
stem cells. Science. 284:143-147.
Poznansky MC, Evans RH, Foxall RB et al. (2000). Effcient
generation of human T cells from a tissue-engineered
thymic organoid. Nat. Biotechnol. 18:729-734.
Prochazka M, Gaskins HR, Shultz LD, Leiter EH (1992).
The nonobese diabetic scid mouse: model for sponta-
neous thymomagenesis associated with immunodef-
ciency. Proc. Natl. Acad. Sci. USA. 89:3290-3294.
Prockop DJ, Gregory CA, Spees JL (2003). One strategy for
cell and gene therapy: harnessing the power of adult
stem cells to repair tissues. Proc. Natl. Acad. Sci. USA.
100:11917-11923.
Rabin L, Hincenbergs M, Moreno MB, et al. (1996). Use
of standardized SCID/hu Thy/Liv mouse model for
preclinical effcacy testing of anti-human immunode-
fciency virus type 1 compounds. Antimicrob. Agents
Chemother. 40:755-762
Ramasamy R, Fazekasova H, Lam EW, Soeiro I, Lombardi
G, Dazzi F (2007). Mesenchymal stem cells inhibit
dendritic cell differentiation and function by preventing
entry into the cell cycle. Transplantation. 83:71-76.
Reya T, Morrison SJ, Clarke MF, Weissman IL (2001).
Stem cells, cancer, and cancer stem cells. Nature.
414:105-111.
Ringden O, Uzunel M, Rasmusson I, et al. (2006).
Mesenchymal stem cells for treatment of therapy-
resistant graft-versus-host disease. Transplantation.
81:1390-1397.
Saito M, Iwawaki T, Taya C, et al. (2001). Diphtheria toxin
receptor-mediated conditional and targeted cell ablation
in transgenic mice. Nat. Biotechnol. 19:746-750.
Saito Y, Kametani Y, Hozumi K, et al. (2002). The in vivo
development of human T cells from CD34+cells
in the murine thymic environment. Int. Immunol.
14:1113-1124.
Vol -1, Issue - 1, Jan - Apr 2011 21
Sandhu J, Shpitz B, Gallinger S, Hozumi N (1994). Human
primary immune response in SCID mice engrafted
with human peripheral blood lymphocytes. J. Immunol.
152:3806-3813.
Sandhu J S, Gorczynski R, Shpitz B, Gallinger S, Nguyen
HP, Hozumi N (1995). A human model of xenogeneic
graft-versus-host disease in SCID mice engrafted with
human peripheral blood lymphocytes. Transplantation.
60:179-184.
Santini SM, Lapenta C, Logozzi M, et al. (2000). Type I
interferon as a powerful adjuvant for monocyte-derived
dendritic cell development and activity in vitro and in
Hu-PBL-SCID mice. J. Exp. Med. 191:1777-1788.
Santini SM, Spada M, Parlato S, et al. (1998). Treatment
of severe combined immunodefciency mice with
anti-murine granulocyte monoclonal antibody improves
human leukocyte xenotransplantation. Transplantation.
65:416-420.
Sato T, Laver JH, Aiba Y, Ogawa M (1999). NK cell colony
formation from human fetal thymocytes. Exp. Hematol.
27:726-733.
Scaramuzzino DA, McNiff JM, Bessen DE (2000).
Humanized in vivo model for streptococcal impetigo.
Infect. Immun. 68(5):2880-2887.
Segre JA, Nemhauser JL, Taylor BA, Nadeau JH, Lander ES
(1995). Positional cloning of the nude locus: genetic,
physical, and transcription maps of the region and
mutations in the mouse and rat. Genomics. 28:549-559.
Seita J , Ema H, Ooehara J , et al. (2007). Lnk negatively
regulates self-renewal of hematopoietic stem cells by
modifying thrombopoietin-mediated signal transduction.
Shinkai Y, Rathbun G, Lam KP, et al. (1992). RAG-2-
defcient mice lack mature lymphocytes owing
to inability to initiate V(D)J rearrangement. Cell.
68:855-867.
Shresta S, MacIvor DM, Heusel JW, Russell JH, Ley TJ
(1995). Natural killer and lymphokine-activated killer
cells require granzyme B for the rapid induction of
apoptosis in susceptible target cells. Proc. Natl. Acad.
Sci. USA. 92:5679-5683.
Shultz LD, Banuelos S, Lyons B, et al. (2003). NOD/LtSz-
Rag1nullPfpnull mice: a new model system to increase
levels of human peripheral leukocyte and hematopoietic
stem cell engraftment. Transplantation. 76:1036-1042.
Shultz LD, Ishikawa F, Greiner DL (2007). Humanized mice
in translational biomedical research. Nat. Rev. Immunol.
7:118-130.
Shultz LD, Lyons BL, Burzenski LM, et al. (2005). Human
lymphoid and myeloid cell development in NOD/
LtSz-scid IL2R gamma null mice engrafted with
mobilized human hemopoietic stem cells. J. Immunol.
174:6477-6489.
Shultz LD, Saito Y, Najima Y, et al. (2010). Generation of
functional human T-cell subsets with HLA-restricted
immune responses in HLA class I expressing NOD/
SCID/IL2rnull humanized mice. Proc. Natl. Acad. Sci.
USA. 107:13022-13027.
Shultz LD, Schweitzer PA, Christianson SW, et al. (1995).
Multiple defects in innate and adaptive immunologic
function in NOD/LtSz-scid mice. J. Immunol.
154:180-191.
Singh SK, Hawkins C, Clarke ID, et al. (2004). Identifcation
of human brain tumour initiating cells. Nature.
432:396-401.
Sorrentino BP (2004). Clinical strategies for expansion
of haematopoietic stem cells. Nat. Rev. Immunol.
4:878-888.
Steinman RM, Mellman I (2004). Immunotherapy:
bewitched, bothered, and bewildered no more. Science.
305:197-200.
Strom SC, Davila J, Grompe M (2010). Chimeric mice
with humanized liver: tools for the study of drug
metabolism, excretion, and toxicity. Methods Mol. Biol.
640:491-509.
Suematsu S, Watanabe T (2004). Generation of a synthetic
lymphoid tissue-like organoid in mice. Nat. Biotechnol.
22:1539-1545.
Suemizu H, Monnai M, Ohnishi Y, Ito M, Tamaoki N,
Nakamaura M (2007). Identifcation of a key molecular
regulator of liver metastasis in human pancreatic
carcinoma using a novel quantitative model of metas-
tasis in NOD/SCID/gc null (NOG) mice. Int. J .Oncol.
31:741-751.
Sugiyama T, Kohara H, Noda M, Nagasawa T (2005).
Maintenance of the hematopoietic stem cell pool by
CXCL12-CXCR4 chemokine signaling in bone marrow
stromal cell niches. Immunity 25:977-988.
Sun Z, Denton PW, Estes J D, et al. (2007). Intrarectal
transmission, systemic infection, and CD4+T cell
depletion in humanized mice infected with HIV-1. J.
Exp. Med. 204:705-714.
Tanaka T, Kitamura F, Nagasaka Y, Kuida K, Suwa H,
Miyasaka M (1993). Selective long-term elimination
of natural killer cells in vivo by an anti-interleukin 2
receptor beta chain monoclonal antibody in mice. J.
Exp. Med. 178:1103-1107.
Vol -1, Issue - 1, Jan - Apr 2011 22
Toma C, Pittenger MF, Cahill KS, Byrne BJ , Kessler PD
(2002). Human mesenchymal stem cells differentiate to
a carbiomyocyte phenotype in the adult murine heart.
Circulation. 105:93-98.
Traggiai E, Chicha L, Mazzucchelli L, et al. (2004).
Development of a human adaptive immune system
in cord blood cell-transplanted mice. Science.
304:104-107.
Uze G, Lutfalla G, Gresser I (1990). Genetic transfer of
a functional human interferon alpha receptor into
mouse cells: cloning and expression of its cDNA. Cell.
60:225-234.
van der Loo JC, Hanenberg H, Cooper RJ, Luo FY, Lazaridis
EN, Williams DA (1998). Nonobese diabetic/severe
combined immunodefciency (NOD/SCID) mouse as
a model system to study the engraftment and mobili-
zation of human peripheral blood stem cells. Blood.
92(7):2556-7250.
van Halteren AG, Kardol MJ, Mulder A, Roep BO (2005).
Homing of human autoreactive T cells into pancreatic
tissue of NOD-scid mice. Diabetologia. 48:75-82.
van Hensbergen Y, Schipper LF, Brand A, et al. (2006). Ex
vivo culture of human CD34+cord blood cells with
thrombopoietin (TPO) accelerates platelet engraftment
in a NOD/SCID mouse model. Exp. Hematol.
34:943-950.
Vosshenrich CA, Garcia-Ojeda ME, Samson-Villeger SI,
et al. (2006). A thymic pathway of mouse natural
killer cell development characterized by expression of
GATA-3 and CD127. Nat. Immunol. 7:1217-1224.
Wang G, Bunnell BA, Painter RG, et al. (2005). Adult stem
cells from bone marrow stroma differentiate into airway
epithelial cells: potential therapy for cystic fbrosis.
Warner JK, Wang JC, Hope KJ, Jin L, Dick JE (2004).
Concepts of human leukemic development. Oncogene.
23:7164-7177.
Watanabe S, Terashima K, Ohta S, et al. (2007).
Hematopoietic stem cell-engrafted NOD/SCID/IL2R
null mice develop human lymphoid systems and induce
long-lasting HIV-1 infection with specifc humoral
immune responses. Blood. 109:212-218.
Wills-Karp M (1999). Immunologic basis of antigen-induced
airway hyper-responsiveness. Annu. Rev. Immunol.
17:255-281.
Yahata T, Ando K, Nakamura Y, et al. (2002). Functional
human T lymphocyte development from cord blood
CD34+cells in nonobese diabetic/Shi-scid, IL-2
receptor gamma null mice. J. Immunol. 169:204-209.
Yahata T, Yumino S, Seng Y, et al. (2006). Clonal analysis
of thymus-repopulating cells presents direct evidence
for self-renewal division of human hematopoietic stem
cells. Blood. 108:2446-2454.
Yamazaki S, Iwama A, Morita Y, Eto K, Ema H, Nakauchi
H (2007). Cytokine signaling, lipid raft clustering, and
HSC hibernation. Ann. NY Acad. Sci. 1106:54-63.
Yeoman H, Gress RE, Bare CV, et al. (1993). Human
bone marrow and umbilical cord blood cells generate
CD4+and CD8+single-positive T cells in murine
fetal thymus organ culture. Proc. Natl. Acad. Sci. USA.
90:10778-10782.
Yokoyama WM, Kim S (2006). Licensing of natural killer
cells by self-major histocompatibility complex class I.
Immunol. Rev. 214:143-154.
Yoshida A, Tanaka R, Kodama A, Yamamoto N, Ansari AA,
Tanaka Y (2005). Identifcation of HIV-1 epitopes that
induce the synthesis of a R5 HIV-1 suppression factor
by human CD4+T cells isolated from HIV-1 immunized
hu-PBL SCID mice. Clin. Dev. Immunol. 12:235-242.
Yoshida A, Tanaka R, Murakam M, et al. (2003). Induction
of protective immune responses against R5 HIV-1
infection in the hu-PBL-SCID mice by intra-splenic
immunization with HIV-1-pulsed dendritic cells:
possible involvement of a novel factor of human CD4+
T cell origin. J. Virol. 77:8719-8728.
Yoshino H, Ueda T, Kawahata M, et al. (2000). Natural killer
cell depletion by anti-asialo GM1 antiserum treatment
enhances human hematopoietic stem cell engraftment
in NOD/Shi-scid mice. Bone Marrow Transplant.
26:1211-1216.
Yoshizato K, Tateno C (2009). In vivo modeling of
human liver for pharmacological study using
humanized mouse. Expert Opin. Drug Metab. Toxicol.
5:1435-1446.
Zhang J, Niu C, Ye L, et al. 2003. Identifcation of the
haematopoietic stem cell niche and control of the niche
size. Nature 425:836-841.
Zhang L, Kovalev GI, Su L (2007). HIV-1 infection and
pathogenesis in a novel humanized mouse model.
Blood. 109:2978-2981.
Zijlstra M, Li E, Sajjadi F, Subramani S, Jaenisch R (1989).
Germ-line transmission of a disrupted 2-microglobulin
gene produced by homologous recombination in
embryonic stem cells. Nature. 342:435-438.
Zumbach A, Marbet GA, Tsakiris DA (2001). Infuence of
the genetic background on platelet function, micropar-
ticle and thrombin generation in the common laboratory
mouse. Platelets 12:496-502.
Vol -1, Issue - 1, Jan - Apr 2011 23
Ravindranath H Aladakatti received his MPhil, PhD from the Karnataka University,
Dharwad. Subsequently worked as Post Doctoral Fellow in DBT-PDF Programme,
Department of Molecular Reproduction, Development and Genetics, Indian
Institute of Science and in Human Genome Project, Centre for Human Genetics
Lab, Bangalore. His research interests includes studying the early mammalian
development, management of male (in)fertility by means of phytotherapy approach
and environmental toxicology. He has been bestowed with Scientist of the Year
(2008) award from National Environmental Science Academy (NESA), New-Delhi
and Best Poster Presentation and Gold Medal at ISSRF Meeting - 2007, New-Delhi.
He is member of several societies and published several articles in national and
international journals. Currently he is serving as Scientifc Ofcer at the Central Animal
Facility, Indian Institute of Science, Bangalore.
Ravindranath
H Aladakatti
Efect of graded doses of nimbolide on biochemical
and sperm functional parameters in male albino rats
Ravindranath H. A
1*
, Sukesh B
2
, Umadevi C. J
3
Murigendra. B. H
4
.
1
Central Animal Facility, Indian Institute of Science, Bangalore-560012, India

2
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science
Bangalore-560012, India
3
Department of Zoology, Govt. First Grade College, Sira, Karnataka, India
4
KLES KidneyFoundation & Diabetic Centre, KLES Dr. Prabhakar Kore Hospital & MRC, Belgaum, India
Abstract
Nimbolide, a major component of Azadirachta indica leaves, has been shown to exhibit numerous
biological activities without any toxicity when given through intragastric route to experimental animals. The
aim of the present study was to examine the effect of nimbolide on biochemical parameters, sperm functional
parameters and fertility performance in treated animals. Wistar strain male albino rats were administered
subcutaneously with graded concentrations of nimbolide (0.5, 1.0 and 1.5 mg/kg body weight in suspension
of 50% DMSO) followed by maintaining suitable control group for 24 days. Five animals from each group
were used for fertility test. The control and treated animals were sacrifced 24 h after the last dose; repro-
ductive organs were dissected out and used for biochemical analysis and cauda epididymal fuid for sperm
analysis. The present study revealed that there was no signifcant differences in their body weight, however, a
decrease in their weights of reproductive organs were observed. The biochemical analysis showed a general
decrease in the total protein content, acid phosphatase and increase in the total free sugar, activities of alkaline
phosphatase and lactate dehydrogenase. Sperm analysis revealed a decrease in sperm functional parameters
with increased abnormal sperms in a dose dependent manner and fertility performance test showed decrease
in number of implantation in female rats when mated with treated male groups.
Keywords: nimbolide, reproductive organs, biochemical parameters, sperm analysis, fertility
Ravindranath H. Aladakatti, Central Animal Facility, Indian Institute of Science
Bangalore-560012, Karnataka, India
Phone: +91-80-22932457, Fax: +91-80-23606569, email : ravindranath@caf.iisc.ernet.in
Research
Vol -1, Issue - 1, Jan - Apr 2011 24
Effect of nimbolide in rats, Ravindranath H.A.
Introduction
Azadirachta indica (commonly called as neem) products
has been widely used in agriculture as an alternative to
synthetic pesticides. A number of chemical components
have been isolated from neem plant extract. Many of the
secondary compounds of neem have been identifed (Van
der Nat et al. 1991), purifed and some have been tested
for their effects on mammals. These include azadirachtin,
nimbolide, nimbinin, solannin, deacetylazadirchtinol,
nimbin, nimbidinin and meliantriol. Among the various
constituents of neem, limonoids are the major compounds.
These compounds are identifed to possess a wide range of
biological activities. Phytochemical studies on the products of
neem tree have revealed the presence of various isoprenoids,
and non-isoprenoids such as proteins, carbohydrates,
sulphurous compounds and polyphenols. Nimbolide (Gibbs,
2000; Fig.1), a major component of A. indica leaves, has been
shown to exhibit numerous biological activities such as anti-
feedent (Suresh et al. 2002), anti-malarial (Rochanakij et al.
1985) and antimicrobial activities (Rojanpo et al. 1985). It
has also exhibited signifcant anti-cancer properties (Cochen
et al. 1996). It has been shown that nimbolide, when given
through an intragastric route to experimental animals, did
not show any toxicity (Glinsukon et al. 1986). A number of
studies have been carried out on various biological activities
of neem extracts and a few reports are available on clinical
studies with the extracts or the compounds and their medicinal
applications.
Fig.1.Chemical structure of nimbolide
(Ref. Glinsukon et al. 1986)
In order to establish their potential use, it was envisaged to
evaluate the antiandrogenic/antifertility nature of the active
principle, nimbolide. The present study was designed to
determine the effect of nimbolide on androgen dependent
reproductive organs. The present was aimed at determination
of some of the androgen dependent biochemical parameters
like protein, free sugar content, acid phosphatase (ACP),
alkaline phosphatase (ALP) and lactate dehydrogenase
(LDH) in reproductive organs, sperm functional parameters
and fertility in both the control and treated rats.
Materials and Methods
Animals
Colony bred healthy adult male albino rats (Wistar strain)
weighing 200 g were utilized for the experiments. All animals
were obtained from the animal facility, KLE Societys JNM
College, Belgaum. They were housed at a temperature of 22
+2
0
C with 12:12 light and dark cycle. They were maintained
on a standard rat pellet diet and water was given ad libitum.
The animals were acclimatized to the laboratory conditions
before conducting experiments and proper care was provided
to the experimental animals as per the Committee for the
Purpose of Control and Supervision on Experiments on
Animals (CPCSEA) guidelines. Necessary approval from the
Institutional Animal Ethics Committee (IAEC) was obtained
before undertaking animal experimentation.
Chemicals
Technical nimbolide was procured from the SPIC Ltd.,
Chennai, India. All other reagents were procured from Sigma
or Hi Media Lab Pvt. Ltd., Mumbai, India.
Treatment
The different concentrations of nimbolide (0.5, 1.0 and
1.5 mg/kg body weight) were dissolved in 50% Dimethyl
sulfoxide (DMSO) and administered to rats by subcutaneous
injection. Forty male rats were divided into four groups of
10 males each. The rats in group A were administered with
50% DMSO (vehicle control; 5 ml/kg body weight), while
the three groups (B, C and D) were administered with
graded doses of nimbolide (0.5, 1.0 and 1.5 mg/kg body
weight, respectively). The DMSO and the graded doses of
nimbolide were administered subcutaneously daily for 24
days. Twenty-four hours after the last dose, out of 10 animals,
fve animals from each group were used for fertility test and
the remaining fve animals from each group were sacrifced
by cervical dislocation. The testis, epididymis (caput and
cauda), vas deference, seminal vesicle and ventral prostate
were dissected out, blotted free of mucus and weighed to the
nearest milligram were used for determination of biochemical
and sperm functional parameters.
Biochemical analysis
A 100 mg of each tissue was quantitatively homogenized in
1.0 ml of 0.1 M Tris-Hcl buffer (pH 7.2), 0.1 M phosphate
buffer (pH 7.2) or distilled water and then centrifuged at 8000
g for 15 minutes at 4C. The supernatants were collected
and used for various biochemical analyses using U.V.
Spectrophotometer (Hitachi, Japan).
i) Estimation of total protein
The total protein level in the testis was estimated by
the method of Lowry et al. (1951) using Bovine Serum
Albumin as standard. The optical density (OD) of the
resultant colour was read at 660 nm and expressed as mg
protein per gram wet tissue.
ii) Estimation of total free sugar
The total free sugar content of the tissue was estimated by
the method of Folin and Wu (Oser, 1965). The OD was
recorded at 420 nm and expressed as mg sugar per gram
wet tissue.
iii) Estimation of acid phosphatase (ACP) and alkaline
phosphatase (ALP)
The enzyme assays were carried out according to the
Vol -1, Issue - 1, Jan - Apr 2011 25
MeO2C
method described by Andersch and Szezypinski (1947).
The OD of the resultant colour was read on a colorimeter
at 400 nm and both phosphatase activities were expressed
in terms of mMoles of P-nitrophenol formed per hour per
gram protein.
iv) Estimation of lactate dehydrogenase (LDH)
The LDH levels in testis were determined by the method
of King (1965). The intensity of the colour was measured
at 440 nm and expressed as Mole/gram/h.
Sperm analysis
For standard sperm analysis, the cauda epididymis from each
animal was chopped into phosphate buffered glucose saline
(PBGS: composition: Nacl 50 mM/l; Na2 HPO
4
200 mM/l;
glucose 200 mM/l; KH2PO4 26 mM/l). The debris was
removed and a clear suspension of the epididymis was used
for sperm analysis. The following parameters were deter-
mined: (i)Total number of sperms per ml; ( ii) Total number
of motile sperms per ml; (iii) Normal and abnormal sperm
count (relative percentage); and (iv) Forward velocity of the
sperm (m/sec).
The total sperm count was calculated by the method of Besley
et al. (1980), using Neubauer haemocytometer. To increase
the accuracy of sperm count, the epididymal fuid was diluted
with a spermicidal solution, prepared by dissolving 5 g of
NaHCO3 and 1 ml of 40 % formaldehyde in 100 ml of normal
saline. A twenty times dilution was made by using WBC
pipette, which was thoroughly mixed and one drop was added
to both sides of Neubauer haemocytometer. The sperms were
allowed to settle down in the haemocytometer by keeping
them in a humid chamber for one hour. The total number of
sperms were counted in all the major squares and calculated
as follows.
Total no. of
sperms/ml
=
Total no. of sperms
per square (X)
x
dilution
factor (20)
Total volume
per square (10-4)
Similarly, the total number of motile sperms was calculated,
using PBGS instead of spermicidal solution.
The relative proportions of the normal and abnormal sperms
were calculated by smear preparation according to the method
of Bauer et al. (1974). Equal volume of cauda epididymal
fuid and 5 % sodium bicarbonate were taken in a centrifuge
tube, mixed well and centrifuged for 5 minutes at 4000 g.
The supernatant was discarded and to the precipitate 5 ml
of normal saline was added, mixed well and centrifuged
again. The procedure was repeated 2 to 3 times and a clear
precipitate was obtained. To the fnal precipitate, few drops
of normal saline were added, mixed thoroughly and a smear
was prepared on a clean slide. The smear was dried at room
temperature, fxed by heating it over the fame for two to
three seconds. Then the smear was fushed with 95% alcohol,
drained and dried. It was stained in Ziehl Neelson's Carbol
Fuchsin diluted with equal volume of 95% alcohol for 3
minutes and counter stained with 1:3 (v/v) aqueous solution
of Loeffer's methylene blue for 2 minutes. After staining,
the smear was rinsed in water and dried in air. The abnormal
sperms like double tailed, detached head, detached tail, mid
piece bending and irregular head were determined. The
relative proportion of the normal and abnormal sperms from
the smear was expressed in terms of percentage.
To assess the forward velocity of sperms, the method of
Ratnasoorya (1984) was adopted. The epididymal plasma
was suspended in PBGS, cleared the tissue debris and a clear
solution was used for the assessment of average forward
velocity of sperms. The assessment was made under light
microscope, ftted with a movable mechanical stage and a
calibrated ocular micrometer at 400 X magnifcation. A drop
of sperm suspension was transferred to a clean glass slide and
the initial place and time of each sperm was recorded. The
time taken for forward movement of sperm from the initial
place within microscopic feld was recorded using a stop
watch. The procedure was repeated for 10 spermatozoa in
each sample and the average forward velocity of sperm was
calculated and expressed as m / sec.
Fertility test
To assess the fertility rate with reference to the number of
implantations, the remaining fve males in each group were
paired with female of proven fertility exhibiting regular
estrous cycles. The appearance of sperm in the vaginal smear
next morning confrmed the mating and was considered as Day
1 of the pregnancy. After 8 days, the females were laparoto-
mized and the numbers of implantations were recorded.
Statistical analysis
Results are expressed as the mean value +standard error
of mean (SEM). All data were subjected to either Students
t-test or one-way analysis of variance (ANOVA) followed by
multiple t-tests.
Results
Body and organ weights
The body weight of rats did not differ in different groups of
animals. In group B rats (0.5 mg/kg body weight), the weights
of testis and other accessory structures remain unchanged
excluding seminal vesicle and ventral prostate (P 0.05)
when compared to controls. Whereas, rats in group C (1.0
mg/kg body weight), exhibited a decrease in the weight of
testis, epididymis (both caput and cauda) and vas deference (P
0.05) and signifcant decrease in weights of seminal vesicle
and ventral prostate (P 0.001). In group D (1.5 mg/kg body
weight), the weights of seminal vesicle and ventral prostate
decreased signifcantly (P 0.001). The testis and other
accessory structures were also exhibited a signifcant decrease
in their weights (P 0.01) when compared to controls (group
A, Table 1).
Androgen dependent biochemical parameters
The protein, free sugar content, ACP, ALP and LDH in the
testis, epididymis (caput and cauda), vas deferens, seminal
vesicle and ventral prostate were determined in the control
and nimbolide treated rats.
Vol -1, Issue - 1, Jan - Apr 2011 26
Table 3: Effect of nimbolide on various biochemical parameters in caput and cauda of
epididymis in rats
Group
Protein
(mg/g)
Sugar
(mg/g)
ACP
(mM/g/hr)
ALP
(mM/g/hr)
LDH
(M/g/hr)
Caput Cauda Caput Cauda Caput Cauda Caput Cauda Caput Cauda
Group A
50% DMSO
(control; 5 ml/
kg b.w.)
46.93
1.34
36.41
2.19
0.62
0.03
0.72
0.02
10.91
0.06
8.01
0.06
1.01
0.05
1.25
0.04
302.3
4.87
374.4
4.91
Group B
Nimbolide
(0.5 mg/kg b.w.)
42.74
4.29*
26.74
4.36**
1.24
0.03
3.22
0.03**
7.76
0.03*
5.41
0.03*
2.63
0.03*
3.19
0.03**
361.3
4.79*
845.2
4.76**
Group C
Nimbolide
(1.0 mg/kg b.w.)
32.45
4.84**
26.51
4.83**
1.97
0.06*
3.47
0.05**
6.44
0.05**
4.30
0.05**
3.35
0.04**
3.75
0.05**
534.3
4.91***
918.7
4.89***
Group D
Nimbolide
(1.5 mg/kg b.w.)
18.62
4.84***
22.67
4.85**
2.15
0.06**
5.29
0.06***
5.55
0.05**
4.36
0.05**
4.21
0.05**
4.59
0.04**
743.5
4.92***
974.2
4.90***
Data are mean S.E.M. of 5 replicates. ** Signifcant difference at P 0.01 level
*Signifcant difference at P 0.05 level ***Signifcant difference at P 0.001 level
Table 4: Effect of nimbolide on various biochemical parameters in vas
deferens of rats
Group
Protein
(mg/g)
Sugar
(mg/g)
ACP
(mM/g/h)
ALP
(mM/g/h)
LDH
(M/g/h)
Group A
50% DMSO
(control;
5 ml/kg b.w.)
46.53
2.16
1.86
0.02
4.89
0.05
1.03
0.05
363.7
4.82
Group B
Nimbolide
(0.5 mg/kg b.w.)
45.68
4.39
4.38
0.03*
4.66
0.03
1.04
0.03
618.9
4.85**
Group C
Nimbolide
(1.0 mg/kg b.w.)
44.49
4.85
4.26
0.06*
4.31
0.05
1.26
0.17
784.3
5.66***
Group D
Nimbolide
(1.5 mg/kg b.w.)
42.43
4.93*
4.54
0.06*
4.09
0.05*
1.17
0.05
906.5
4.97***
Data are mean S.E.M. of 5 replicates. **Signifcant difference at P 0.01 level
Protein content in testis and other reproductive
organs
The protein content in testis reduced signifcantly in group D
(P 0.01) and group C (P 0.05). There was no difference in
the protein content of group B rats when compared to control
(Table 2). In epididymis of group D rats, the protein content
of caput was signifcantly reduced (P 0.001). However,
the caput of group C and cauda of groups B, C and D, the
protein content was reduced signifcantly (P 0.01). In group
B, caput exhibited reduction in its content (P 0.05) when
compared to control (Table 3). In vas deferens, the protein
content decreased in group D (P 0.05) and no changes were
observed in groups B and C when compared to control (Table
4). In seminal vesicle, the protein content reduced signifcantly
in groups D (P 0.001), B and C (P 0.01) respectively when
compared to control (Table 5). In ventral prostate, the protein
content reduced signifcantly in all the treated groups B, C and
D (P 0.001) when compared to control (Table 6).
Sugar content in testis and other reproductive
organs
The free sugar content in testis increased signifcantly in
groups D (P 0.01) and C (P 0.05). No difference was
observed in the sugar content of group B when compared to
control (Table 2). In epididymis of group D, the sugar content
of cauda was increased signifcantly (P 0.001). In groups
D and C, the sugar content of caput increased signifcantly
(P 0.01; P 0.05 respectively) and in cauda of groups B
and C, the sugar content increased signifcantly (P 0.01).
However, there was no difference in caput of group B when
compared to control (Table 3). In vas deferens, the sugar
content was increased in all treated groups (P 0.05) when
compared to control (Table 4). In seminal vesicle, the sugar
content increased signifcantly in all treated groups (P 0.01)
when compared to control (Table 5). In ventral prostate, the
sugar content increased signifcantly in group B (P 0.01) and
groups C and D (P 0.001) respectively when compared to
control (Table 6).
Acid phosphatase concentration (ACP) in testis
and other reproductive organs
In testis, the ACP concentration reduced signifcantly in groups
D (P 0.01) and C (P 0.05) respectively. No difference in the
ACP concentration was observed in group B when compared
to control (Table 2). The concentration of ACP reduced
signifcantly in epididymis of group B (P 0.05) and groups
C and D (P 0.01, respectively) when compared to control
(Table 3). In vas deferens, the concentration reduced in group
D (P 0.05) and no difference was observed in groups B and
C when compared to control (Table 4). In seminal vesicle,
the concentration was reduced signifcantly in groups D (P
0.01) and C (P 0.05) respectively. However, there was no
difference in group B when compared to control (Table 5). In
ventral prostate, the concentration was reduced signifcantly
in groups B and C (P 0.05) and D (P 0.01) when compared
to control (Table 6).
Table 1: Effect of nimbolide on body weight (g) and reproductive organ
weights (mg) in rats
Group
Body
weight
Testis
Epididymis
Vas
deferens
Seminal
vesicle
Ventral
prostate
Caput Cauda
Group A
50% DMSO
(control; 5 ml/
kg b.w.)
205.00
3.76
695.05
2.57
137.28
2.87
116.51
2.04
168.88
3.26
601.33
7.26
381.92
12.58
Group B
Nimbolide
(0.5 mg/kg b.w.)
193.61
2.48
712.48
4.12
133.21
2.32
112.65
2.98
163.20
2.89
580.16
10.22*
363.61
10.45*
Group C
Nimbolide
(1.0 mg/kg b.w.)
202.14
3.77
686.41
6.10*
130.12
2.12*
107.63
2.62*
156.30
4.02*
542.43
14.03***
334.53
9.97***
Group D
Nimbolide
(1.5 mg/kg b.w.)
198.20
2.06
661.71
8.54*
113.41
1.80**
96.85
3.68**
140.23
3.55**
517.33
9.61***
310.01
11.32***
Data are mean S.E.M. of 5 replicates. ** Signifcant difference at P 0.01 level
Table 2: Effect of nimbolide on various biochemical parameters of the testis
in rats
Group
Protein
(mg/g)
Sugar
(mg/g)
ACP
(mM/g/h)
ALP
(mM/g/h)
LDH
(M/g/h)
Group A
50% DMSO
(control;
5 ml/kg b.w.)
37.30
1.08
0.60
0.03
2.96
0.06
1.22
0.04
268.28
4.92
Group B
Nimbolide
(0.5 mg/kg b.w.)
35.63
4.36
0.89
0.03
2.13
0.03
2.69
0.03*
381.90
4.79**
Group C
Nimbolide
(1.0 mg/kg b.w.)
31.49
4.92*
2.18
0.06*
1.55
0.05*
4.19
0.04**
670.27
3.53***
Group D
Nimbolide
(1.5 mg/kg b.w.)
26.41
4.89**
3.08
0.06**
0.83
0.05**
5.92
0.04**
826.22
4.21***
Vol -1, Issue - 1, Jan - Apr 2011 27
Alkaline phosphatase concentration (ALP) in
testis and other reproductive organs
In testis, the ALP concentration increased signifcantly in
group B (P 0.05) and groups C and D (P 0.01) when
compared to control (Table 2). In epididymis of groups C and
D, the ALP concentration in caput and cauda signifcantly
increased (P 0.01). However, in group B, the ALP concen-
tration in caput and cauda increased signifcantly (P 0.05; P
0.01) when compared to control (Table 3). In vas deference,
no difference was observed in all groups when compared
to control (Table 4). In seminal vesicle, the concentration
increased signifcantly in group D (P 0.01) and groups B and
C (P 0.05) when compared to control (Table 5). In ventral
prostate, the concentration increased signifcantly in group D
(P 0.01) and group C (P 0.05). However, there was no
difference in group B when compared to control (Table 6).
Lactate dehydrogenase concentration (LDH) in
testis and other reproductive organs
The LDH concentration in testis increased signifcantly in
group B (P 0.01) and groups C and D (P 0.001) when
compared to control (Table 2). In epididymis of groups C
and D, the concentration of LDH in both caput and cauda
increased signifcantly (P 0.001). However, caput and
cauda in group B showed increased LDH concentration (P
0.05; P 0.01) when compared to control (Table 3). In vas
deferens, the concentration increased signifcantly in group B
(P 0.01) and groups C and D (P 0.001) when compared
to control (Table 4). In seminal vesicle, the concentration
increased signifcantly in all treated groups (P 0.001) when
compared to control (Table 5). In ventral prostate, the concen-
tration increased signifcantly in all groups (P 0.001) when
compared to control (Table 6).
Sperm parameters and fertility in rats
Analysis of sperm parameters, such as total sperm count, total
number of motile sperms, forward velocity of the sperm and
percentage of abnormal sperm in cauda were carried out in the
control and all nimbolide treated animals. The total number of
sperms/ml in epididymal fuid was 56.20 X 10
4
, total number
of motile sperms/ml in epididymal fuid was 51.80 X 10
4

with a speed of 127.2 m/sec and a total of 11.20 % abnormal
sperms were recorded in control group. Where as in the
treated animals (groups B, C and D), the abnormal sperms in
cauda epididymal fuid were dose dependent and a signifcant
difference (P 0.01 or P 0.001) in total sperm count, total
number of motile sperms and forward velocity of sperm were
observed when compared to control (Table 7). Different doses
of nimbolide were used to achieve the fertility inhibition. In
group B, no fertility inhibition was observed. However, group
C and D rats showed 30% and 70% fertility inhibition (P
0.01) respectively when compared to control group (Table 8).
Table 8: Effect of nimbolide on implantations in
female rats mated with treated male rats
Group No. of implantations
Group A
50% DMSO
(control; 5 ml/kg b.w.)
10.66 0.95
Group B
Nimbolide
(0.5 mg/kg b.w.)
9.20 0.71
Group C
Nimbolide
(1.0 mg/kg b.w.)
7.40 0.25*
Group D
Nimbolide
(1.5 mg/kg b.w.)
3.80 0.29**
Data are mean S.E.M. of 5 replicates. * Signifcant difference at P 0.05 level
**Signifcant difference at P 0.01 level
Table 5: Effect of nimbolide on various biochemical parameters in
seminal vesicle of rats
Group
Protein
(mg/g)
Sugar
(mg/g)
ACP
(mM/g/h)
ALP
(mM/g/h)
LDH
(M/g/h)
Group A
50% DMSO
(control;
5 ml/kg b.w.)
50.49
1.04
0.66
0.03
4.43
0.06
1.18
0.05
356.6
4.80
Group B
Nimbolide
(0.5 mg/kg b.w.)
44.74
4.37**
3.41
0.03**
3.69
0.03
2.83
0.03*
876.7
4.81***
Group C
Nimbolide
(1.0 mg/kg b.w.)
39.44
4.97**
4.05
0.06**
3.12
0.05*
3.17
0.04*
984.6
4.89***
Group D
Nimbolide
(1.5 mg/kg b.w.)
23.67
4.76***
4.25
0.06**
2.67
0.05**
3.82
0.05**
988.6
4.83***
Table 6: Effect of nimbolide on various biochemical parameters in ventral
prostate of rats
Group
Protein
(mg/g)
Sugar
(mg/g)
ACP
(mM/g/h)
ALP
(mM/g/h)
LDH
(M/g/h)
Group A
50% DMSO
(control;
5 ml/kg b.w.)
70.65
1.05
0.76
0.02
5.54
0.03
1.05
0.04
372.3
4.89
Group B
Nimbolide
(0.5 mg/kg b.w.)
48.73
4.37***
4.99
0.03**
3.25
0.03*
1.84
0.03
717.1
4.80***
Group C
Nimbolide
(1.0 mg/kg b.w.)
44.41
4.94***
6.26
0.06***
3.10
0.04*
2.22
0.04*
851.4
4.91***
Group D
Nimbolide
(1.5 mg/kg b.w.)
43.48
4.84***
6.63
0.05***
2.36
0.05**
3.16
0.05**
915.4
4.90***
Table 7: Effect of nimbolide on various sperm parameters of cauda
epididymal fuid in rats
Group
Sperm count
(Total No. x
10
4
/ml)
Motile sperm
(Total No. x
10
4
/ml)
Forward
velocity
(m/sec)
Abnormal
sperms (%)
Group A
50% DMSO
(control; 5 ml/kg b.w.)
56.20 3.07 51.80 2.91 127.2 3.07 11.20 3.07
Group B
Nimbolide
(0.5 mg/kg b.w.)
46.00 5.01** 36.20 4.90** 113.6 4.80* 14.00 4.14
Group C
Nimbolide
(1.0 mg/kg b.w.)
41.60 4.80** 29.40 4.80*** 102.2 4.90** 32.60 4.80***
Group D
Nimbolide
(1.5 mg/kg b.w.)
38.20 3.07*** 24.60 2.82*** 86.60 4.80*** 58.20 3.10***
Data are mean S.E.M. of 5 replicates ** Signifcant difference at P 0.01 level
* Signifcant difference at P 0.05 level ***Signifcant difference at P 0.001 level
Vol -1, Issue - 1, Jan - Apr 2011 28
Discussion
It is known that monitoring body weight provides information
on the general health status of animals, which is important
for the interpretation of reproductive effects (US EPA,
1996). The accessory system of male ducts and glands are
morphologically and physiologically dependent upon the
production of androgens (Williams-Ashman and Reddy,
1972). In the present study, a decrease in the mean testis
and other accessory organ weights may due to dwindling of
androgen levels. Decrease in protein and ACP concentration
and an increase in total free sugar, ALP and LDH in the
testis and other reproductive organs like epididymis, vas
deferens, seminal vesicle and ventral prostate on treatment
with nimbolide are suggestive of antiandrogenic properties
as the above mentioned biochemical parameters are androgen
sensitive. These observations are similar to those found in
studies of Gupta et al. (2007); Parandin et al. (2008); Kuang
et al. (2009) and Aladakatti et al. (2010). They reported that
the structural and functional integrity of the reproductive
organs depends on the circulating level of the androgen, and
any small change in the androgen level results in the reduction
in the biochemical parameters of organs leading to reduction
of fertility.
It has been reported that protein level is directly correlated with
the secretory concentration of the testis and accessory glands,
which in turn depends on the androgen levels (Jones, 1977).
The reduction in protein content observed in the present study
may be attributed to the reduction in secretory concentration
of the testis and other organs because of the androgen depri-
vation which indirectly indicates the anti-androgenic property
of this active constituent. It is generally known that any inter-
ference in the normal reproductive physiology would result in
decrease of carbohydrate metabolizing enzymes like pyruvate
kinase, glucose-6-phosphate dehydrogenase and 6-phospho-
gluconate dehydrogenase (Aruldhas et al. 1982a; 1982b). Any
decrease in the levels of such enzymes of pentose phosphate
pathway would result in under utilization of sugar and hence
its accumulation in the target organs (Verma et al. 1980; J oshi
et al. 1996; Aladakatti et al. 2010). Taking into consideration
the above reports, it was presumed that a signifcant increase
in the total free sugar content of testis and other organs may be
due to a decrease in the carbohydrate metabolizing enzymes
of pentose phosphate pathway, resulting in accumulation of
sugar in the target organs.
Both ACP and ALP are sensitive functional indicators of the
reproductive status and thus responsible for the secretory
concentration of target organs (Mann et al. 1981). The
effects of androgen on the target organs are mediated through
ACP and ALP. In the present study, a decline in the ACP
and increase in ALP concentration in the testis and other
reproductive organs refecting decreased androgen output.
This agrees with the observations of aqueous extract of
Chromolaena odoratum and lyophilized A.indica leaf powder
in rats (Yakubu et al. 2007; Aladakatti et al. 2010). An increase
in LDH level observed in the testis and other reproductive
organs of treated animals suggests the elevation of substrate
lactate level as LDH is one of the key enzymes in Embden-
Mayeroff pathway of carbohydrate metabolism and the low
concentration of the enzyme has been used as a marker for
active spermatogenesis. Increase in LDH concentration level
has a direct effect on testicular functions such as sperm count
and production, as well as sperm morphology (Sinha et al.
1995; Pant and Srivastava, 2003). In the present study, a
considerable elevation in the epididymal and other organs
LDH concentration in treated rats indicate the production of
substrate lactate, suggesting altered physiological /metabolic
concentration which may have a defnite infuence on
androgen-regulated glycolytic enzyme activities in the male
accessory organs, thereby indirectly affecting the secretory
activities of these tissues.
In the present study, nimbolide treatment resulted in low
sperm count, motility and sperm speed in dose dependent
manner. It is likely that any contraceptive agent that affects
sperm motility would infuence spermatozoa indirectly
through disruption of epididymal epithelial cell function or
act directly on the spermatozoa by affecting their enzymes
(Cooper and Yeung, 1999). In this study, although an increase
in abnormal sperm count and inhibition of sperm motility
suggest that this nimbolides target is within the internal milieu
of the epididymis. The signifcant alterations in epididymal
epithelium in the cauda epididymis of treated rats indicate
that it is less likely that nimbolide affects the sperm motility
by altering the epididymis itself (Aladakatti et al. 2001;
Ghodesawar et al. 2003). The inhibition of fertility by 70% in
nimbolide treated rats seemed to depend on the graded doses
administered subcutaneously for 24 days. The male fertility
potency resulting after the treatments, indicate that for a 100%
non-successful contraceptive effect, it is apparently necessary
that rats be subjected to further treatment with high doses or
increasing duration of treatment. As in this study, it has been
shown that known graded doses of nimbolide with time period
(24 days), some of the androgen dependent biochemical
parameters exhibited considerable changes which in turn
reduced the sperm count, motility and sperm speed due to
androgen depletion at the target level, particularly in the cauda
epididymis thereby affecting physiological maturation of the
sperm. This study suggests that using nimbolide in effective
dose manner, may be a potential candidate as an antifertility
drug for the induction of infertility in humans.
In conclusion, subcutaneous administration with graded
concentrations of nimbolide (0.5, 1.0 and 1.5 mg/kg body
weight in suspension of 50% DMSO) to male rats resulted
in dose-dependent effects on biochemical parameters of
reproductive organs, sperm functional parameters and fertility
potency. It can be suggested that a change in the chemical
composition of reproductive organs in the present study
is probably due to a defciency in the level of circulating
androgen; the results indirectly refect the antiandrogenic
property of the nimbolide. Further, sperm functional and
fertility observations, it can be suggested that changes in
sperm parameters may be a general disturbance of proteins and
alteration in the epididymal milieu probably due to androgen
defciency consequent upon the antiandrogenic/antifertility
action of nimbolide. It is clear from our investigations that the
nimbolide possesses an anti-androgenic property that could
be used in future to regulate male fertility. Further research
is required on the impact of nimbolide on circulating levels
of FSH, LH and testosterone and the reversibility are needed.
Vol -1, Issue - 1, Jan - Apr 2011 29
Acknowledgements
The authors are acknowledging the University Grants
Commission for funding the research and research facilities
from KLES Kidney Foundation & Diabetic Centre, KLES Dr.
Prabhakar Kore Hospital & MRC, Belgaum, India.
References
Aladakatti RH, Ghodesawar MG, Mukhtar Ahmed,
Sannadurgappa D (2010). Effect of lyophilized
Azadirachta indica leaf powder on biochemical
parameters of testis and epididymis in albino rats. Int.
J.Biol. Chem. Sci. 6: 75-87.
Aladakatti RH, Nazeer Ahamed R, Ahmed M, Ghodesawar
MG (2001). Sperm parameters changes induced by
Azadirachta indica in albino rats. J. Basic Clin.
Physiol. Pharmacol. 12: 6976.
Andersch MA, Szezypinski AJ (1947). Use of P-nitro Phenyl
phosphate as the substrate in determination of serum
acid phosphatase. J. Clin. Pathol. 17: 571-574.
Aruldhas MM, Valivullah HM, Govindarajulu P (1982a).
Specifc effect of thyroid hormone on testicular
enzymes involved in carbohydrate metabolism II.
Hyperthyroidism. Biochem. Biophysics. Acta. 715:
121-125.
Aruldhas MM, Valivullah HM, Govindarajulu P (1982b.)
A Specifc effect of the thyroid on testicular
enzymes involved in carbohydrate metabolism I.
Hypothroidism. Int. J. Androl. 5: 196-204.
Bauer JD, Ackerman PG, Toro G (1974). Clinical Laboratory
methods. St.ouis Missouri, Mosby Co., USA.
Besley MA, Eliarson R, Gallegos AJ , Moghissi KS, Paulsen
CA, Prasad MRN (1980). Laboratory manual for the
examination of human semen and semen cervical mucus
interaction. WHO Press, Singapore.
Cohen E, Quisted GB, Jefferies PR (1996). Nimbolide is the
principle cytotoxic component of neem seed insecticide
preparations. Pesti.Sci. 48:135140.
Cooper TG, Yeung CH (1999). Approaches to post-testicular
contraception. Asian J. Androl. 1: 2936.
Ghodesawar MG, Nazeer Ahamed R, Ahmed AW, Aladakatti
RH (2004). Ultrastructural changes in cauda epididy-
midal epithelial cell types of Azadirachta indica leaf
treated rats. Ind. J. Exp. Biol. 42:1091-1095.
Gibbs JB (2000). Mechanism-based target identifcation and
drug discovery in cancer research. Sci.. 287: 1969-1973.
Glinsukon T, Somjaree R, Piyachaturawat P, Thebtaranonth
Y (1986). Acute toxicity of nimbolide and nimbic acid
in mice, rats and hamsters. Toxicol. Lett. 30: 159166.
Gupta RS, Kachhawa JB, Sharma A (2007). Effect of
methanolic extract of Dendrophthoe falcata stem on
reproductive function of male albino rats. J. Herb.
Pharmacother. 7:1-13.
Jones R (1977). Effects of testosterone, testosterone
metabolites and anti-androgens on the function of
the male accessory glands in the rabbit and rat. J.
Endocrinol. 74(1):7588.
Joshi AR, Ahamed RN, Pathan KM, Manivannan B (1996).
Effect of Azadirachta indica leaves on testis and its
recovery in albino rats. Ind.J.Exp Biol. 34: 10911094.
King J (1965). Practical clinical enzymology. D. Van
Norstrand Co. London.
Kuang NZ, He Y, Xu ZZ, Bao L, He RR, Kurihara H (2009).
Effect of pomegranate peel extracts on experimental
prostatitis rats. Zhong Yao Cai. 32(2):235-239.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951).
Protein measurement with the Folin-phenol reagent. J
Biol Chem. 193: 265-275.
Mann T, Lutwak Mann C (1981). Male reproductive function
and Semen. Springer-verlag, Newyork.
Oser BL (1965). Hawk's Physiological Chemistry. (14th ed).
McGraw-Hill Book Co: New York.
Pant N, Srivastava SP (2003). Testicular and spermatotoxic
effects of quinalphos in rats. J Appl Toxicol. 23:
271-274.
Parandin R, Sadeghipour HR, Haeri Rohani SA (2008).
Evaluation of Antifertility Effect and Recovery of
the Seed Oil Constituents of Iranian Species of Melia
Azadrach L. in Male Rats J. Reprod. Contracept,
19(3):161-166.
Ratnasooriya WD (1984). Effect of Atropine on fertility of
female rat and sperm motility. Ind. J. Exp.Boil. 22:
463-466.
Rochanakij S, Thebtaranonth Y, Yenjai C, Yuthayong Y
(1985). Nimbolide, a constituent of Azadirachta indica,
inhibits Plasmodium falciparum in culture. Southeast
Asian J. Trop. Med. Public Health. 16: 66-72.
Rojanpo W, Suwanno S, Somjaree R, Glinsukon T,
Thebtaranont Y (1985). Mutagenic and antibacterial
concentration testing of nimbolide and nimbic acid. J.
Sci. Soc.Thail.11: 177-178.
Sinha N, Narayan R, Shanker R, Saxena DK (1995).
Endosulfan-induced biochemical changes in the testis of
rats. Vet Hum Toxicol. 37(6):547-549.
Suresh G, Geetha Gopalakrishnan, Wesley SD, Pradeep
Singh ND, Malathi R, Rajan SS (2002). J. Agric. Food
Chem. 50: 4484 -4490.
United State Environmental Protection Agency-US EPA
(1996). Reproductive toxicity assessment guidelines,
61. United State Environmental Protection Agency-US
EPA. pp 5627356322.
Van der Nat J M, Van der Sluis WG, De Silva KTD,
Labadie RP (1991). Ethnopharmacognostical
survey of Azadirachta indica A. Juss (Meliaceae).
J.Ethnopharmacol. 35: 124.
Verma OP, Joshi BC, Kumar S, Chattarjee SN (1980).
Antifertility effects of Malvaviscus conzatti Green
fower extract (SC) on male albino mice. Ind. J. Exp.
Biol. 18: 561-564.
Williams-Ashman HG, Reddy AH (1972). Androgenic
regulation of tissue growth and function. In Biochemical
actions of hormones. G. Litwack (edn.) Vol. II,
Academic Press, New York.
Yakubu MT, Akanji MA, Oladiji AT (2007). Evaluation
of antiandrogenic potentials of aqueous extract of
Chromolaena odoratum (L.) K. R. leaves in male rats.
Andrologia. 39: 235-243.
Vol -1, Issue - 1, Jan - Apr 2011 30
Introduction
Afatoxin B1 (AFB1) is a natural toxin produced as a
secondary metabolite by the mould Aspergillus avus and one
of the most potent hepatocarcinogens known. Afatoxicosis
in poultry, ducks, laboratory animals and swine is a major
concern in areas of the world where temperature and humidity
are conducive to growth of the mould, such as India, Africa
and Southeast Asian countries. Acute or chronic toxicosis
can result from exposure to feed or bedding contaminated
with toxins that may be produced during growth of various
saprophytic or phytopathogenic fungi or moulds on cereals,
hay, straw, pastures, or any other fodder or feed ingredients
like maize, sorghum, groundnut, pea nut etc.
Analysis of AFB1 metabolites in the bile of rats indicated
that a major product of metabolism was the glutathione
conjugate of the AFB1-8,9-epoxide (Degen and Neumann,
1978). These conjugates are formed by the action of a
glutathione S-transferase, identifed as GSTA5 (Hayes et al.
N. Prakash, MVSc., PhD., FNASc (AW), completed post-graduation in the year 1990 and PhD in
Veterinary Pharmacology in the year 1994 from Veterinary College, University of Agricultural
Sciences, Bangalore. Served as Assistant Professor at the Department of Veterinary Pharmacology
and Toxicology, Veterinary College, Bidar from 1994 to 2004 and presently working as Associate
Professor and Head at the Department of Pharmacology & Toxicology at Shimoga Veterinary
College, Karnataka Veterinary & Fisheries Sciences University. Recipient of the Karnataka State
Government Certifcate of Merit and the University Gold Medal for master and doctoral degree
programmes respectively. Also recipient of young scientist award, ISSAR (1997) and best research
paper awards in the year 2003 (IDA) and 2009 from ISVPT. Current area of research includes
pharmacokinetics of veterinary drug formulations, residue toxicology of foods of animal origin.
He is also serving as nominee of the CPCSEA, New Delhi. Prakash N.
Dietary supplementation of curcumin ameliorate
afatoxn B
1
induced oxidative stress in mice
Abstract
Studies were carried out to evaluate the effect of dietary supplementation of curcumin on toxicopathology of
afatoxicosis in laboratory mouse following short-term exposure to afatoxin B1. Sexually matured Swiss albino
mice of either sex weighing between 28-30g were divided into two groups consisting of six animals each.
Animals in group I (positive control) were fed with afatoxin B1 mixed in standard rodent chow (7.5 ppb) and
group II (treatment) received afatoxin B1 (7.5 ppb) along with curcumin (1g/kg feed) for a period of 14 days.
At term, oxidative stress and hepatotoxicity were assessed by determining tissue and serum specifc enzymes
and by histopathology. Short term exposure to afatoxin-B1 signifcantly (P<0.05) decreased hepatic GSHr
levels on account of oxidative stress as evidenced by elevated TBARS levels (group I). However, concomitant
administration of curcumin has signifcantly (P<0.05) reduced the TBARS and protected depletion of hepatic
GSHr levels. Curcumin supplementation (group II) has markedly reduced the toxicopathological lesions in
liver induced by afatoxin B1 and consequent reduction (P<0.05) in serum levels of both aminotransferase
(s) and phosphatase. Thus, the present study demonstrated the potential benefts of dietary supplementation of
curcumin in ameliorating afatoxin induced oxidative stress in mice
Keywords: afatoxin-B1, curcumin, lipid peroxidation, mice and toxicopathology
N. Prakash, Department of Veterinary Pharmacology & Toxicology, Veterinary College, Shimoga-577204
Phone: 91+ 8182-651005, Cell: 09448569467, email: pnadoor@redifmail.com.
Curcumin ameliorate aatoxin induced oxidative stress in mice, Prakash N.
Prakash N
1
*, Sunilchandra U
1
, Vijaykumar M
1
, Pavithra B.H
1
, Naghabhushan V
2
and Ramachandra S.G
3
.
1
Department of Pharmacology and Toxicology, Veterinary College, Bidar -585 401
Karnataka Veterinary, Animal & Fisheries Sciences University, Bidar, India
2
Department of Animal Nutrition, Veterinary College, Bidar-585 401
3
Central Animal Facility, Indian Institute of Science, Bangalore-560 012, India
Vol -1, Issue - 1, Jan - Apr 2011 31
1991). Other metabolic pathways were also demonstrated,
including the reduction of the cytotoxic AFB1 dialdehyde to
AFB1-dialcohol catalyzed by AKR7A1 (Ellis et al. 1993).
As both the glutathione conjugate and the dialcohol products
were less toxic than the parental compounds, these were
deemed to represent detoxifcation steps, and the GSTA5 and
AKR7A
1 enzymes were seen to be pivotal in protecting cells
against AFB
1 toxicity.
Curcumin is a yellow colored pigment present in the rhizome
of Curcuma longa (turmeric), a perennial herb belonging
to Zingiberaceae family. Curcumin is non-toxic, highly
promising natural antioxidant, hepatoprotective, hypocho-
lesteremic, anticarcinogenic and antimutagenic compound
having a wide spectrum of biological applications. It is
expected that curcumin may become a novel agent in the near
future to control various diseases, including infammatory
disorders, carcinogenesis and oxidative stress-induced patho-
genesis in man and animals (Chattopadhyay et al. 2004).
Materials and methods
Swiss albino mice of either sex (n=12; 28-30g) were
maintained in the laboratory animal house of veterinary
college, Bidar were procured for the experiment. They were
acclimatized to laboratory conditions (temperature: 241.0C
and humidity 605 %). Animals were fed with standard rodent
chow (Amruth Feeds Ltd., Pune, India) and given drinking
water ad libitum. The experimental protocol met the national
guidelines on the proper care and use of animals in laboratory
research. Six mice (group I) were fed with afatoxin B
1
(7.5 ppb) containing diet delivered through standard rodent
chow and the remaining six animals (group II) received
afatoxin B1 (7.5 ppb) and curcumin (Hi-Media
, Mumbai,
India) at 1g/kg feed given concomitantly. Body weights
of the animals were recorded at weekly interval. On Day 14
of the experiment, all the animals in both the groups were
fasted overnight and sacrifced. Blood samples were collected
to harvest serum samples which were stored at -20C until
analysis. Liver tissues from individual animals were dissected
out, weighed and frozen immediately in liquid nitrogen until
analysis for thiobarbituric acid reacting substance (TBARS)
and reduced glutathione (GSH
r) levels. Representative liver
samples were also stored in formal-Bouins fuid for histo-
logical examination.
Serum enzymes:
Serum levels of aspartate aminotransaminase (AST) and
alanine aminotransaminase (ALT) were estimated according
to Reitman & Flankel et al. (1957). Membrane bound alkaline
phosphates levels was determined by IFCC(1983) method.
TBARS and GSHr in hepatic tissue:
Frozen hepatic tissues were trimmed from extraneous material
using chilled saline solution and homogenized in 0.25M
ice-cold sucrose solution (10%w/v) using pestle and mortar.
The homogenates were centrifuged at 700xg for 10 min. to
remove cell debris and the supernatant of the hepatic tissue
was used for the estimation of TBARS and GSH
r levels.
Histopathology:
Formal-Bouins fxed tissues were dehydrated in graded
alcohols, processed to prepare paraffn embedded
blocks and sections of 5-8 m thickness were obtained.
Haematoxylineosin (H&E) stained tissues were examined
under light microscopy (Sheenan, 1973).
All the values were expressed as Mean SE. Studentst test
was applied to compare the difference between two groups
with respect to various parameters studied. A difference of
P<0.05 was considered as statistically signifcant.
Results
There was no mortality or signifcant change in the body
weight between the two groups of experimental animals.
Serum levels of aminotransferases (AST and ALT) and
alkaline phosphatase (ALP) were signifcantly (P<0.05)
reduced in curcumin treated (group II) group (Fig.1).
Fig . 1. Serum levels of transaminases (AST & ALT ) and alkaline phosphatase
(ALP) in control and curcumin treated group (U/ml) at term.
.
Fig . 2 TBARS (nmoles/g) and reduced GSHr (nmoles/g) levels in hepatic
tissue at term.
Fig.3. Afatoxin induced hepatic lesions characterized by extensive haemor-
rhages, infltration of infammatory cells and swelling of nucleus (H & E, 40X)
Fig.4. Hepatocytes with milder congestion and normal cellular
architecture in curcumin treated group (H & E, 40X)
Vol -1, Issue - 1, Jan - Apr 2011 32
At termination of the experiment (Day 14), TBARS
levels was signifcantly (P<0.05) elevated in the group I
(positive control) due to afatoxin induced oxidative stress
when compared with group II which received curcumin
concomitantly with dietary afatoxin exposure. Further,
co-administration of curcumin with afatoxin B
1 (group II)
has signifcantly (P<0.05) protected the depletion of hepatic
GSHr levels in mice (Fig.2)
Histological examination of liver revealed extensive hemor-
rhages, infltration of infammatory cells and swelling of the
nucleus in control group (Fig.3), while milder degenerative
changes without loss of hepatic architecture was observed in
curcumin treated group (Fig.4).
Discussion
Chemical induced cellular alteration varies from simple
increase in the metabolism to death of the cell (Giray et al.
2001). Alterations in the transaminases and or phosphatase
activity are related to the intensity of the cellular damage. The
increase in serum transaminase activity (AST and ALT) along
with the increase in ALP activity in group I (positive control)
is due to the afatoxin B
1 induced pathological changes in the
hepatic tissue. A signifcant reduction in serum enzyme levels
(AST, ALT and ALP ) in the group II animals was due to the
ameliorating and hepatoprotective role exerted by curcumin.
The antioxidant activity of curcumin was reported as early as
in 1970s (Sharma, 1976). It acts as a scavenger of oxygen free
radicals (Joe and Lokesh, 1994). It can protect haemoglobin
from oxidation (Pulla Reddy and Lokesh, 1992).
Curcumin also lowers the reduction of reactive oxygen species
(ROS) in vivo (Joe and Lokesh, 1994) and also decreases lipid
peroxidation in liver microsomes, erythrocyte membranes and
brain homogenates which is brought about by maintaining the
activities of antioxidant enzymes like superoxide dismutase,
catalase and glutathione peroxidase (Pulla Reddy and
Lokesh,1994). In the present study, the TBARS levels were
comparatively lesser in the treatment group compared to the
control group and the free radical scavenger enzyme reduced
glutathione (GSHr) levels were also signifcantly higher in
curcumin treated animals as compared to positive control
group. It is important to note that oxidative status in animals
may vary according to husbandry practices; however we did
not monitor stress level under our laboratory conditions.
Acute toxicity was initially attributed to mainly genotoxic
effects of the epoxide, dependent on the formation of DNA
adducts which at high levels lead to cell death. However, a
dialdehyde metabolite of AFB
1that rapidly forms from the
epoxide, can form adducts with proteins, and these were
proposed to contribute to the acute toxicity (Neal et al. 1981).
In addition, such cellular necrotic damage caused by AFB
1
dialdehyde may lead to compensatory liver hyperplasia and
may promote the incorporation of mutations into the DNA
of dividing cells and hence contribute towards carcinoge-
nicity initiated by the AFB
1-exo-epoxide (Roebuck, 2004).
Histological studies of hepatic tissue in the control group
revealed extensive hemorrhages, infltration of infammatory
cells and swelling of nucleus suggestive of extensive damage
by the epoxides of afatoxin B
1 where as the changes were
milder in the treatment group, with normal hepatic cellular
architecture. Thus, dietary supplementation of curcumin had
ameliorating role in afatoxin induced oxidative stress and
toxicopathology in mice.
Acknowledgements
The authors are thankful to Harsha Kumar for his assistance
in the preparation of afatoxin B
1 pre-mixed feed for the
experimental animals.
References
Chattopadhyay I, Biswas K, Bandyopadhyay U, Banerjee
RK (2004). Turmeric and curcumin: Biological actions
and medicinal applications. Curr. Sci. 87:1-10.
Degen GH, Neumann HG (1978). The major metabolite of
afatoxin B
1 in the rat is a glutathione conjugate. Chem.
Biol. Interact. 22:239255.
Ellis EM, Judah DJ, Neal GE, Hayes JD (1993). An
ethoxyquin-inducible aldehyde reductase from rat liver
that metabolizes afatoxin B
1 defnes a subfamily of
aldo-keto reductases. Proc. Natl. Acad. Sci. USA. 90:
1035010354.
Giray B, Gurbay A, Hineal F (2001). Cypermethrin induced
oxidative stress in rat brain and liver is prevented by Vit
E or allopurinol. Toxicol.Let. 118:139-146.
Hayes J, Judah D, McLellan L, Kerr L, Peacock S , Neal G
(1991). Ethoxyquin-induced resistance to afatoxin-B
1
in the rat is associated with the expression of a novel
alpha class glutathione s-transferase subunit, Yc2 for
afatoxin B1-8,9-epoxide. Biochem. J. 279: 385398.
IFCC : International Federation of Clinical Chemistry
(1983). Methods for alkaline phosphatase. Clin. Chem.
Acta.135:339-367.
Joe B, Lokesh BR (1994). Role of capsaicin, curcumin and
dietary n-3 fatty acids in lowering the generation of
reactive oxygen species in rat peritoneal macrophages.
Biochim. Biophys. Acta.1224: 255263.
Neal GE, Judah DJ, Stirpe F, Patterson DSP (1981). The
formation of 2,3-dihydroxy- 2,3,dihydroafatoxin B1
by liver microsomes isolated from certain avian and
mammalian species. Toxicol. Appl. Pharmacol. 58:
431437.
Pulla Reddy ACH, Lokesh BR (1992). Studies on spice
principles as antioxidant in the inhibition of lipid
peroxidation of rat liver microsomes. Mol.Cell.
Biochem. 111:117- 124.
Pulla Reddy ACH, Lokesh BR (1994). Effect of dietary
turmeric (Curcuma longa) on iron-induced lipid
peroxidation in the rat liver. Food Chem. Toxicol.32:
279283.
Reitman S, Frankel SA (1957). Colorimetric method for
determination of glutamic oxaloacetic and glutamic
pyruvic transminase. Am. J. Clin. Patho. 28: 56- 63
Roebuck BD (2004). Hyperplasia, partial hepatectomy and
the carcinogenicity of afatoxin B
1. J. Cell. Biochem.
91:243249.
Sharma OP (1976). Antioxidant activity of curcumin
and related compounds. Biochem. Pharmacol. 25:
18111812
Sheenan DC (1973). Theory and practice of histotechnology,
CV Mosby Co., London. pp:115.
Curcumin ameliorate aatoxin induced oxidative stress in mice, Prakash N.
Vol -1, Issue - 1, Jan - Apr 2011 33
Srinivasan K completed his post graduation in Biotechnology from Bharathidasan
University, Trichirappalli, Tamil Nadu and currently pursuing PhD in Biotechno
-logy at Tamil University, Thanjavur. He is recipient of gold medal in National Level
Biotechnology Talent Search Examination conducted by Department of Biotech-
nology, Govt. of India. Currently working as Senior Research Fellow at the Indian
Institute of Crop Processing Technology under the Ministry of Food Processing
Industries, New Delhi.
Srinivasan K.
Evaluation of certain bioactive compounds
using in ovo diabetic model
Srinivasan K
1
, Venkateswaran G
2
and Muthukumar S. P
3
.
1 &3
Biochemistry & Nutrition,
2
Food Microbiology
Central Food Technological Research Institute, Mysore, Karnataka, India
Abstract
Mammalian models are frequently used for drug research and delivery systems. However, valid mammalian
models are expensive, time consuming and not easy to set up and evaluate. Furthermore, they are often linked
with ethical and legal aspects. Many micro, macro nutrients and other photochemical from food materials
are found to possess many desirable health benefts. A large number of studies involving animals have
been carried out to explore these benefcial properties. As an alternative, chick embryo developed through
incubation of fertile chicken eggs has been used as a novel system to develop suitable models for biochemical
and neutraceutical research. The in ovo technique is accepted as an alternative to traditional mammalian
models and this can be suitably used in our routine biochemical tests, wherever the animal trials are not
feasible. In view of the above, the development and evaluation of certain bioactive components using in ovo
technique as a diabetic model was carried out in our laboratory. Bixin decreased signifcantly the plasma
glucose, triglycerides, cholesterol, urea, creatinine and blood urea nitrogen, while liver glycogen levels in
treated diabetic chick embryos increased when compared to control diabetic chick embryos.

Key words: in ovo, chicken embryo, diabetes, bioactive compounds, animal model
Srinivasan K. , Indian Institute of Crop Processing Technology, Pudukkottai Road ,
Thanjavur 631 005, Tamil Nadu, India ,
Phone: 09842639565, email: srinigene@gmail.com
Introduction
For the evaluation of novel compounds in new drugs discovery
and development, mammalian animal models like rats, mice,
rabbits and Guinea pigs are frequently used because of its
suitability for a wide variety of experimental designs, its
docility and ease of handling and care, its short gestation
period, wide dietary preferences and availability. However,
valid mammalian models are expensive, time-consuming, and
not easy to set up and evaluate. Furthermore, they are often
linked to administrative burden with respect to ethical and
legal issues. In ovo model, using chicken embryo as an animal
model is a way of reducing the usage of mammalian models.
Chick embryo development, which lasts for 21 days before
hatching, is a well-known animal model, which has been
extensively studied from Aristotles time that opened hens
eggs daily to examine progressive stages of embryogenesis
until the modern molecular era (Angelica et al. 2007). Diabetes
model can be created by treatment of streptozotocin (STZ) in
chick embryos and this model can be used to predict the effect
of drug (Yuji et al. 2005). The in ovo model forms an alternate
system for evaluating the novel drugs in screening procedures
of formulation candidates, thus establishing an intermediate
step between in vitro cellular tests and preclinical mammalian
models (Gardner et al. 2004).
We have developed a diabetic model using chick embryo and
Vol -1, Issue - 1, Jan - Apr 2011 34
In ovo diabetic model, Srinivasan K .
evaluated the antidiabetic activity of bioactive compounds
like curcumin, capsaicin, quercetin and bixin
Collection and handling of fertile eggs
Fertile eggs from 32 weeks old Giriraja (meat type multiple
cross) chicken were collected from the Regional Poultry Farm,
Mallavalli, Karnataka. Hatching eggs were incubated at 37.5
0.5 C and relative humidity of about 60-70%. Turning of
the eggs was done twice a day for physiological development
and to avoid the adhering of embryo to the membrane. Eggs
were kept vertically (broad end up) and a proper temperature
and humidity was provided for the respiration of embryos.
Chemicals
Streptozotocin was obtained from Sigma Aldrich (USA).
Curcumin, capsaicin, quercetin and bixin were obtained from
authorized chemical suppliers. All chemicals used were of the
highest purity commercially availableand of analytical grade.
Development of in ovo diabetic model
On the ninth day of incubation, eggs were transilluminated
with a special egg tester (candling) to monitor the fertility rate
and adequate development. Embryonated eggs were further
kept under incubation and infertile eggs were discarded.
Fertile eggs were grouped according to its mean weight into
six groups. The group I served as control while group II served
as a diabetic control. For developing the diabetic model, 0.3
mg of streptozotocin was dissolved in physiological saline
(0.85% NaCl) and sterilized through a membrane flter. A
dosage of 0.2 ml of fltrate was injected into the albumen
of each egg on 14
th
day of incubation through the short end
of eggs. After checking for the viability and development
on 17
th
day of incubation, the eggs in group III to VI were
injected with 0.1 l of curcumin at 250 mg/kg of egg weight,
capsaicin at 50 mg/kg of egg weight, quercetin at 50 mg/kg
of egg weight and bixin at 10 mg/kg of egg weight in 30%
ethanol respectively through the air sac of the eggs. Prior to
injection, the blunt end of the egg was sterilized with 70%
ethanol. A suitable scalpel was used to create a single hole,
without penetrating the chorio-allantoic membrane. The hole
was sealed with an adhesive sticker or wax. Eggs were set in
an incubator (temperature : 37 0.5 C; humidity : 65-75%).
Drugs like curcumin, capsaicin, quercetin and bixin were
dissolved in 30% ethanol in distilled water and were sterilized
through a membrane flter. Each drug (0.1 ml) was injected
through the air sac to eggs of respective groups and was
incubated for 6 h.

Collection of samples
Relatively straight courses of major veins were delineated
by transillumination and marked on the eggshell on the
17
th
day of incubation. Then shell membrane was rendered
transparent with saline water and a vessel was punctured
with a thin cannula. Finally, the shell fragment was removed.
Approximately 300 l of blood were extracted for the
evaluation of sample. Blood was collected in 1% EDTA
through the vessels using a tuberculin syringe. Their organs
like brain, liver, and heart were removed and rinsed in saline
water. They were weighed and stored using aluminium foil at
-70 C for further studies.
Estimation of glucose in blood
(Enzymatic method)
The plasma glucose level was measured using commercially
available spectrophotometric enzymatic assay kits.
Estimation of liver glycogen
Liver glycogen was estimated by chemical method based on
the principle that glycogen is hydrolyzed to glucose and the
glucose thus formed is estimated by the standard method.
Estimation of plasma cholesterol
The plasma cholesterol was measured by using FeCl
3
H
2
SO
4
method (Albert et al. 1953).
Estimation of triglycerides
The plasma triglyceride levels were estimated by using
chemical method that involves the extraction and saponifcation
of triglycerides, oxidation of glycerol moiety to formaldehyde
and the conversion of formaldehyde to a yellow-colored
compound, 3,5 diacetyl-1-4 dihydrolulidine and the intensity
of which is determined spectrophotometrically (Raghuraman
et al. 1983).
Estimation of urea and creatinine
Urea and creatinine were estimated by using diagnostic kit
Histological analysis
For the diagnosis of tissue damage in heart, brain, liver and
pancreas, histology was done by sequential process of fxation,
tissue processing and histological staining (Lillie, 1965).
All the results were subjected to statistical analysis and one
way analysis of variance was used to determine the statistical
signifcance.
Results
Effect of different compounds on plasma
glucose level
Plasma glucose levels of chick embryo were measured using
spectrophotometer in normal, diabetic and treated chick
embryos (Table 1).
Vol -1, Issue - 1, Jan - Apr 2011 35
Table 1. Effect of different compounds
on plasma glucose levels in chick embryos
Group Compound Plasma glucose (mg/dl)
I Control 114 7.6
II Diabetic 202 9.2
III Curcumin 143 7.9
IV Capsaicin 138 7.6
V Quercetin 150 8.2
VI Bixin 124 7.5
The plasma glucose concentration in control group which was
treated with normal saline was 114 7.6 mg/dl on day 0 and
202 9.2 mg/dl in the diabetic chick embryos on day 17. The
bixin showed a better hypoglycemic activity and the glucose
level was 124 7.5 mg/dl compared to normal value of 114
7.6 mg/dl. Curcumin and quercetin showed higher glucose
values i.e. 143 7.9 mg/dl and 150 8.2 mg/dl respectively
and in capsaicin treated animals, it was 138 7.6 mg/dl.
Effect of different compounds on liver glycogen
level
Liver glycogen values are provided in Table 2. From the
table, it is evident that liver glycogen content in the diabetic
chick embryo was 75% of the normal chick embryo. Diabetic
chick embryos showed a lower glycogen level (18.44 1.5
mg/dl) compared to control and treated chick embryos. The
glycogen level in curcumin treated group (23.05 2.1 mg/dl)
was comparable to control group (24.011.8 mg/dl), capsaicin
group (26.90 1.9 mg/dl), quercetin group (27.09 2.23 mg/
dl) and bixin group (24.59 1.9 mg/dl).
Table 2. Effect of different compounds on liver lycogen levels
in chick embryos
Group Compound Liver glycogen (mg\dl)
I ontrol 24.01 1.8
II Diabetic 18.44 1.5
III Curcumin 23.05 2.1
IV Capsaicin 26.90 1.9
V Quercetin 27.09 2.2
VI Bixin 24.59 1.9
Effect of different compounds on plasma
cholesterol and triglycerides
The effect of different compounds on plasma cholesterol and
triglycerides is presented in Table.3. The cholesterol level of
diabetic chick embryos increased signifcantly (276 10.2
mg/dl) compared to the control group (197 8.6 mg/dl). The
plasma cholesterol levels were low in chick embryos treated
with capsaicin (153 7.9 mg/dl) and bixin (166 7.6 mg/
dl) compared to curcumin (197 8.9 mg/dl) and quercetin
(191 8.9 mg/dl). The plasma triglyceride levels in quercetin
(320 13.1 mg/dl) and bixin (312 12.2 mg/dl) were
comparable to control group (300 12.9 mg/dl). However,
plasma triglyceride levels in diabetic group (560 17.5 mg/
dl), curcumin group (480 14.2 mg/dl) and capsaicin group
(640 18.6 mg/dl) remained high.
Table 3. Effect of different compounds on plasma cholesterol
and triglycerides in chick embryos
Group Compound
Cholesterol
(mg/dl)
Triglyceride
(mg/dl)
I Control 197 8.6 300 12.9
II Diabetic 276 10.2 560 17.5
III Curcumin 197 8.9 480 14.2
IV Capsaicin 153 7.9 640 18.6
V Quercetin 191 8.9 320 13.1
VI Bixin 166 7.6 312 12.2
Effect of different compounds on urea, creatinine
and blood urea nitrogen
Urea and creatinine concentrations in plasma were measured
by using commercial kit and the nitrogen level of plasma
was calculated from urea level by the formula (Table.4).
The plasma urea level in chick embryos with diabetes was
signifcantly high (63.46 3.2 mg/dl) than the normal chick
embryos (24.56 1.8 mg/dl). The urea levels in treatment
groups were comparable to control group embryos. The
blood urea nitrogen values were increased in diabetic and
reduced in treated groups. There was no difference between
the plasma creatinine levels in control, diabetic and treated
chick embryos. The BUN levels were high in diabetic group
embryos compared to control and other treated groups.
Table 4. Effect of different compounds on urea, creatinine and
blood urea nitrogen in chick embryos
Group Compound
Urea
(mg/dl)
Creatinine
(mg/dl)
Blood
Urea
Nitrogen
(mg/dl)
I Control
24.56
1.8
0.55
0.11
11.46 0.99
II Diabetic
63.46
3.2
0.50
0.06
29.63 2.0
III Curcumin
26.53
1.9
0.62
0.08
12.38 0.95
IV Capsaicin
30.08
2.1
0.53
0.12
14.04 1.2
V Quercetin
20.76
1.4
0.54
0.10
9.69 0.8
VI Bixin
18.46
1.1
0.66
0.12
8.62 0.78
Histological analysis
The organs like liver, pancreas, heart and brain were collected
and processed for the analysis of histological changes. The
heart and brain did not show any change in control, diabetic
and treated groups. The liver of diabetic embryos showed
Vol -1, Issue - 1, Jan - Apr 2011 36
Fig.1 Effects of different compounds on liver of chick embryos
LIVER
CONTROL CURCUMIN CAPSAICIN
DIABETIC QUERCETIN BIXIN
The liver of diabetic chick embryos shows vacuolar regions which is absent in normal liver. Treated
groups showing a marginal improvement in vacuolar lesions
Fig. 2 : Effects of different compounds on pancreas of chick embryos
PANCREAS
CONTROL CURCUMIN CAPSAICIN
DIABETIC QUERCETIN BIXIN
The beta cells in pancreas of diabetic group shows degenerative changes when compared to control
and other treated groups.
In ovo diabetic model, Srinivasan K .
Vol -1, Issue - 1, Jan - Apr 2011 37
vacuolar regions, which was not observed in the normal liver.
The curcumin treated liver showed a marginal improvement
in vacuolar lesions. The capsaicin treated animals showed
minimal vacuolations when compared to diabetic group. The
quercetin and bixin showed no vaculations and were almost
like normal liver (Fig.1).
The pancreas from normal embryos showed alpha and beta
cells. In diabetic group, the beta cells showed tendency
towards degeneration of pancreatic cells. In all the treatment
groups, the pancreas showed no abnormal deviation (Fig. 2).
Discussion
It is evident from the results that signifcant reduction in
plasma glucose levels in all treated group. The bixin showed
a better hypoglycemic activity than capsaicin. Curcumin and
quercetin showed a moderate hypoglycemic activity. Present
fndings on chick embryos are in agreement with the earlier
results on hypoglycemic activity of curcumin in rats (Halim
and Ali, 2002).
These compounds might enhance glucose utilization because
it signifcantly decreased the blood glucose level in diabetic
chick embryos. It may be due to restoration of insulin
response. This could be due to potentiation of the insulin
effect of plasma by increasing the pancreatic secretion of
insulin from existing -cells or its release from bound insulin.
The effect of different compounds on liver glycogen levels
established a signifcant role in the alteration of liver glycogen.
Liver glycogen levels in diabetic group were low compared
to controls. The treatment of diabetic chick embryos with
the capsaicin and quercetin resulted in increase in the liver
glycogen but not signifcantly compared to control group.
The administration of bioactive compounds signifcantly
decreased the plasma cholesterol and triglycerides in
diabetic chick embryos. In the present study, diabetic chick
embryos showed an increased cholesterol level compared to
controls. Plasma cholesterol levels in curcumin and quercetin
treated groups were comparable to control group. Other two
compounds showed a reduced value more or less comparable
to normal group. In consistence with the present data, other
workers have also reported reduction of plasma cholesterol
levels. According to these investigators, the triglyceride
lowering effect appears to be due to inhibition of fatty acid
synthesis. The triglyceride level of diabetic group was high
compared to the normal turmeric (Curcuma longa), as well
as its active constituent curcumin, inhibits lipid peroxidation.
Curcumin also decreases serum cholesterol levels in
hyperlipidaemic rats (Halim and Ali, 2002). Consistent with
this, the in ovo treatment of these bioactive compounds
reduced the lipid peroxidation products (Eidi and Eidi, 2006).
Thus, the triglyceride levels and cholesterol levels reduced
in treated chick embryos compared to the diabetic chick
embryos.
Our data showed that urea levels were increased in diabetic
chick embryos. This may be due to metabolic disturbance
in diabetes, refected in high activities of xanthine oxidase,
lipid peroxidation and increased triglycerides and cholesterol.
Our data showed that bioactive compounds decreased the
plasma urea levels in diabetic chick embryos. The plasma
urea levels in diabetic group were high compared to control
group. Elevation of the plasma urea and creatinine are related
to renal dysfunction in diabetic hyperglycemia (Eidi and
Eidi, 2006). The histological analysis of heart and brain did
not show any change in control, diabetic and treated groups.
The liver of diabetic embryos showed vacuolar regions
and treated groups were almost like control group. The
pancreas from normal embryos showed alpha and beta cells.
In diabetic group the beta cells showed tendency towards
degeneration of pancreatic cells when compared to normal
and in treatment groups. The present study indicated that
the bioactive test compounds signifcantly decreased plasma
glucose, triglycerides, cholesterol, urea, creatinine and blood
urea nitrogen, while increased liver glycogen levels in treated
diabetic chick embryos as compared to control diabetic chick
embryos.
References
Albert Z, Bennie Z, Albert J B (1953). A new method for the
determination of serum cholesterol. J. Lab. Clin. Med.
41: 486-492.
Angelica V, Magali Zeisser-Laboube, Norbert L, Robert
G, Florence Delie (2007). The chick embryo and
its chorioallantoic membrane (CAM) for the in vivo
evaluation of drug delivery systems. Adv. Drug Del.
Rev. 59:1162 - 1176.
Eidi M, Eidi A (2006). Antidiabetic effect of garlic (Allium
sativum L.) in normal and STZ-induced diabetic rats.
Phytomedicine.13: 624-629.
Gardner CR, Almarsson O, Chen H et al. (2004). Application
of high throughput technologies to drug substance and
drug product development. Comput. Chem. Eng. 28
943953.
Halim EM, Ali H (2002). Hypoglcemic, Hypolipidemic and
Antioxidant properties of combination of curcumin from
Curcuma Longa Linn, and partially purifed product
from Abroma Augusta Linn. In STZ induced Diabetes.
Indian J. Clin. Biochem. 17(2): 33-43.
Lillie RD (1965) Histopathologic Technic and Practical
Histochemistry. 3
rd
Edition. McGraw- Hill Book
company, New York.
Raghuraman N, Madhavan KN, Kalyanasundarm S (1983).
A Manual of Laboratory Techniques. National Institute
of Nutrition. Indian Council of Medical Research. pp
78-79 and 92-93.
Yuji Y, Takashi S, Motoko K (2005). Experimental diabetes
model in chick embryos treated with STZ. Biol Pharm.
Bull. 28:1986-1988.
Vol -1, Issue - 1, Jan - Apr 2011 38
Jojoba meal in rabbits, Kiran L.
Kiran received his BVSc., and AH in 2003 and MVSc., in 2005 in Animal Nutrition from the
Veterinary College, Bidar, Karnataka Veterinary, Animal & Fisheries Sciences University,
Bidar, India. Currently he is working as Veterinary Ofcer in the Department of Animal
Husbandry and Veterinary Sciences, Karnataka.
Kiran L
Efect of graded levels of Jojoba meal in the
complete feed of rabbits on voluntary intake and
nutrient utilization
Abstract
The study was conducted to evaluate the supplementation of Jojoba meal (Simmondisa Chinensis) on feed
consumption, body weight changes, feed effciency, and digestibility of nutrients in rabbits. A total of forty
New Zealand white rabbits (4 week old) were randomly allotted to four dietary treatment groups of ten rabbits
in each group. The control group (T0) was fed with complete feed with no Jojoba oil meal and test groups
were fed complete feed with 5, 15 and 25% Jojoba oil meal in T1, T2 and T3 groups respectively. The daily dry
matter intake in test groups were signifcantly (P 0.05) reduced as compared to the control group. The average
weekly body weight in test diet groups found signifcantly (P 0.05) reduced consistently leading to reduction
of body weight. Digestibility of either proximate principles (Dry matter, Organic matter, Crude fbre, Nitrogen
free extractives) or fbre fractions (Neutral detergent fbre and Acid detergent fbre) was unaffected by jojoba
supplementation whereas crude protein and ether extract digestibility and intake of total digestible nutrients
and digestible crude protein were signifcantly lower in treated groups as compared to control group of rabbits.
Key words : jojoba meal, rabbits, nutrients
V. Nagabhushana, Associate Professor, Department of Animal Nutrition
Veterinary College, Vinobanagar, Shimoga-577204
Phone : 09902204994, 08182 248004, email : nagabhushana.kuliyadi@gmail.com
Kiran L, Nagabhushana V, Prakash N, Jadhav N.V, Madhavaprasad C.B,
Ashok Pawar and Shettar V.B.
Veterinary College, Bidar, -585 401
Introduction
Jojoba (Simmondsia chinensis) is an evergreen native oil
seed shrub of the Sonora deserts of Arizona, California in the
United States and Mexico (Hogan, 1979). In India, it is grown
in some parts of Kutch in Gujarat, Jodhpur and Jaipur in
Rajasthan. Jojoba oil meal, a high protein residue (Ngoupayou
et al. 1985), which remains after oil extraction is a potential
unconventional protein feed supplement. But, the toxic and
anti-nutritional substances make the meal unsuitable for
livestock feeding. Boiled Jojoba meal is bitter in taste and has
low acceptability in animal diets. Toxicity of the incriminating
factors such as simmondsin, simmondsin-2-ferulate (Swingle
et al.1985) and related cyanomethylene glycosides however
has never been fully proved nor understood the physiological
mechanisms by which these factors elicit adverse effects.
Although short time consumption of Jojoba meal produced
detrimental effect in animals, long time feeding had shown
gradual adaptation to the meal indicating a scope for its use in
animal feeding under some circumstance. Hence, additional
research is needed to revalidate the dose response effect and
to suggest appropriate feeding system for diets containing
Jojoba meal in farm animals.
Vol -1, Issue - 1, Jan - Apr 2011 39
Further, possibility of partial replacement of costly protein
sources with locally available protein supplements has to
be explored for its nutritive value and after appropriate dose
response study for safe level for sustained production. Very
few animal experiments have been conducted to ascertain
the feeding value as well characterization of toxic effects
of Jojoba meal. Keeping this in view, the present study was
conducted to evaluate the nutritive value of Jojoba meal in
broiler rabbits.
Jojoba meal was procured from Association of the Rajasthan
Jojoba plantation and Research project, Jaipur, Rajasthan.
Forty weaned (4 weeks old) New Zealand white rabbits of
comparable body weights were selected for the study. They
were randomly allotted to four dietary treatment groups
consisting of ten each namely, T
0
(basal diet with no Jojoba),
T
1
(basal diet with 5% Jojoba), T
2
(basal diet with 15% Jojoba)

and T
3
(basal diet with 25% Jojoba). Basal diet was prepared in
the form of complete ration using locally available ingredients
such as maize, sunfower cake, groundnut cake, wheat bran,
red gram pod husk, mineral mixture, vitamin mixture and salt.
Experimental diets, T
1,
T
2
and T
3
were prepared by replacing 5,
15 and 25% Jojoba meal. The rabbits were kept in individual
cages (15 x 18 x 11) and maintained in a well ventilated
laboratory animal house.
Samples of feed collected during the experiment were pooled
every week for further analysis. The samples were analyzed
for proximate principles as per the method described in
A.O.A.C. (1990). Calcium and phosphorus were estimated
according to Talapatra et al. (1940). The forage fber fractions
were estimated according to the method described by Goering
and Vansoest (1970) and toxic content of feed were estimated
according to method described by Van Boven et al. (1993).
Feeding cum growth trial was conducted for a period of 13
weeks. Individual experimental rabbits were weighed before
the start of experiment and subsequently at weekly interval
till the end of experiment. Average daily gain of body weight
was calculated. A digestion trial was conducted for a period of
fve days after the completion of three weeks of feeding trial
to study the nutrient utilization in experimental animals.
Statical analysis
The data generated in the present study with regards to dry
matter (DM) intake, digestibility of nutrients and growth rate
were subjected to analysis of variation according to Snedecor
and Cochran (1985) and results were interpreted accordingly.
Results
Chemical composition of Jojoba oil meal and
experimental diets
The Jojoba oil meal used in the preparation of complete
ration of experimental rabbits in the present study comprised
27.49% crude protein (CP), 11.59% ether extract (EE), 7.37%
crude fber (CF) and total ash (TA) 3.05% (Table 1). The
hexane extract of the oil meal (10.75%) when re-extracted
with acetone, the waxy material obtained which is said to be
containing mixed toxicants was about 5.83%.
Table 1: Chemical composition of J ojoba meal (% DM)
Parameters Per cent
Organic matter 96.95
Crude protein 27.49
Crude fat 11.59
Crude fber 7.37
Total ash 3.05
Neutral detergent fber 28.34
Acid detergent fber 26.21
Mixed toxicant 5.83
Hexane extract 10.75
The CP content in four experimental diets (T
o
, T
1
, T
2
and T
3
)
were 17.24, 17.78, 18.37, 18.57% respectively. The mixed
toxicant level contributed from different levels of Jojoba oil
meal included in the complete ration was 1, 3.79 and 5.78 %
in T
1
, T2and T
3
rations respectively.
Effect of Jojoba oil meal on voluntary feed intake
The average dry matter (DM) intake (g/day) at the end of the
frst week was 21.2, 10.99, 11.52 and 13.16 in T
o
, T
1
, T
2
and
T
3
respectively (Table 2). There was a signifcant (P<0.05)
reduction in DM intake irrespective of the level of Jojoba in
the test diets. The same trend continued till the end of 4
th
week
and the control group animals consumed 26.28 g/day while
the test groups consumed 11.81 to 15.81 g/day.
Table 2: Mean dry matter intake (DMI g/day) in experimental
rabbits
Weeks
Control Treatment groups
T
0
T
1
T
2
T
3
1 21.20
a
10.99
b
11.52
b
13.16
b
2 25.42
a
18.79
b
17.48
b
13.96
b
3 26.33
a
17.32
b
16.68
b
20.23
b
4 26.28
a
11.81
b
15.58
c
15.81
c
5 44.65
a
17.05
b
23.75 -
6 44.22
a
23.03
b
- -
7 43.92
a
29.00
b
- -
8 41.32
a
28.12
b
- -
9 51.21
a
19.97
b
- -
10 60.24
a
27.97
b
- -
11 46.35
a
17.75
b
- -
12 58.46
a
12.40
b
- -
13 55.60 - - -
MeanSE 41.941.20 19.510.72 17.000.82 15.791.3
Mean value with different superscripts in a row differ
signifcantly (P<0.01)
Table 3: Mean weekly body weights (g) in experimental rabbits
Weeks
T
0
T
1
T
2
T
3
0 293.50 298.00 293.50 293.80
1 360.50 314.00 319.00 303.50
2 418.00 345.00 338.75 294.17
3 469.00
a
333.33
b
307.50
b
285.00
b
4 496.00
a
335.00
b
326.67
b
275.00
b
5 524.00
a
312.50
b
250.00
b
-
6 552.00 350.00 - -
7 575.00 365.00 - -
8 607.00 355.00 - -
9 640.00
a
350.00
b
- -
10 672.00 320.00 - -
11 700.00
a
305.00
b
- -
12 788.75
a
300.00
b
- -
13 816.25 - - -
MeanSE 608.6151.65 356.9035.14 305.9011.78 290.294.31
Mean value bearing different superscripts in a row differ
Vol -1, Issue - 1, Jan - Apr 2011 40
Effect on body weight
The initial body weights (g) of the experimental animals
were 293.50, 298.00, 293.50 and 293.80 in T
0
, T
1
, T
2
and
T
3
respectively (Table 3). The weekly mean body weight
gradually increased in control group while in the test diet
group, body weight reduced signifcantly (P<0.05).
Feed conversion effciency
The effciency of feed conversion in control group was initially
low (0.77) and subsequently the effciency was increased to
2.73 at the end of the experiment. Since the test diet groups
were showing negative growth as well as feed intake, the feed
conversion effciency values were not calculated.
Digestibility of dietary nutrients
The average digestibility of dietary nutrients such as dry
matter, organic matter (OM), crude fbre, nitrogen free
extractives (NFE), neutral detergent fbre (NDF) and acid
detergent fbre (ADF) did not differ signifcantly between the
groups except CP and EE (Table 4). The animals in groups
T
1
, T
2
and T
3
were shown to have signifcantly (P<0.05) lower
digestibility for CP and EE.
Table 4:Percent digestibility of nutrients (MeanSE) in
experimental animals
Parameters T
0
T
1
T
2
T
3
Dry matter
79.120.54 81.140.76 80.030.78 80.900.58
Organic matter
79.080.54 80.420.41 79.970.78 78.591.05
Crude protein
77.34
a
0.59 72.01
b
1.12 71.85
b
1.43 70.80
b
1.09
Ether extract
89.50
a
0.33 79.17
b
0.68 80.72
b
2.69 78.17
b
0.78
Crude fber
56.691.14 51.853.76 50.104.17 47.892.39
Nitrogen free
extract
84.930.39 85.050.45 84.432.22 77.923.51
Neutral detergent
fber
61.171.05 57.051.25 56.603.72 54.615.70
Acid detergent
fber
61.390.60 59.651.98 55.781.63 57.521.24
Mean value with different superscript in a row differ
The digestible crude protein (DCP) intake in control group
(T
0
) was signifcantly ( P< 0.05 ) higher ( 3.75 g/day ) as
compared to T
1
(2.83), T
2
(2.87 ) and T
3
(1.77). Similarly,
total digestible nutrients (TDN) intake was 22.06 g/day in T
0

which was signifcantly (P<0.05) high as compared to T
1

(16.78), T
2
(18.56) and T
3
(10.29). However neither DCP nor
TDN content of the experimental diets differs signifcantly.
Discussion
Chemical composition of Jojoba oil meal and
experimental diets
The Jojoba oil meal was having the crude protein content of
27.49% (Table 1) which is comparable to the traditional oil
such as sunfower cake. Hence, complete diet in the present
study were prepared by partial replacement of high protein
groundnut cake with a Jojoba oil meal at 0, 5, 15 and 25% with
sunfower cake to make the complete diets iso-nitrogenous.
The diets used in the present study were adequate in providing
required nutrients as per the standard recommendation (NRC,
1977).
The mixed toxicants mainly comprising simmondsin and
simmondsin 2 ferulate, in Jojoba oil meal was also comparable
to the values reported by Ngoupayou et al. (1985) and Swingle
et al. (1985). The ether extract content of Jojoba oil meal used
in the present study was comparatively at higher range i.e.
11.59% as the Jojoba oil meal procured for the present study
might be screw pressed, contrary to solvent extraction. The
crude fbre content of the test ingredient (Jojoba) was also
comparable to the value obtained by Swingle et al. (1985).
Effect of Jojoba oil meal on voluntary feed intake
Rabbits in Jojoba oil meal supplemented groups had shown
severe inhibition of dry matter intake (Table 2) as compared
to the control group. The voluntary intake was uniformly
reduced in all the Jojoba supplemented groups. The results
indicate that Jojoba oil meal has severely reduced the
voluntary feed intake in rabbits. Similarly dry matter intake
reduction has been reported by Ngouypayou et al. (1982) in
chicks; Ngouypayou et al. (1985) in rabbits; Cokelaere et
al. (1993a) in rats; Vermaut et al. (1997) in chicken ; Flo et
al.(1999) in rats and Trei et al. (1979) in sheep and cattle. The
reduction in dry matter intake in the present study could be
attributed to bitter favors of Jojoba beans (Booth et al. 1974)
and anorexic effect of simmondsin due to its effect on satiety
center (Flo et al.1998).
Effect on body weight
The body weight of rabbits in test groups were signifcantly
(P<0.05) reduced as compared to control group (Table 3).
Like voluntary intake, the body weight was also uniformly
reduced in all the Jojoba supplemented groups, indicating
that the Jojoba oil meal has got a profound adverse impact
on normal growth. This is evident from the Table 3 which
shows signifcantly negative body weight that is affecting
the cumulative average weekly gain of rabbits in test diet
groups. Similar fndings were also reported by Ngoupayou
et al. (1982) in chicks; Ngoupayou et al. (1985) in New
Zealand white rabbits; Arnouts et al. (1993) in broiler breeder
pullets; Vermaut et al. (1997) in chicken; Booth et al. (1974);
Cokelaere et al. (1993a,b) and Flo et al. (1999) in rats; and
Roeline et al. (2000) in dogs. Severe voluntary feed intake
reduction appears to be the direct cause for the reduced body
weight gain in the present study. The results are in agreement
with Arnouts et al. (1993) and Cokelaere et al. (1995) who
have attributed the body weight reduction to inhibition of
appetite linked with simmondsin content of Jojoba oil meal.
Feed conversion effciency
It is evident from the previous section that voluntary feed
intake was reduced and concomitant reduction of daily gain
in weight. Moreover, mortality in the experimental animals
was recorded in test diet groups from second week onwards.
Therefore feed conversion effciency of control diet could not
be compared with the test groups.
Digestibility of dietary nutrients
The digestibility of dry matter, organic matter and fbre
content was not altered by the inclusion of Jojoba oil meal in
the present study (Table 4). However, digestibility of crude
protein and ether extract fractions were signifcantly (P 0.05)
Jojoba meal in rabbits, Kiran L.
Vol -1, Issue - 1, Jan - Apr 2011 41
reduced in Jojoba fed group as compared to control group. The
reason for no effect on digestibility on the above parameters
may be due to compensatory effect of experimental animals to
overcome from the availability of less feed, at digestion level,
due to depressed voluntary feed intake to fulfll the critical
need of nutrients. However, the reason for the decreased CP
intake may be attributed to the presence of additional toxicant
such as trypsin inhibitor present in Jojoba meal as reported
by Swingle et al. (1985). The digestible CP intake and also
TDN intake were reduced in the experimental rabbits in the
present study. Decrease in digestible nitrogen values was also
observed in rats (Ngoupayou et al. 1985) sheep (Swingle et al.
1985) and dog (Roeline et al. 2000).
Conclusions
From the foregoing, it is concluded that the supplementation
of Jojoba oil meal as low as 5% in diet may severely reduce dry
matter intake with concomitant loss of body weight leading to
high mortality in broiler rabbits. Nutrient utilization was also
affected in terms of reduced DCP and TDN intake. Jojoba oil
meal, a new unconventional protein supplement found to be
of limited nutritional value in rabbit production particularly
beyond 5% inclusion levels.
Acknowledgements
The authors are thankful to the Association of the Rajasthan
Jojoba plantation and Research project, Jaipur, Rajasthan for
providing Jojoba meal for the present study.
Reference
A. O. A. C. (1990). Offcial Methods of Analysis, 15
th
edition.
Association of Offcial Analytical Chemists, Washington
DC.
Arnouts S, Buyse J , Cokelaere MM. and Decuypere E
(1993). Jojoba meal (Simmondsia chinensis) in the
diet of broiler breeder pullets: Physiological and
Endocrinological effects Poul. Sci. 72 : 1714 1721.
Booth AN, Elliger CA, Waiss AC (1974). Isolation of a toxic
factor from Jojoba meal. Life Sci. 15: 1115 1120.
Cokelaere MM, Buyse J , Daenens P, Decuypere E, Kuhn E
R, Vanboven M (1993a). The infuence of Jojoba meal
supplementation on growth and organ function in rats.
J. Agri. Food Chem. 41: 1444 1448.
Cokelaere MM, Daenens P, Vanboven M, Kuhn E,
Decuypere E, Darras V (1993b). Infuence of long term
Simmondsin administration on thyroid hormone levels
in adult rats. J. Agri. Food Chem.. 41 (9): 1449 1451.
Cokelaere MM, Decuypere E, Vermaut S, Daenens P,
Vanboven M (1995). Evidences for a satiating effect of
defatted Jojoba meal. Ind. Crops Prod. 4 (2): 91 96.
Flo G, Vermaut S, Darras VM, Vanboven M, Decuypere E,
Daenens P, Kuhn ER, Cokelaere M (1998). Jojoba meal
and Simmondsin: role in food intake regulation. J. Agri.
Food Chem. 2 : 653 671.
Flo G, Vermant S, Darass VM, Vanboven M, Decuypere
E, Kuhn E, Daenens P, Cokelaere MM (1999). Effects
of Simmondsin on food intake, growth and metabolic
variables in lean (+1) and obese (fa/fa) Zucker rats. Br.
J.Nutri. 81 : 159 167.
Goering HK, Vansoest PJ (1970). Forage fbre analysis. In:
Agricultural Handbook. 379 USDA, Washington, DC.
pp 8 12.
Hogan L (1979). Jojoba: A new crop for arid regions. In: G.
A. Richie (Ed.) New Agricultural corps. AAAS selected
symposium, 38: 177 205.
Ngoupayou JDN, Maiorino PM, Reid BL (1982). Jojoba
meal in poultry diets. Pou. Sci. 61: 1692 1696.
Ngoupayou JDN, Maiorino M, Schur WA, Reid BL (1985).
Jojoba meal in rabbit diets. Nutri. Repo. Int. 31 (1):
11 19.
NRC (1977). Nutrient requirement of domestic animals:
Nutrient requirements of rabbits. 2
nd
revised Edn.
National Research Council, Washington D C.
Roeline H, Vermaut S, Flo G, Cokelaere MM, Decuypere
E (2000). Digestive performance of dogs fed a Jojoba
meal supplemented diet. Ind. Crops Prod. 12 (3) : 159
- 163.
Snedocor GW, Cochran WG (1985). Statistical methods, 8
th
Edition IOWA State University Press, Ames, IOWA,
50010
Swingle RS, Garcia MR, Delfno FJ, Prouty FL (1985). An
evaluation of Lactobacillus acidophilus treated jojoba
meal in Beef cattle diets. J. Agri. Food Chem. 60(3):
832 839.
Talapatra SK, Ray SN, Sen KC (1940). Estimation of
phosphorus, chlorine, calcium, magnesium, sodium and
potassium in feeding stuffs. Indian J ournal of Veterinary
Sciences and Animal Husbandry. 10: 243.
Trei JE, Nelson EA, Verbiscar AJ, Banigan TF (1979).
Evaluation of deoiled non-detoxifed Jojoba meal with
lambs. In: Proc. West. Sec. Am. Soc. Anim. Sci. 30: 239-
242.
Van Boven M, Blaton N, Cokelaere MM, Daenens P
(1993). Isolation, purifcation and stereochemistry of
Simmondsin. J. Agri. Food Chem. 41 : 1605 1607.
Vermaut S, Coninck KD, Flo G, Cokelaere, MM, Onagbesan
M, Decuypere E (1997). Effect of deoiled Jojoba meal
on feed intake in chickens: satiating or taste effect? J.
Agri. Food Chem. 45(8): 3158 3163.
Vol -1, Issue - 1, Jan - Apr 2011 42
Uday kumar received his MBBS from the Gandhi Medical College, Hyderabad in
1986 and MD in Pathology from the Kakatiya Medical College, Warangal in 1991.
Currently he is working as Scientist E (Deputy Director) at the National Institute
of Nutrition, Hyderabad. He has been working as Pathologist and toxicologist for
the past 20 years in the felds of nutrition and toxicology. His main research inter-
est includes nutrition and cancer interaction, apoptosis and toxicology. Currently,
undergoing training in Allergencity assessment of genetically modifed foods at
University of Nebraska, Lincoln, USA. He is serving as CPESEA nominee and Inspec-
tor for various Govt. organizations and institutes.
Uday Kumar P.
Efect of varying doses of Cisplatin on rat
intestinal cell apoptosis
Uday Kumar P*, Vijayalakshmi B*, Kalyanasundaram S
+
, Raghunath M
#
and Sesikeran B*
* Division of Pathology,
#
Biochemistry Division,
+
National Center for Lab Animal Sciences.
National Institute of Nutrition, Hyderabad, India.
Abstract
The purpose of this study was to (a) determine the dosage of cisplatin required to induce intestinal epithelial
cell apoptosis, without causing lethality or necrosis and (b) to assess the biochemical changes in intestinal
mucosa with different doses of cisplatin administration. A total of 9 WNIN weanling male rats were divided
into three groups (n=3). Groups I and II rats were administered low and high doses of cisplatin intraperitoneally
(3 and 12 mg/kg body weight) for three weeks, based on computation from human dose while control rats
were administered saline intraperitoneally. Morphometric apoptotic counts increased signifcantly in both
villus and crypt regions of the jejunum in animals of group I but not in animals of group II as compared
to control. These changes in apoptotic index correlated with enhanced caspase-3 activity and DNA ladder
pattern studies. Increased cell death also resulted in loss of functional integrity of the jejunal mucosa and these
events were linked to increased oxidative stress and altered antioxidant enzyme activities. A weekly dose of
3 mg cisplatin/kg body weight appeared to induce intestinal epithelial cell apoptosis in WNIN strain, without
causing mortality and could be used as a model for determining the therapeutic and cytoprotective potential of
novel test candidates.
Key words: apoptosis, cisplatin, epithelial cells, oxidative stress
Uday Kumar Putcha, Scientist E, Div. of Pathology, National Institute of Nutrition, Hyderabad 500 007
Phone : 91 040 27197309, email : putchaaudaykumar@yahoo.com
Effect of cisplatin on intestinal apoptosis, Uday Kumar P.
Introduction
Injury to normal but rapidly proliferating cells in the bone
marrow and intestine often complicates the treatment of patients
with neoplastic disease (Van Hayen et al. 1998; Wadler et al.
1998). Systemic chemotherapy exerts cytoablative actions via
several different mechanisms, ultimately leading to cell cycle
arrest and/or apoptosis and produces changes in the structure
of the intestinal mucosa (Baskerville and Bater-Hatton, 1997;
Levin, 1968; Slavin et al. 1978) that are associated with
increased permeability of the intestine (Siber et al. 1980).
Cisplatin (cis-diamminodichloroplatinum, II) is one of the
most frequently used anticancer drugs. The therapeutic
effcacy of cisplatin derives from its ability to form complexes
with DNA (Cohen and Lippard, 2001), which exert their
cytotoxicity by directly inhibiting DNA and RNA synthesis and
inducing apoptosis (Meyn et al. 1995; Sorenson et al. 1990).
In addition, cisplatin has been shown to induce production of
reactive oxygen species (ROS) that are important mediators of
stress response in many cell types (Adler et al. 1999; Benhar
et al. 2001). Also, it has been shown that mitochondrial
density within a cell could also determine its response to
Vol -1, Issue - 1, Jan - Apr 2011 43
cisplatin exposure (Miyajima et al. 1999). In fact, increased
intracellular reduced glutathione (GSH) concentrations are
found in cells resistant to cisplatin (Godwin et al. 1992).
However, cisplatin-induced apoptosis also involves events
that are not ROS-dependent (Miyajima et al. 1997).
Despite the extensive use of cisplatin in cancer chemotherapy,
there are few animal models currently available to assess
the effects, if any, of micronutrient defciencies on cisplatin
induced apoptosis and the probable biochemical/molecular
mechanisms underlying such an effect.
The present study was conducted to arrive at the optimum
dose of cisplatin in WNIN rats that would induce apoptosis
in intestinal mucosal cell, without causing mortality in the rat.
This study helps in looking into cisplatin induced apoptosis
which could be used to study the effects of compounds
including micronutrients, their defciencies and intervention
on intestinal mucosal cell apoptosis.
Animals and study design
The animal study protocols were approved by the Scientifc
Advisory Committee as well as Institutional Animal Ethics
Committee. Nine weanling male, WNIN rats were obtained
from and acclimatized in National Center for Laboratory
Animal Sciences for 1 week and maintained at 24 2 C, 50-
60% relative humidity, with a 12 h light-dark cycle. They were
housed in polypropylene cages with stainless steel top grill
with food and water spouts and closed bottom. Autoclaved
paddy husk was used for bedding. They were fed for 17 weeks
on a casein based (20 % protein) control diet, till achieving
adulthood.
After 17 weeks, the animals were divided into three groups
(n=3, each group). Group I and II were administered cisplatin
intraperitoneally, once weekly, at a dose of 3 and 12 mg/
kg body weight for three weeks and the control group was
administered the vehicle (saline). This dosage of cisplatin was
computed taking into consideration the human therapeutic
dosage of 50-120 mg/m
2
body surface. Cisplatin (1 mg/ml)
was diluted in 0.9% saline to obtain appropriate dosage before
administration. The intraperitoneal dose of cisplatin gives
blood levels equivalent to a routine intravenous administration
of the drug (Reed and Kohn, 1990).
The rats had free access to feed and water. Their food intake
(daily) and body weights (weekly) were recorded during the
course of the experiment. The animals were observed for
survival, body weights, hemoglobin, RBC, WBC and platelet
counts after 7 days of last dose.
At the end of feeding and treatment regimen, venous blood
was collected from all rats after a 17 h fasting, through orbital
sinus vein puncture (Riley, 1960) into heparin containing vials.
Rats from each group were euthanized in a CO
2
chamber. A
20-cm segment of the jejunum, beginning 12-cm distal to the
ligament of Treitz, was immediately excised via a midline
abdominal incision and freed from mesentery and fat. It was
then processed for evaluating the changes in apoptotic rates.
Chemicals and Reagents
Cisplatin was obtained from Dabur Pharmaceuticals, India.
The substrate for caspase-3 (Ac-DEVD-pNA) was procured
from Calbiochem, San Diego, CA, USA. RNase, proteinase K,
Nonidet NP-40, agarose, lys, ala- 7-amido-4-methyl coumarin
were procured from Sigma chemical company, St Louis, MO,
USA. All other chemicals were of the highest analytical grade
procured from local sources.
Processing of the jejunum
A 20-cm segment of jejunum was gently washed with ice-cold
phosphate buffered saline (PBS), divided randomly into three
segments of 5, 5 and 10 cm each and processed by standard
procedures (Bodiga et al. 2005). They were used respectively
for light microscopic observations, isolation of epithelial cells
and extraction of DNA and determination of enzyme activities,
parameters of oxidative stress and antioxidant status (Bodiga
et al. 2005; Vijayalakshmi et al. 2005).
Detection of Apoptosis
Samples of rat jejunum fxed in buffered formalin for 24 h
were dehydrated, embedded in paraffn and the number of
apoptotic cells per 1000 cells were scored in 4-mm thick
serial sections using a light microscope, after staining with
hematoxylin and eosin. Apoptotic bodies were identifed by
the presence of shrunken/pyknotic nuclei with surrounding
rim of irregular cytoplasm and vacuolation all around (Kerr et
al. 1972). Also, the DNA isolated from the jejunal epithelial
cells was resolved on an agarose gel and the DNA fragments
visualized under a UV trans-illuminator after ethidium
bromide staining (BioRad) (Gong et al. 1994). Caspase-3
activity was also determined according to previously reported
method (Henkels and Turchi 1999).

Oxidative stress, antioxidant status
The frozen samples of the mucosal scrapings were processed
and the activities of caspase-3, Cu, Zn-SOD, glutathione
peroxidase, catalase were determined as described by us
earlier (Bodiga et al. 2005). Tissue oxidative stress was
quantifed by the estimation of TBARS and protein carbonyls
(Bodiga et al. 2005; Vijayalakshmi et al. 2005).
Functional integrity of villus
Activities of alkaline phosphatase and lys, ala dipeptidyl
amino peptidase were measured in 12000g supernatant, as
markers of functional integrity of the mucosal membrane
(Vijayalakshmi et al. 2005).
All the results were expressed as mean SE. Data was
analyzed statistically by one way analysis of variance
(ANOVA) followed by Post Hoc multiple comparison tests of
signifcance using the SPSS package (Version 10.0, Chicago,
USA). Since no heterogeneity of variance was observed with
any of the parameters tested. Differences among the groups
were tested by the parametric, least signifcant difference
(LSD) test. The differences were considered signifcant only
if P 0.05.
Animals treated with cisplatin at dose of 3 mg/kg body weight
Vol -1, Issue - 1, Jan - Apr 2011 44
tolerated the dose well and survived till the end of 3 weeks,
while those treated with 12mg/kg body weight did not tolerate
the dose as seen in the form of weakness, lethargy and gross
morbidity Therefore, group II rats were sacrifced after 2
weeks, before mortality occurred.
Results
Jejunal mucosal apoptosis
Apoptotic index measured in jejunal mucosa showed a
signifcant increase in the percentage of apoptotic cells in
both villus and crypt regions (Figs.1 and 2) in group I while
a single dose of 12 mg/kg body weight of cisplatin in group
II did not show any increase in the apoptotic index in either
villus or crypt regions (Table 1). Caspase-3 activity was
found to correlate with the induction of apoptosis. Enhanced
caspase activity was observed in group I rat intestine, while
group II has not shown increased activation, compared to
controls (Table1). The electrophoretic separation of DNA
isolated from the intestinal mucosal cells showed shearing
of DNA and not the ladder pattern characteristic of apoptosis
(data not given).
Table 1 : Cisplatin effect on the apoptotic rates of
epithelial cells
Group
(n=3)
Villus
Apoptotic
Index
Crypt
Apoptotic
Index
Caspase-3
activity
(mmoles of
p-NA/mg
protein)
Mean SE Mean SE Mean SE
Control 3.37
a
0.35 0.19
a
0.01 0.045
a
0.003
Group I 6.43
b
0.15 2.30
b
0.26 0.177
b
0.015
Group II 2.63
a
0.52 0.20
a
0.00 0.057
a
0.012
Values with different superscripts are signifcantly different
(P <0.05)
Tissue oxidative stress
Enhanced protein oxidation was observed signifcantly in
group I and group II as compared to control, but the extent
of oxidation seems to be relatively less in group II compared
to group I. The thio barbituric acid reacting substance
(TBARS) were observed signifcantly in group I compared
to other groups while glutathione was signifcantly reduced in
experimental groups compared to controls (Fig. 1).
Tissue antioxidant status
Accompanying changes in antioxidant enzyme showed that
cytosolic SOD activity was signifcantly increased in group
I and II (but lesser in group II) as compared to controls.
Catalase and glutathione peroxidase (GPX) showed a reverse
trend, where the activity was found to be signifcantly reduced
in both experimental groups in comparison to control group
(Fig. 2).
Functional integrity of the jejunal villi
Lys-ala-dipeptidyl aminopeptidase activity was signifcantly
less in group I but comparable in group II to controls.
Signifcantly decreased activity of alkaline phosphatase
(maximal towards to the upper half of the villus) was seen in
both experimental groups as compared to controls (Fig. 3).
Discussion
Essentially, group I rats received a cumulative dose of 9 mg
cisplatin/kg body weight during the 3-week period and the
tissues were available for all the analysis. Although group
II rats were sacrifced within 2 weeks to get the tissues for
analysis, the data may not be comparable to group I, because
they were not time-matched. In line with earlier reports
that chemotherapy causes intestinal damage with apoptosis
in intestinal crypts, and hypoproliferation resulting in crypt
hypoplasia (Keefe et al. 2000). Our present study also
revealed that the crypt region showed greater sensitivity to
cisplatin. We observed that cisplatin could induce apoptosis
in both differentiated (villi) and proliferative (crypt) regions
of the intestinal epithelium indicating that its action is not cell
cycle specifc.
Fig 1. Effect of cisplatin on oxidative stress in IECs.
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
Group II Group I Control
G
S
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(
m
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l
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s
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g

p
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t
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S

(
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0.0
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2.5
3.0
0.0
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1.0
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2.0
2.5
3.0
3.5
4.0
a
a
a
a
b
b
b
b
c

Values with different superscripts are signifcantly different (P < 0.05)
Vertical bars are means with standard error (n=3 per group). Levels of lipid peroxides (TBARS) do not show signifcant
difference while glutathione (GSH) and protein carbonyls are signifcantly different.
Vol -1, Issue - 1, Jan - Apr 2011 45
Caspase 3 is an effector caspase activated by many apoptotic
stimuli. The present study also showed increased effector
caspase activation in rats treated with cisplatin confrming
that intestinal epithelial cells (IEC) death in cisplatin treated
rats is truly apoptotic and not necrotic, the sensitivity of IECs
to cisplatin induced apoptosis was further assessed by DNA
fragmentation in these cells. Despite a constitutive low rate of
apoptosis in IECs of control rats, no DNA fragmentation was
detectable. Cisplatin treatment per se signifcantly increased
DNA fragmentation. This could be due to DNA breakage as a
result of drug interactions with DNA.
There is a balance maintained between oxidative stress and
antioxidant enzymes for normal functioning of the cells. To
cope with increased oxidative stress, antioxidant enzymes
are generally up-regulated. It is well known that cisplatin is
a redox cycler that generates toxic reactive oxygen species
and causes oxidative injury to various cells and tissues (Chu,
1994; Masuda et al. 2003). The signifcantly higher levels of
TBARS and protein carbonyls (oxidative products of lipids
and proteins) in the IECs of cisplatin treated rats indicates
increased oxidative stress and the data suggests that cisplatin
treatment substantially increased the ROS formation in the
intestinal mucosa (Benhar et al. 2001). Enhanced protein
oxidation was observed in group I and II as compared to
controls. However, it was lesser in group II than group I
because of the short-term exposure to the drug.
Endogenous antioxidant systems are of singular importance
in limiting oxidative cellular damage. Elevated intracellular
levels of GSH, the cellular antioxidant involved in free
radical scavenging activity, is associated with resistance to
chemotherapeutic agents (Zhang et al. 1998). The GSH levels
in the IECs treated with cisplatin in our study were decreased
Fig 2. Antioxidant enzymes status in control and cisplatin treated groups
0
1
2
3
4
5
6
7
8
9
S
O
D

(
U
/
m
g

p
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0.02
0.04
0.06
0.08
0.10
0.12
0.14
c
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(
U
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0.06
0.08
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0.14
0.16
0.18
0.20
G
P
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)
c
c
c
a
a
a
b
b
b
Values with different superscripts are signifcantly different (P <0.05)
Vertical bars are means with standard error (n=3 per group) Superoxide dismutase (SOD), catalase and GPX levels are
signifcantly different between groups.
Fig 3. Effect of cisplatin on IEC marker enzyme activities
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0
2
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8
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12
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16
18
20
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p
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a
b
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a
Values with different superscripts are signifcantly different (P < 0.05)
Vertical bars are means with standard error (n=3 per group). No change in lys-ala-di-peptyl amino peptidase but
signifcant differences seen with alkaline phosphatase between the groups.
Vol -1, Issue - 1, Jan - Apr 2011 46
markedly. Cisplatin treatment increased SOD and decreased
catalase and GPX levels than control rats. These changes in
the antioxidant enzymes observed in this study are in general
agreement with earlier reports where up-regulation/over
expression of SOD and catalase has been shown to decrease the
toxicity of cisplatin (Davis et al. 2001) and down-regulation
of GPX was associated with mitochondrial dysfunction in the
kidney epithelial cells (Sugiyama et al. 1989). The increased
SOD activity in cisplatin treated animals, could be in
response to increased generation of superoxide (O
2
-
) radicals
known to occur during the interaction between cisplatin
and DNA and are involved in the toxic effects of this drug
(Masuda et al. 2003). These toxic effects can be decreased
by overexpression of Mn-SOD and catalase enzymes (Davis
et al. 2001). However, in the current study the increased SOD
activity could not protect the cells probably because there was
a decrease in catalase and GPX activities. These changes in
antioxidant enzyme activities may enhance the accumulation
of hydroxyl radicals causing damage and apoptosis.
It appears that the possible increase in the accumulation
of H
2
O
2
or R
2
O
2
as a consequence of the imbalance in the
antioxidant enzyme activities could enhance the intrinsic
sensitivity of the intestinal mucosa to cisplatin-induced
apoptosis. It is known that along with O
2
radicals, OH
radicals also play role in cisplatin toxicity (Masuda et al.
2003). Thus increased oxidative stress seems important in
cisplatin induced apoptosis/cytotoxicity.
We intended to look into the integrity of the mucosal cells,
which is compromised upon exposure to chemotherapeutic
agents due to increased oxidative stress. Cisplatin
administration signifcantly altered mucosal marker enzyme
activities in IECs of control rats indicating altered structural
and functional integrity of mucosa. These results indicate
that cisplatin per se increases oxidative stress in general and
lipid peroxidation in particular, which damage the membranes
involving membrane fuidifcation (Rebillard et al. 2007).
It could also result in loss of barrier integrity which is a
risk factor for infection (Khan and Wingard, 2001). Severe
oxidative damage to intestine could be the causative agents for
the death of the animals (Arivarasu et al. 2007).
Among different doses of cisplatin tested, 3mg/kg bodyweight
could effectively induce apoptosis without causing lethality.
This is in contrast to earlier studies, wherein single dose
administration of 5mg/kg cisplatin induced apoptosis, but it
was on a smaller magnitude and transient, peaking at 36 h
and reaching control by 48 h (Tamaki et al. 2003). The results
of this preliminary study demonstrate that intraperitoneally
administered cisplatin at a dose of 3.0 mg/kg body weight
for three weeks (once in a week) is safe,

well tolerated, and
effective in inducing apoptosis in both villus and crypt regions.
Acknowledgements
The authors acknowledge the scientifc suggestions given by
Drs. K. M. Nair, Scientist E and G. Bhanuprakash Reddy,
Scientist D of National Institute of Nutrition, Hyderabad. We
place on record the appreciation of the technical help rendered
by Mr. B. Giribabu.
References
Adler V, Yin Z. Tew KD, Ranai Z (1999). Role of redox
potential and reactive oxygen species in stress signaling.
Oncogene 18: 6104-6111.
Arivarasu NA, Fatima S, Mahmood. (2007). Effect of
cisplatin on brush border membrane enzymes and anti-
oxidant system of rat intestine. Life Sci. 81(5): 393-398.
Baskerville A, Batter-Hatton D (1977). Intestinal lesions
induced ex perimentally by methotrexate. Br. J. Exp.
PathoI. 58: 663-669
Benhar M, Dalyot L Engelberg D, Levitzki A (2001).
Enhanced ROS production in oncogenically transformed
cells potentiates c-J un N-terminal kinase and p38
mitogen-activated protein kinase activation and
sensitization to genotoxic stress. Mol. Cell Biol. 21:
6913-6926.
Benhar M, Dalyot L, Engelberg, Levitzki A (2001).
Enhanced ROS production in oncogenically transformed
cells potentiates c-J un N-terminal kinase and p38
mitogen-activated protein kinase activation and
sensitization to genotoxic stress. Mol. Cell Biol. 21 :
6913-6926.
Bodiga VL, Boindala S, Putcha U, Subramaniam K,
Manchala R (2005). Chronic low intake of protein or
vitamins increases the in testinal epithelial cell apoptosis
in Wistar/NIN rats. Nutri. 21: 949-960.
Chu G. (1994). Cellular responses to cisplatin. The roles of
DNA binding proteins & DNA repair. J. Biol. Chem.
269: 787-790.
Cohen SM, Lippard SJ (2001). Cisplatin: from DNA damage
to can cer chemotherapy. Prog. Nucleic Acid Res. Mol.
Bio. 67: 93-130.
Davis CA, Nick HS, Agarwal A (2001). Manganese
superoxide dis mutase attenuates Cisplatin-induced renal
injury: importance of superoxide. J. Am. Soc. Nephrol.
12: 2683-2690.
Godwin AK. Meister A, ODwyer PJ , Huang CS, Hamilton
TC, Anderson M (1992). High resistance to cisplatin
in human ovar ian cancer cell lines is associated with
marked increase of glu tathione synthesis. Proc. Natl.
Acad. Sci. USA. 89: 3070-3074.
Gong JP, Traganos F, Darzynkiewicz Z (1994). A selective
procedure for DNA extraction from apoptotic cells
applicable for gel electrophoresis and fow cytometry.
Anal. Biochem. 218: 314-316.
Henkels KM, Turchi JJ (1999). Cisplatin induced apoptosis
proceeds by caspase 3 dependent and independent
pathways in cisplatin resistant and sensitive human
ovarian cancer cell lines. Cancer res. 59: 3077-3083.

Vol -1, Issue - 1, Jan - Apr 2011 47
Keefe DM, Brealey J, Goland GJ, Cummins AG (2000).
Chemother apy for cancer causes apoptosis that precedes
hypoplasia in crypts of the small intestine in humans.
Gut. 47: 632-637.
Kerr JF, Wyllie AH, Currie AR (1972). Apoptosis: A basic
biological phenomenon with wide ranging implications
in tissue kinetics. Br. J. Cancer. 26: 239 - 257.
Khan SA, Wingard JR (2001). Infection and mucosal injury
in cancer treatment. J. Nat. Cancer Inst. Monographs.
29 : 31-36.
Levin RJ (1968). Anatomical and functional changes of the
small in testine induced by 5-fuorouracil. J. Physiol.
197: 73P-74P.
Masuda H, Tanaka J, Takahama U (2003). Cisplatin
generates superoxide onion by interaction with DNA
in a cell free system. Biochem. Biophys. Res. Commun.
1994 : 1175-1180.
Meyn RE, Stephens LC, Hunter NR, Milas L (1995).
Kinetics of cisplatin-induced apoptosis in murine
mammary and ovarian adenocarcinomas. Int. J. Cancer.
60: 715-729.
Miyajima A, Nakashima J, Tachibana M, Nakamura K, Hay-
akawa M, Murai M (1999). N-acetylcysteine modifes
cis-dichlorodi ammineplatinum induced effects in
bladder cancer cells. Jpn. J. Cancer Res. 90: 565-570.
Miyajima A, Nakashima J, Yoshioka K. Tachibana M, Tazaki
H, Murai M (1997). Role of reactive oxygen species in
cis-dichlorodiam mineplalinurn induced cytotoxicity on
bladder cancer cells. Br. J. Cancer. 76: 206-210.
Rebillard A, Tekpli X, Meurette O, Sergent O, LeMoigne G,
Muller, Vernhet L, Gorria M, Chevanne M, Christmann
M, Kaina B (2007). Cisplatin-Induced Apoptosis
Involves Membrane Fluidifcation via Inhibition of
NHE1 in Human Colon Cancer Cells. Cancer Res.
67(16): 7865 - 7874.
Reed E, Kohn KW (1990). Platinum analogues. in: Cancer
Chemotherapy. Principles and Practice. BA Chabner
and JM Collins (eds.), Philadelphia: J. B. Lippincott. pp.
465-490.
Riley V (1960). Adoptation of orbital bleeding techniques to
rapid serial blood studies, in Proceedings of Society of
Experimental Biology Medicine, 104 751-754.
Siber GR, Mayer RF, Levin MJ (1980). lncreased
gastrointestinal absorption of large molecules in
patients after S-fuorouracil therapy for metastatic colon
carcinoma. Cancer Res. 40: 3430-3436.
Slavin RE, Dias MA, Saral R (1978). Cytosine arabinoside
induced gastrointestinal toxic alterations in sequential
chemotherapeu tic protocols: a clinical-pathologic study
of 33 patients. Cancer. 42: 1747-1759.
Sorenson CM, Barry MA, Eastman A (1990). Analysis of
events asso ciated with cell cycle arrest at G2 phase and
cell death induced by cisplatin. J. Natl. Cancer Inst. 82:
749-755.
Sugiyama S, Hayakawa M, Kato T, Hanaki Y, Shimizu K,
Ozawa T (1989). Adverse effects of anti-tumor drug.
cisplatin, on rat kidney mitochondria: disturbances in
glutathione peroxidase activity. Biochem. Biophys. Res.
Commun. 159: 1121-1127.
Tamaki T, Naomoto Y, Kimura S, Kawashima, Shivakawa Y,
Shigemitsu, Yamatsuji T, Herisa M, Gunduz M, Tanaka
M. (2003). Apoptosis in normal tissues induced by anti-
cancer drugs. J. Int. Med. Res. 31(1): 6-16.
Van Hayen JP, Bloch F, Attar A, Levoir D, Kreft C, Molina
T, Bruneval P (1998). Diffuse mucosal damage In the
large intestine associated with Irinotecan (CPT-11). Dig.
Dis. Sci. 43: 2649-2451.
Vijayalakshmi B, Sesikeran B, Udaykumar P, Kalyanasun-
daram S, Raghunath M (2005). Effects of vitamin
restriction and supplementation on rat intestinal
epithelial cell apoptosis. Free Radic. Biol. Med. 38:
1614-1624.
Wadler S, Benson AB 3rd, Engelking C, Catalano R, Field
M, Kornblau SM, Mitchell E, Rubin J , Trotta P, Vokes
E (1998). Rec ommended guidelines for the treatment
of chemotherapy induced diarrhea. J. Clin. Oncol. 16:
3169-3178.
Zhang K, Mack P, Wong KP (1998). Glutathione-related
mechanisms in cellular resistance to anticancer drugs.
Int. J. Onco1. 12: 871-882.
Vol -1, Issue - 1, Jan - Apr 2011 48
Vinay T. received his BVSc and AH from Veterinary College Bidar in 2005 and MVSc.,
in Veterinary Pharmacology and Toxicology from the Veterinary College Bangalore
in 2007. Worked as Veterinary Offcer in the Department of Animal Husbandry and
Veterinary Services, Government of Karnataka for a short duration. Currently, he is
pursuing PhD in Veterinary Pharmacology and Toxicology at Veterinary College,
Bangalore.
Vinay T.
Sub chronic toxicity study of Fusarium xylarioides culture
fltrate from ground nut hay in rats
Sub chronic toxicity of Fusarium xylarioides, Vinay T.
Sridhar N.B., Assistant Professor, Department of Veterinary Pharmacology and Toxicology
Veterinary College, Hebbal, Bangalore-560024, Karnataka
Phone : 9448059777, email: sridhar_vet@rediffmail.com
Abstract
Sub chronic toxicity study of culture fltrate of Fusarium xylarioides isolated from the fungal contaminated
ground nut hay was carried out in rats. The fungi was isolated from the contaminated groundnut hay which
caused mycotoxicosis in cross bred cattle, exhibited the clinical signs of colic, tenesmus, ruminal atony,
anorexia, bleeding from nostrils, rectum and fy bite site. The rats were gavaged with culture fltrate at the
dose level of 0.25, 0.5, 1 and 2 ml daily for 90 days. Clinical signs observed were diarrhea, weakness,
severe arching of back, swollen forehead and conjunctival hemorrhage. Cutaneous hemorrhagic patches on
back, scrotum, abdomen, ears and legs region seen. There was a signifcant increase (P 0.05) in serum
concentrations of creatinine, urea nitrogen, ALT and AST indicated the renal and hepatic damage which was
confrmed by histopathology. There were lesions in brain and GI tract of the treated rats. The present study
indicated the toxic feature of the culture fltrate of Fusarium xylarioides isolated from ground nut hay.
Key words: sub chronic toxicity, rat, Fusarium xylarioides, groundnut hay,
Vinay T
1
, Shridhar N.B
1
*, Narayanaswamy H.D
2
, Shrikrishna I
3
and J ayakumar K
1
.
1
Department of Veterinary Pharmacology and Toxicology,
2
Department of Veterinary Pathology
3
Departement of Veterinary Microbiology
Veterinary College, Hebbal, Bangalore 560 024, Karnataka Veterinary,
Animal and Fisheries Sciences University, Karnataka, India.
Introduction
Cross bred cattle fed with contaminated groundnut hay for duration
of 2-3 months exhibited the clinical signs of loss of body condition,
colic, tenesmus, ruminal atony and anorexia which was noticed in
Pattanayakanahalli village of Tumkur District in Karnataka, India.
There was bleeding from nostrils, rectum and fy bite site of the
affected animals. Liver function tests of these animals revealed liver
damage. The detailed clinical investigation and history revealed that
the groundnut hay was fed to these animals and revealed blackish
specks or spots indicative of fungal growth. The clinical signs were
observed especially during winter season.
Fungal infected hay can infect dairy cattle, especially during stressful
periods when they are immune suppressed, causing a disease
referred to as mycosis. Molds also produce secondary metabolites
or poisons called mycotoxins that affect animals when they consume
mycotoxin contaminated feeds (Pier, 1992). Mycotoxicosis is well
documented in poultry. However, there is scanty information on cattle
mycotoxicosis.
In the present study, sub chronic toxicity of Fusarium xylarioides
isolated fromfungal affected groundnut hay was evaluated in rats.
Collection of material:
Fungal contaminated groundnut hay which was fed to the ailing
animals was obtained fromPattanayakanahalli village of Sira taluk,
Tumkur District, Karnataka. Groundnut hay was cultured on
potato dextrose agar. Among the grown fungal isolates, Fusarium
xylarioides (F. xylarioides) was mass cultured on potato dextrose
broth (Fig. 1). The fungus was identifed and confrmed at National
Facility, Agharkar Research Institute, Pune. After confrmation of
complete growth of the fungus, the supernatant was discarded and
the fltrate was used for gavaging the rats. The fungal culture fltrate
was analyzed for the presence of afatoxins (B1, B2, G1 and G2),
Vol -1, Issue - 1, Jan - Apr 2011 49
ochratoxin, T2, citrinin, sterigmatocystin and zeralenone by TLC
method.
Experimental design:
Apparently healthy young Wistar albino rats aged fve weeks
weighing 100 10 g were used. Animals were divided into 5 groups
with 12 animals in each group and housed in standard polypropylene
cages during the experiment. Group I served as control which was
gavaged with 2 ml of potato dextrose broth where as group II, III,
IV and V were gavaged with F. xylarioides culture fltrate at the dose
rate of 0.25, 0.5, 1 and 2 ml daily respectively for 90 days. The rats
were weighed individually at the beginning of the study and once in
a week for three weeks and at fortnight interval till day 90. All the
rats were observed daily for the clinical signs of toxicity, morbidity
and mortality.
Clinical biochemistry:
The blood samples were obtained by retro-orbital plexus puncture
method on day 0, 7, 14, 21, 35, 50, 75 and 90 and the serum
was used to estimate alanine aminotransferase (ALT), aspartate
aminotransferase (AST), creatinine (CRT) and blood urea nitrogen
(BUN) using Semi-Automatic Biochemical Analyzer (ARTOS,
Bangalore) and commercially available diagnostic kits (Swemed
Diagnostics, Bangalore).
Gross and histopathological studies:
Necropsy of the rats were conducted which succumbed during the
experiment and the survived rats were sacrifced at the end of the
study. Organs were weighed and representative tissue samples of liver,
kidney, brain, intestines and stomach were collected in 10 % normal
buffer formalin solution (NBF) and were subjected to histopathology
(Luna, 1968).
Statistical analysis:
The data was analyzed by one-way ANOVA with Tukeys post test
using GraphPad Prism Software (Trial version 4.03 for Windows)
GraphPad Software, San Diego, California, USA.
Results
In the present study, one of the predominant fungal species isolated
fromthe fungal contaminated ground nut hay was F. xylarioides.
Perusal of the literature revealed no reports of the same fungal species
identifed on groundnut hay. The fungal culture fltrate was negative
for all the nine mycotoxins analyzed.
There was a signifcant increase (P0.001) in serum ALT and AST
concentrations in the samples of day 21, 35, 50, 75 and 90 (Table 1 and
2). The serum creatinine concentration in rats increased signifcantly
(P0.001) from day 35 to 90 and serum urea nitrogen concentration
increased signifcantly (P0.001) from day 50 to day 90 (Table 3 and
4) in groups treated with 1 and 2 ml of culture fltrate.
Clinical signs in rats observed were diarrhea, weakness, reduced feed /
water intake, loss of body weight, recumbency, severe arching of back,
swollen forehead and torticollis. The rats lost balance of hind limbs
and rarely the forelimbs. Hemorrhages were seen on conjunctiva, sub
cutis on back, scrotum, abdomen, ears and legs region (Fig 2 and 3).
Fig 1.Growth of Fusarium xylarioides in potato dextrose broth

Table 1: The effect of Fusarium xylarioides culture fltrate on serum ALT concentration (U/L)
Treatment
Days
0 7 14 21 35 50 75 90
Group I
(PD broth, Control)
24.7 1.89 27.251.52 30.441.20 32.911.48 35.580.40 38.221.10 40.621.37 37.571.86
Group II (0.25 ml) 24.231.56 28.171.14 31.690.84 35.381.19 39.321.12 45.221.71 57.622.36*** 52.612.42**
Group III (0.5 ml) 23.511.85 27.861.44 31.361.43 35.271.59 40.591.44 46.763.45 62.305.96*** 55.973.16***
Group IV (1 ml) 24.920.89 28.311.04 32.251.75 37.632.30 46.614.91 56.983.83*** 68.127.60*** 62.626.84***
Group V (2 ml) 23.501.98 28.651.49 34.672.69 42.303.00 53.004.38*** 63.276.65*** 83.857.45*** 73.326.78***
Values are mean SE, n = 12, *** P 0.001, * * P 0.01
Table 2: The effect of Fusarium xylarioides culture fltrate on serum AST concentration (U/L)
Treatment
Days
0 7 14 21 35 50 75 90
Group I
(PD broth, Control)
90.162.95 93.673.29 98.213.07 103.412.52 107.022.32 111.372.69 120.684.30 114.223.77
Group II (0.25 ml) 87.673.26 91.562.39 97.082.77 101.752.60 114.05.13 135.667.92** 164. 5.59*** 157.095.11***
Group III (0.5 ml) 88.563.77 93.922.29 99.322.52 104.82.79 122.944.92 149.978.39*** 179.588.74*** 171.468.43***
Group IV (1 ml) 90.923.47 97.232.02 109.452.07 128.094.71** 143.135.49***171.868.34*** 200.589.55*** 189.086.82***
Group V (2 ml) 90.032.68 97.601.80 115.382.95 137.095.34***161.756.47***188.947.35*** 224.9711.99*** 214.3712.35***
Vol -1, Issue - 1, Jan - Apr 2011 50
Table 3: The effect of Fusarium xylarioides culture fltrate on serum creatinine concentration (mg/dl)
Treatment
Days
0 7 14 21 35 50 75 90
Group I
(PD broth, Control)
0.450.03 0.540.034 0.600.026 0.660.02 0.710.020 0.800.031 0.730.02 0.670.014
Group II (0.25 ml) 0.440.038 0.520.035 0.640.049 0.860.040 0.870.023 0.950.057 0.900.052 0.860.046
Group III (0.5 ml) 0.450.030 0.540.037 0.640.038 0.910.049 0.960.057 1.08.058** 0.980.060 0.920.0602
Group IV (1 ml) 0.450.032 0.570.017 0.710.04 0.970.029** 1.050.049** 1.150.043** 1.070.04** 0.980.0431**
Group V (2 ml) 0.470.032 0.610.020 0.740.049 1.160.04*** 1.220.054*** 1.300.069*** 1.170.05*** 1.050.028**
Table 4 : The effect of Fusarium xylarioides culture fltrate on serum urea nitrogen concentration (mg/dl)
Treatment
Days
0 7 14 21 35 50 75 90
Group I
(PD broth, Control)
35. 00.83 36.0 0.89 38.930.82 41.080.72 42.821.37 44.0 1.48 45.71.26 42.050.70
Group II (0.25 ml) 35.331.12 37.300.66 38.23.63 40.160.55 44.750.83 47.561.11 49.51.26 46.321.42
Group III (0.5 ml) 34.991.13 36.570.82 38.640.87 41.680.94 46.301.77 50.641.43** 54.851.71*** 52.702.08***
Group IV (1 ml) 35.281.05 37.910.85 41.301.22 44.561.54 50.661.55** 55.931.76*** 60.122.11*** 56.652.40***
Group V (2 ml) 35.311.25 38.311.17 43.061.39 48.951.41** 55. 2.97*** 62.543.28*** 69.873.70*** 65.973.01***
Values are mean SE, n =12, *** P 0.001,* * P 0.01.
Fig 2: Hemorrhagic patches on ears in rats treated with
Fusarium xylarioides culture fltrate
Fig 3: Hemorrhagic patches on legs and scrotum in rats
treated with Fusarium xylarioides culture fltrate
The gross changes in the liver comprised of hemorrhage, congestion
and the typical histopathological lesions confrmed the liver damage
due to F.xylarioides in all treated groups of rats. This was further
supported by the presence of severe congestion, centrilobular
necrosis, vacuolar degeneration, mild biliary hyperplasia, fbrotic
change at periportal areas and karyomegaly in some of the hepatocytes
(Fig 4 and 5). Further, kidney showed histopathological lesions
like, congestion along with vacuolar degeneration, necrosis of the
tubules and fbrosis in the interstitium (Fig 6). In the present study,
the lesions in the brain comprised of congestion of blood vessels,
perivascular cuffng with mononuclear cells, multiple focal areas of
necrosis with infltration of few infammatory cells and occasional
glial cell aggregation (Fig 7). In the present study, histopathological
lesions observed in the brain included necrosis, liquefaction and
hemorrhages. Further, severe congestion of intestinal mucosa and
hemorrhage was observed. Congestion of blood vessels of stomach
was also observed in all groups of the rats.
Discussion
The gross changes in the liver comprised of hemorrhage, congestion
and the typical histopathological lesions confrmed that the liver
damage is due to F.xylarioides treatment. The result of the present
study is in accordance with the fndings of Sharma et al. (1983).
The elevated serum AST and ALT concentration compared to
control group is suggestive of possible role of mycotoxins present
in gavaged culture fltrate. This was further supported by the gross
and histopathological lesions in the treated groups, by the presence
of lesions like severe congestion, centrilobular necrosis, vacuolar
degeneration, mild biliary hyperplasia, fbrotic change at periportal
areas and karyomegaly in some of the hepatocytes. Such hepatic
damage in rats and mice due to mycotoxicosis was also reported by
many workers with elevation of serumAST concentration (Kellerman
et al. 1990; Voss et al. 1995; Fodor et al. 2006).
Sub chronic toxicity of Fusarium xylarioides, Vinay T
Vol -1, Issue - 1, Jan - Apr 2011 51
Fig 4: Section of liver from rat treated with Fusarium xylarioides culture fltrate showing extensive with loss of nor-
mal architecture. Note hepatomegaly in some of hepatocytes adjacent to normal cells (b) ( H&E x 500)
(a) Control (b) Experimental
Fig 5: Section of liver from rat treated with Fusarium xylarioides culture fltrate showing prominent biliary hyperpla-
sia in periportal region (b) (H&E x 500)
Fig 6: Section of kidney from rat treated with Fusarium xylarioides culture fltrate showing severe congestion of
inter tubular vessels and focal areas of tubular necrosis with loss of architecture (b) (H&E x 500)
Fig 7: Section of brain from rat treated with Fusarium xylarioides culture fltrate showing severe congestion of
blood vessels and perivascular mononuclear cuffng (b) (H&E x 500)
Vol -1, Issue - 1, Jan - Apr 2011 52
The elevated serumcreatinine and urea nitrogen concentration in
comparison to control concentration is suggestive of the possible
role of toxins in causing kidney damage. This was further supported
by histopathological lesions like, congestion along with vacuolar
degeneration, necrosis of the tubules and fbrosis in the interstitium.
Similar fndings were also reported by Fodor et al. (2006). The
changes in the brain described as necrosis, liquefaction and
hemorrhages in the present study were similar to those of earlier
reports of Uhlinger (1997).
T2 toxin is a very potent mycotoxin in cattle which was associated
with gastroenteritis and intestinal hemorrhages (Petrie et al. 1977).
However T2 toxin was negative in the screening of the culture fltrate
which indicates either the concentration of T2 might be too low to
be detected by TLC method or rats might be susceptible to such low
concentration. Congestion of blood vessels of stomach seen in all rats.
Similar fndings were reported by Junsuk et al. (1999) in rats which
were fed with fungal contaminated the diets.
Further studies are essential to confrm the changes seen under
natural disease process in large animals by considering many factors
including dose, concentration of the fungal culture extract and the
formin which the test material is administered.
Conclusions
In Fusarium xylarioides culture fltrate administered group, rats
exhibited diarrhoea, severe arching of back and paraplegia. The
clinical symptoms like swollen forehead, conjunctival hemorrhages,
cutaneous hemorrhagic patches on back, scrotum, abdomen, ears and
legs were observed along with pronounced nervous disorders. The
present study revealed the hepatotoxic and nephrotoxic properties of
the culture fltrate of F. xylarioides in rats at the dose tested (0.25,
0.5, 1 and 2 ml).
Acknowledgements
The authors are thankful to Dr. S.C. Chandrashekhar, Associate
Professor, Department of Plant Pathology, GKVK, UAS, Bangalore
and to Agarkar Research Institute, Pune for isolating and identifying
the fungi. The authors are also thankful to the Government of
Karnataka for funding the project entitled Obscure Diseases of
Cattle and Buffaloes of Karnataka: Studies on Cause and Cure.
References
Fodor J, Meyer K, Riedlberger M, Bauer J, Posa R (2006).
The distribution and elimination of fumonisins after oral
administration of Fusarium verticillioides fungal culture.
Magy. Allatorv. Lapja. 128: 334-342.
Junsuk P, Kyungrim L, Jincheol K, Sunhee L, Jeongah S, Yinwon
L (1999). A hemorrhagic factor (apicidin) produced by toxic
Fusarium isolates fromsoybean seeds. Appl. Env. Microbiol.
4:126130.
Kellerman TS, Marasas WFO, Thiel PG, GelderblomWCA,
Cawood MJ, Coetzer AW (1990). Leukoencephalomalacia
in two horses induced by oral dosing of fumonisin B1.
Onderstepoort J. Vet. Res. 57: 269-275.
Luna LG (1968). Manual of histopathological staining methods of
the armed forces institute of pathology. 3rd Edn., McGraw Hill
Book Co, New York. pp 25-78.
Pier AC (1992). Major biological consequences of afatoxicosis in
animal production. J. Anim. Sci. 70: 3964-3970.
Petrie L, Robb J, Stewart AF (1977). The identifcation of T-2 toxin
and its association with a hemorrhagic syndrome in cattle. Vet.
Rec. 101: 326-326.
Sharma RB, Kumar A, Roy AN (1983). Nature of hepatic damage
induced by culture fltrates of seed-borne fungi in the
laboratory rat. Toxicol. Lett. 19: 1-2.
Uhlinger C (1997). Leukoencephalomalacia in the veterinary clinics
of North America. Equine Pract. 13: 13 20.
Voss KA, Chamberlain WJ , Bacon CW, Herbert RA, Walters
DB, Norred WP (1995). Sub chronic feeding study of the
mycotoxin fumonisin B1 in B6C3F1 mice and Fischer 344 rats.
Fundamental App. Toxicol. 24: 102 110.
Sub chronic toxicity of Fusarium xylarioides, Vinay T
Vol -1, Issue - 1, Jan - Apr 2011 53
Mr. Prakash received his MSc., in Applied Zoology from the Kuvempu University,
Shankaraghatta, Shimoga, Karnataka in 2004 and just submitted the thesis for
award of PhD from the Kuvempu University, Shimoga. Currently, working as
Associate Research Scientist at the Department of Mutagenicity and Teratology,
Advinus Therapeutics Ltd., Bangalore. Served as lecturer at the Department of
Zoology, Sahyadri Science College, Shimoga for two years. He has been nominated
by the CPCSEA as member of IAEC in few organizations.
Prakash G.
Protective efect of cafeine against cyclophospha-
mide induced in vivo genotoxicity
Prakash G
1
*, Ingle A.D
2
and Hosetti B.B
1
.
1
Department of Applied Zoology, Kuvempu University, Shankaraghatta, 577 451,
Karnataka, India.
2
Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata
Memorial Centre (TMC), Kharghar, Navi Mumbai- 410 210, MS, India.
Abstract
Cyclophosphamide is a DNA non-intercalating agent with wide application against several human neoplasms.
It is found that caffeine alters protection against cytogenotoxicity of different chemicals and physical mutagens
including cyclophosphamide. Modulation of the clastogenic induced effect of cyclophosphamide by three
different doses of caffeine (25, 50 and 100 mg/kg) was assessed through the chromosomal aberration and
mitotic index studies in bone marrow cells. Micronucleus level were decreased signifcantly (28.8%) in 100 mg
caffeine treated group at 30 h post treatment. Results revealed that caffeine decreased the cyclophosphamide
induced chromosomal aberration and mitotic index in bone marrow cells. In the present study, the percentage
of dividing cells were reduced more in the mice pre-treated with CAF at the dose rate of 100 mg/kg than
with lower doses of CAF in all the three anticancer drugs tested. Thus, CAF modulation is expected to play
an important role in the development of cancer chemotherapy. The protective effect of caffeine was found to
be dose dependent. However, further study on the mechanism of quenching of free radicals and induction of
apoptosis by caffeine are required to substantiate the results.
Key words: caffeine, chromosomal aberration, cyclophosphamide, cytogenotoxicity, mitotic index.
Prakash G, Associate Research Scientist, DSA, Toxicology, Advinus Therapeutics Ltd., Bangalore
Phone : 9620901066, email : prakash.geriyol@gmail.com
Introduction
Cancer is a scourge affecting mankind from the time
immemorial. Since the last century, treatment of cancer
remains an enigma till today (Srivastava et al. 2002). Since
ancient time, the role of natural products has been recognized
as a source for remedies (Fransworth et al. 1985; Cragg et
al. 1997). Despite major scientifc and technological progress
in combinatorial chemistry of drugs derived from natural
product, it can still make an enormous contribution to drug
discovery today. This is relatively new branch of medicine
that has expanded rapidly over the last twenty years. Caffeine
is well known for its neurostimulator activity. But caffeine
is known to exert protective function against genotoxic/
carcinogenic effects of environmental chemicals in vitro and
in vivo assay systems (Abraham, 1989 and 1991; Aeschacher
and J accud, 1990; IARC, 1991; Nehlig and Debry, 1994;
Stavric, 1992). These fndings indicate that caffeine is a
chemo- preventive drug against environmental mutagens and
carcinogens. The present study was undertaken to evaluate
the modulation or regulation of caffeine (Coffeea arabica)
on cyclophosphamide induced cytogenotoxicity in Swiss
mice. The results were evaluated by analyzing the end points
of number of chromosomal aberrations and mitotic index
(Choudhury et al. 1995).
Vol -1, Issue - 1, Jan - Apr 2011 54
Effect of caffeine on cyclophosphamide induced genotoxicity, Prakash G.
Chemicals and Reagents
Cyclophosphamide sulphate injection was procured from
Cipla India, Pune, whereas Caffeine (CAF) was purchased
from Sigma Co., St. Louis, USA. Colchicine, sodium
chloride, sodium citrate, methanol, glacial acetic acid, ethyl
alcohol, Giemsa stain, Sorensens buffer, sodium dihydrogen
phosphate, disodium hydrogen phosphate were purchased
from HiMedia Chemicals, Mumbai, India.
Animals
Animal experiments were carried out taking appropriate
measures to minimize the pain and discomfort in accordance
with the guidelines of CPCSEA. Experimental procedures
were carried out after obtaining permission from the
Institutional Animal Ethics Committee. Swiss mice (6 weeks
old) were procured from the Animal Facility of SCS College
of Pharmacy, HarapanHalli, Karnataka and were acclimatized
to 25 2
o
C and 12 h-light / dark cycle. The animals were fed
with balanced diet (Sai Feeds, Bangalore, India) and water
was provided ad libitum.
Experimental Protocol
Mice weighing 20- 22 g were selected and divided into
fve groups with 6 (3 males and 3 females) animals in each
group. Among fve groups, group I served as control and
administered orally with 0.5 ml of 0.9% sterile saline; group
II as positive control and administered cyclophosphamide
(40 mg/kg body weight); group III, group IV and group V
were treated with of caffeine at the dose rate of 25, 50 and
100 mg/kg body weight (bw) respectively. After one h of oral
administration, the caffeinated animals were treated with 1.5
mg/kg bw of cyclophosphamide and maintained for a period
of 24 h. Later, all the groups except control were administered
intraperitoneally with 0.02% of colchicine at the rate of l0
mg/kg bw. One h after the treatment, 3 female and 3 male
mice from all groups were sacrifced for the preparation of
slides for chromosomal aberration (CA) and mitotic index
(MI) study from bone marrow. Further, 3 female and 3 male
animals from each group were sacrifced for micronucleus test
(MNT) and polychromatic erythrocytes (PCEs) after 30 h.
Mitotic metaphase chromosome aberration study
For mitotic metaphase chromosomal aberration study, slides
were prepared from bone marrow cells as per the method
described by Choudhury et al. (1995). Briefy, at 24 h post-
treatment, six mice (3 females and 3 males) from each of the 5
groups were injected intraperitoneally with 0.02% colchicine
at the dose rate of 1 ml/100 g bw. After one h, the mice were
sacrifced by cervical dislocation, the femur and humerus
bones were dissected and the adhered muscles were cleaned.
Bone marrow cells from each of the femur and humerus were
extracted separately for each of the animal in 0.9% sodium
citrate pre-incubated at 37
o
C, aspirated thoroughly by Pasteur
pipette to prepare a free cell suspension and incubated for
20 min at 37
o
C. The cell suspension was again centrifuged
at 2000 rpm for 5 min, supernatant was discarded, freshly
prepared fxative (methanol and glacial acetic acid 3:1 v/v)
was added and aspirated thoroughly. The cell suspension
was centrifuged at 2000 rpm for 5 min, the supernatant was
discarded, fresh fxative added, and the cell suspension was
aspirated and stored overnight in the refrigerator. Next day,
the old fxative was replaced by a desired volume of fresh
fxative following centrifugation at 2000 rpm for 5 min. The
fxed cell suspension was dropped on clean, grease free pre-
chilled slides in 50% ethyl alcohol and immediately warmed
to complete the burning of alcohol. The slides were left
overnight in dust free chambers for complete natural drying
and then stained with 3% Giemsa stock diluted in Sorensens
buffer (pH 6.8) for 1 h and the stained slides were washed
thoroughly under running tap water, left for natural drying
before observing under binocular microscope. At least 150
well-spread metaphases from each animal were scanned
and normal and aberrant metaphases with different types
of chromosomal aberrations like chromatid, chromosome
gaps, breaks, fragments, minutes, exchanges, pulverization
etc. were recorded. The percentage of aberrant metaphases
(including metaphases with only chromatid gaps and/ or
chromosome gaps) and the aberrations (excluding gaps)
per hundred metaphases were calculated and tabulated.
The representatives from normal metaphases and aberrant
metaphases with various types of chromosomal aberrations
were photomicrographed.
Mitotic index study
Mitotic index study was undertaken from the same slides
prepared for chromosomal aberration study. At least 1000
cells from each animal were taken into consideration for the
calculation of the percentage of dividing cells.
Micronucleus test
Six mice (3 females and 3 males) from each group were utilized
for MNT from bone marrow. A simple technique of Choudhury
et al. (2000) which is based on the basic principles of Schmid
(1982) was used for this test. Briefy, at 30 h of post-treatment,
the mice were sacrifced by cervical dislocation and the bone
marrow from femur and humerus was extracted in 1% sodium
citrate at 5- 7
o
C and aspirated thoroughly by Pasteur pipette
for the preparation of a cell suspension. The cell suspension
was incubated for 5 min, at 5
o
C and then centrifuged for 5
min, at 1200 rpm. The supernatant was discarded and a desired
volume of 1% sodium citrate at 5
o
C was added and aspirated
gently by a Pasteur pipette to prepare a cell suspension. Small
drops of the cell suspension was taken on clean, grease free,
dry slides and smeared gently by a glass rod. Smeared slides
were kept overnight in a humid chamber at 37
o
C, dipped in
methanol for 5 min, and allowed for natural drying. The slides
were then stained with 3% Giemsa stock diluted in Sorensens
buffer (pH 6.8) for 12 to 15 min, washed in tap water and left
for natural drying before observation. At least 2000 tinge-blue
colored polychromatic erythrocytes (PCEs) were scanned
from each animal (the reddish brick coloured normochromatic
erythrocytes (NCEs) were not taken into consideration). The
Vol -1, Issue - 1, Jan - Apr 2011 55
PCE with micro nucleus (MN) were recorded and the total
MN per thousand PCE was calculated. Representative PCEs
with or without MN were photomicrographed.
Coding and reading of slides
The slides prepared at different end points of various groups
of mice were coded immediately with fve digital random
code numbers obtained from statistics books. After the
completion of observation, slides were decoded and the data
were recorded against the respective groups of mice. The data
generated at different end points for different groups of mice
were pooled group-wise and their averages with standard
deviation were calculated.
The differences between the groups were statistically
evaluated and the signifcance of the differences at different
levels were recorded following the tables of Kastenbaum and
Bowman (1970). In any case, a difference was considered as
statistically signifcant when P 0.05 and P 0.01.
Results
The possible protective effect, if any, of caffeine on
cyclophosphamide induced cytogenotoxicity was investigated.
It was found that the two higher doses of caffeine (50 mg and
100 mg/kg) signifcantly reduced the average percentage
of aberrant metaphases (P <0.01 and P<0.05, respectively)
Table 1: Chromosomal aberrations in bone marrow cells of mice induced by caffeine
and cyclophosphamide at 24 h post-treatment
Chemicals
Number of
metaphases
examined
Number of
aberrant
metaphases
Average
percentage
of aberrant
metaphases
SD
Types and number of chromosomal
aberration Total
number of
aberrations
(excluding
gaps)
Average
percentage
of aberrations
(Excluding
gaps) per 100
metaphases
SD)
Chromatic
Type
Chromosome
type
Frag-
ment/
Minute
Gap Break Gap Break
Normal/Control
1061 30 2.790.57 25 1 4/- - 4 5 0.480.24
Cyclophosphamide/
Control
900 159 17.662.21 **
a
111 75 - - 21/9 114 12.601.16**
b
Caffeine (25 mg) /
Cyclophosphamide
900 137 15.212.12
#

b
95 62 2 4 26/11 118 13.102.51
Caffeine (50mg) /
Cyclophosphamide
900 103 11.441.85**
b
67 46 7 2 19/7 83 9.221.65**
b
Caffeine (100mg) /
Cyclophosphamide
900 124 13.77 2.88 *
b
62 42 3 5 19/15 101 11.221.76
Pooled data of 6 mice (3 females and 3 males), a Signifcant compared to vehicle control
SD = standard deviation b Signifcant compared to CP 40 mg/kg, * P <0 05 ** P <0.01 [two-tail t-test]
Table 2 : Protective effect of caffeine on mitotic index in bone marrow cells of mice
Chemical
Number of cells
examined
Cells in dividing
condition
% of dividing cells MeanSD
Normal/Control 7030 184 2.62 1171.7 2.34
Cyclophosphamide/Control 6594 190 2.88 1099.04.56
Cyclophosphamide/25 mg Caffeine 6237 203 3.25 1039.58.94 *
Cyclophosphamide/50 mg Caffeine 6186 173 2.80 1031.0 8.88**
Cyclophosphamide/100mg Caffeine 6323 167 2.64 1053.8 14.18
Cyclophosphamide 40 mg/kg, Signifcant compared to CP 40 mg/kg, * P < 0.05, ** P< 0.01
Table 3. Induction of micronucleus in polychromatic erythrocytes of mice at 30 h post-treatment with
caffeine and cyclophosphamide
Chemical
Total number of
PCE examined
Number of PCE
with MN
Total number
of MN
Average MN per
1000 PCE S.D
Normal/Control 12593 34 35 2.821.16
Cyclophosphamide/Control 12150 418 458 37.645.60 **
a
Caffeine (25 mg) /Cyclophosphamide 12480 429 446 35.721.57
Caffeine (50 mg) /Cyclophosphamide 12368 387 414 33.442.68
Caffeine (100 mg) /Cyclophosphamide 12555 336 362 28.79 2.57 **
b
Pooled data of 6 mice (3 females and 3 males)
a
Signifcant compared to vehicle control, Micronucleus = MN
SD =standard deviation
b
Signifcant compared to CP, ** P < 0.01 Polychromatic Erythrocytes =PCE
Vol -1, Issue - 1, Jan - Apr 2011 56
induced by cyclophosphamide. The average CAs and excluding
gaps induced by cyclophosphamide was also decreased in
the groups of mice treated with caffeine at 50 and 100 mg/
kg bw respectively and the assumed decrease was statistically
signifcant (P<0.01) in the mice pre-treated with caffeine 50
mg/kg bw alone. Markedly, the reduction in percentage of
aberrant metaphases and CAs were more for the intermediate
dose of caffeine (Table 1). In MI study, the average percentage
of dividing cells induced by cyclophosphamide was decreased
in mice treated with either caffeine 50 or 100 mg/kg bw. But
the decrease in dividing cells was not statistically signifcant
(Table 2). In MNT, only the highest dose of caffeine (100 mg/
kg) signifcantly reduced the cyclophosphamide induced MN
(P < 0.01), whereas the other two doses of caffeine (25 and
50 mg/kg) could only reduce the cyclophosphamide induced
MN, but not as signifcant as the higher dose (Table 3).
Discussion
Irrespective of nature of the genotoxic agents and their
mechanisms of action in the induction of the cytogenotoxic
effects, caffeine is capable of providing protection to the cells
from such effects, may be due to different genotoxic chemicals
without interacting directly with the genotoxic agents.
Therefore, it can be reasonably proposed that CAF does not
give protection from the genotoxicants, rather it provides
protection from their genotoxic effects, could be from the
aftermath actions of the genotoxicants. Cyclophosphamide is
a topoisomerase-II inhibitor and genotoxic to mouse in vivo.
But the cytogenotoxic effects were decreased in caffeine pre-
treated mice. Thus, CAF might not have interacted directly on
drug, but it might have interacted upon a common material
produced by the action, which was instrumental in causing
cytogenotoxic effects. It is known that treatment with
anticancer drugs like ethyl nitrosourea, methylnitrosourea,
ethylmethanesulfonate and methylmethanesulfonate etc.,
creates a state of oxidative stress in the body (Noda and
Wakasugi, 2001). A fall in the plasma antioxidants occurs
following treatment with certain anticancer drugs (Weijl et al.
1998). The reactive oxygen species (ROS) generated by these
drugs leads to lipid peroxidation and DNA lesions, which are
implicated in the cytogenotoxic action of these drugs (Lown
et al. 1982). Caffeine may also play an important role in
protecting the cells against planar aromatic molecules such as
intercalating agents (Traganos et al. 2008). This ROS triggers
apoptosis via p53 and cytochrome release from mitochondria.
The mechanism of action of most of the anticancer drugs
involves active oxygen. Redox control is also involved in
various tissues related to anticancer drug therapy (Noda
and Wakasugi, 2001). Thus, oxidative stress is related to the
treatment of most of the anticancer drugs, if not to all. On
the other hand, CAF readily accepts electrons and acts as a
free radical scavenger. It is a potent scavenger of hydroxyl
radicals, which is comparable with that of other effcient
hydroxyl radical scavengers (Shi et al. 1991; Devasagayam
and Kesavan, 1996). It is a1so an effective inhibitor of lipid
peroxidation (Lee, 2000). The possible mechanism involved
in the antioxidant effects of CAF is the quenching of ROS
(Devasagayam et al. 1996). Contrarily, CAF has a DNA-
repair inhibiting effect and it enhances the cytocidal effects of
anticancer drugs (Tsuchiya et al. 2000). It primarily inhibits
replicative DNA synthesis, potentiates the cytotoxicity by
intervening in DNA repair, overrides G2/M block by leaving
less time for post-replication repair, or makes S-phase
delay (Deplanque et al. 2000). Fernandez et al. (2003) also
reported that the CAF is of critical importance because high
doses of CAF induces apoptosis and low concentrations
can act as antioxidants. There are reports supporting our
fndings that, post-treatment of different concentrations of
caffeine signifcantly reduces the frequency of chromosomal
aberrations induced by gamma-rays in mice (Farooqi and
Kesavan, 1992). However, some investigators have reported
that the caffeine alone induces a concentration and time-
dependent inhibition of DNA synthesis and inhibit the entry
of human fbroblasts into S-phase by 7080% regardless of
the presence or absence of wild type ATM or p53 as well as
it also enhances the inhibition of cell proliferation induced
by ultraviolet radiation in XP variant fbroblasts (Kaufmann
et al. 2003).
The CAF was found weakly clastogenic to mouse bone
marrow cells. Caffeine possibly may have played an
important role in protecting the cyclophosphamide induced
cytogenotoxicity in the somatic and male germ line cells of
mice. Transmission of such effects in the male germ line was
mostly signifcant for the highest dose of caffeine (100 mg/kg
bw) except in the mitotic metaphase chromosomal aberration
study, where caffeine 50 mg/kg bw reduced the toxicity of
cyclophosphamide more effciently. However, pre-treatment
of each of the three doses of CAF has reduced the frequency
of MTX 10 mg/kg-induced aberrant metaphases, CAs, MN
and also the percentage of dividing cells, but signifcant
reduction was only seen in the two higher doses of CAF. Thus,
the higher doses of CAF were shown to protect mouse bone
marrow cells from the cytogenotoxicity of MTX (Ramesh and
Anil, 2004).
From the results of present study, it can be inferred that CAF
met the challenges of the oxidative stress created following the
treatment with the anti-neoplastic drugs in the cells of mice and
thus, protected them to some extent from the cytogenotoxic
effects caused through the generation of free radicals. As the
higher doses of CAF induce apoptosis, thereby eliminating
most of such affected cells and leaving less number of affected
cells for observation. The higherdose of CAF tested here (50
and 100 mg/kg) was found to be more protective may be
because of apoptosis induction and resulting in the incidence
of less number of affected cells than that of the lower dose
of CAF. The potentiation of genotoxicity by the higher dose
of CAF might have also caused the grossly affected cells to
undertake apoptotic pathway with the concomitant decrease in
the effects observed from the surviving cells. Therefore, in the
present MI study the percentage of dividing cells were reduced
more in the mice pre-treated with CAF at the dose rate of 100
mg/kg than with lower doses of CAF. Thus, CAF modulation
is expected to play an important role in the development of
cancer chemotherapy. However, in-depth study is required to
be undertaken to fully understand its potentials.
Effect of caffeine on cyclophosphamide induced genotoxicity, Prakash G.
Vol -1, Issue - 1, Jan - Apr 2011 57
Acknowledgements
We thank Prof. M Krishnamurthi, Mangalore University,
Mangalore, for permitting us to learn the techniques in
his laboratory. We are also thankful to Shilet Mathew for
supervising the animal experiments.
References:
Abrahm SK (1989). Inhibition of in vivo genotoxcity by
coffee. Food Chem. Toxicol. 27:787- 792.
Abrahm SK (1991). Inhibitory effects of coffee in the
genotoxcity of carcinogens in mice. Mutat. Res.
262:109- 114.
Aeschbcher HU, Jaccud E (1990). Inhibition by coffee of
nitrosaurea-mediated DNA damage in mice. Food
Chem. Toxicol. 28: 633- 637.
Choudhury RC, Das Misra, Jagadale MB (2000).
Cytogenetic toxicity of vincristine. J Environ. Pathol.
Toxicol. Oncol. 19(4): 347- 355.
Choudhury RC, Hasan A, Mahanty V (1995). Effects of
mercuric chloride on mitotic, meiotic chromosome and
sperm in mice. Biologisches zentral Blatt. 114 : 369-
377.
Cragg GM, Newman DJ, Snader KM (1997). Natural
products in drug discovery and development. J. Nat.
Prod. 12:6052- 6066.
Deplanque G, Geraline J , Man-Bechrel MC, Cazenave J P,
Berger JP, Klein-Soyer C (2000). Caffeine and the
G2/M block override; a concept resulting a misleading
cell kinetic delay, independent of functional P
53
. Int. J.
Cancer. 94: 363- 369.
Devasayagam TPA, Kesavan PC (1996). Radioprotective
and antioxidant action of caffeine; mechanistic
consideration. Med. J. Exp. Biol. 34: 291- 297.
Devasayagam TPA, Kamath J P, Mohan H, Kesavan PC
(1996). Caffeine as an antioxidant; inhibition of lipid
peroxidation induced by reactive oxygen species.
Biochim. Biophys. Acta. 1282:63-70.
Farnsworth NR, Akerele O, Bingel AS, Soejarto DD, Guo Z
(1985). Medicinal plants in therapy. Bull. World Health
Organ. 63:1- 965.
Farooqi Z, Kesavan PC (1992). Radioprotection by
caffeine pre- and post-treatment in the bone marrow
chromosomes of mice given whole-body gamma-
irradiation. Mutat. Res. 269 (2): 225- 230.
Fernandez MJ, Lopez A, Santa-maria A (2003). Apoptosis
induced by different doses of caffeine on Chinese
hamster ovary cells. J. AppI. Toxicol. 23 (4): 221- 224.
IARC (1991). Monographs on the evaluation of carcinogenic
risks to humans. Coffee, Tea, Mate, Methylxanthines
and Methylglyoxal. International Agency for research on
cancer, Lyon. Vol 51:513
Kastenbaum MA, Bowman KO (1970). Tables for
determining the statistical signifcance of mutation
frequencies. Mutat. Res. 9: 527- 549.
Lee C (2000). Antioxidant ability of caffeine and its
metabolites based on the study of oxygen radical
absorbing capacity and inhibition of LDL peroxidation.
Clinica Chemica Acta. 295:141- 154.
Lown JW, Joshua AV, Chen HH (1982). Reactive oxygen
species leading to lipid peroxidation and DNA lesions
implicated in the cytotoxic action of certain antitumor
antibiotics, In; McBrein, S. C. H., Slater. T. F., (Eds),
Free radicals, lipid peroxidation and cancer academic
press, London New York. pp 305- 328.
Nehlig A, Debry G (1994). Potential genotoxic, mutagenic
and antimutagenic effects of coffee: A review. Mutat.
Res.317: 145- 162.
Noda N, Wakasugi H (2001). Cancer and oxidative stress.
JMAJ 44(12): 535-539.
Ramesh CC, Anil KP (2004). Modulatory effects of caffeine
on methotrexate-induced cytogenotoxicity in mouse
bone marrow. Environ. Toxicol. Pharmacol. 15: 79- 85.
Schmid W (1982). The micronucleus test; An in vivo bone
marrow method, In: Hsu,T.C.(Ed), cytogenetic assay of
environmental mutagens, Oxford and IBH publishing
Co., New Delhi. pp 221- 235.
Shi X, Dalal NS, Jam AC (1991). Antioxidant behavior of
caffeine: Effcient scavenging of hydroxyl radicals.
Food Chem. Toxicol. 29:1- 6.
Srivastava PS (2002). Biotechnological approaches to
potential anti-cancerous herbal drugs of the future.
Vol .V, Special edition on cancer, Ukaaz Publications,
Hyderabad.
Stavric B (1992). An update on research with coffee/caffeine
(1989-1990). Food Chem. Toxicol. 30: 533-555.
Traganos F, Kaminska-Eddy B, Darzynkiewicz Z (2008).
Caffeine reverses the cytotoxic and cell kinetic effects of
Novantrone (mitoxantrone). Cell Prolif. 24: 305- 319.
Tsuchiya H, Yamamoto N, Asada N (2000). Caffeine-
potentiated radio chemotherapy and function saving
surgery for high grade soft tissue sarcoma. Anticancer
Res. 20 (3B):2137-2143.
Weijl NI, Hopman GD, Wipkink-Bakker A, Berger (1998).
Cisplatin combination of chemotherapy induces a fall
in plasma antioxidants of cancer patients. Ann. Oncol.
9:1331-1337.
Vol -1, Issue - 1, Jan - Apr 2011 58
Ingle A.D.received his MVSc., in Veterinary Pathology in 1992 from the Post Graduate Institute,
Punjabrao Krishi Vidyapeeth, Akola, MS. Currently he is working in ACTREC, Tata Memorial Centre,
Navi Mumbai as a Scientifc Ofcer F and Ofcer-in-Charge, Laboratory Animal Facility and
Histopathology Laboratory. Dr. Ingle has wide experience in quality control of laboratory rodents
including health (conventional, ELISA and molecular methods) and genetic monitoring (skin
grafting, biochemical markers, SNP and microsatellite methods). Dr. Ingle is a resource person for
quality control measure of laboratory animals in India. Dr. Ingle has 35 publications to his credit
published in peer reviewed national and international journals. More information on Dr. Ingles
activities can be viewed on ACTREC website at http://www.actrec.gov.in/animal_main.htm
Arvind D Ingle
Hematological values of mice and rat strains :
Perspectives from ACTREC Laboratory Animal Facility
Abstract
Experimental results of the hematological or biochemical data are best interpreted and compared based on
previously reported baseline data for the strains in question. Maintenance and managemental conditions
of the animals varies from organization to organization. Naturally, the baseline data for hematology and
biochemistry may vary amongst the same species or strains of animals. This study was undertaken to establish
hematological reference values in mice and rat strains maintained at Advanced Centre for Treatment, Research
and Education in Cancer (ACTREC), Navi Mumbai and to compare with that of the values reported by other
workers as in the literature. Hematological values were found to be within the normal range when compared
to the values reported in the literature. Slight deviations in these values have been noticed within the strain.
However, they remain within the reported normal range. Most of the time, the normal range values were
so wide that it necessitates the need for the laboratory-specifc baseline data. The wide range may be the
result of genetic changes, change in feed, housing conditions, technique and equipments used or management
conditions. The hematological values of these strains may provide valuable baseline data to the researchers
for future experiments using these inbred strains.
Key words: laboratory animals, hematology, rats, mice
Dr. Arvind D Ingle, Laboratory Animal Facility, ACTREC, Tata Memorial Centre, Kharghar, Navi Mumbai- 410 210
Phone: 91 22 27405047, Fax: 91 22 27405116, email: aingle@actrec.gov.in.
Hematological values in rodents, Ingle A.D.
Ingle A.D, Shinde A.D and Ahire S.D.
Laboratory Animal Facility, ACTREC, Tata Memorial Centre, Kharghar
Navi Mumbai - 410 210
Introduction
Rodents are the most frequently used animals worldwide in
biomedical research. Use of inbred mice and rats in cancer and
biomedical research has made possible many contributions of
fundamental and productive nature that would not have been
possible otherwise. Although several studies were conducted
using different strains of mice and rats, variations in biological
characteristics were extremely common between inbred
strains of mice and rats (Festing, 1979). Studies on rodents
provide clinical toxicity predictions that may be comparable
to studies in other species (Harrison et al. 1978).
Number of mice and rat strains in use for various studies
differs at different organizations. Information concerning
the origin, behavior, physiology, anatomy, reproduction,
disease incidence, hematology and biochemical values of
almost all the strains has been well documented. The values
reported so far in the literature serve as standard baseline data
in research. However, many a times reported values in the
literature do not exactly match during the experimentation
in different laboratories. Slight variation in hematological
indices may be seen in mice of the same strain from different
laboratories, but signifcant difference may also be seen
between mice of different strains. This indicates that, the
normal values may differ from laboratory to laboratory due to
either genetic change, change in environment or feed (Green,
1966). However, the values obtained in one strain are always
under those specifc conditions and should not be construed
to refect data for that strain in all circumstances. Hence, it
was felt necessary to determine the reference values for
hematological parameters under our housing conditions for
mice and rats strains at ACTREC, Navi Mumbai. The present
study deals with the hematological reference values that
Clinical
Vol -1, Issue - 1, Jan - Apr 2011 59
includes hemoglobin (Hb), red blood cells (RBC) count, white
blood cells (WBC) count, packed cell volume (PCV), mean
corpuscular volume (MCV), mean corpuscular hemoglobin
(MCH), mean corpuscular hemoglobin concentration
(MCHC), and differential leucocytes count (DLC).
The mice and rat strains maintained at ACTREC Animal
Facility and used in the present study are listed in Table 1
and 2. Breeding nuclei of these strains have been procured
in the past either from the Jackson Laboratories, USA,
or Charles River Laboratories, USA and then inbred at
ACTREC. For determination of hematological parameters,
6-8 wks old specifc pathogen free animals of each sex (n=5)
were randomly selected. Five animals were housed in each
cage under normal animal housing conditions of 23 2
o
C
temperature, 55 5 % relative humidity and 12 h darkness
and 12 h illumination. The animals were fed on pelleted
feed prepared in-house at the facility as per the nutritional
requirements(CPCSEA guidelines, March 2008) and were
given UV treated drinking water ad libitum. The animals were
free for antibodies of CAR bacillus, Clostridium piliformae
(CP), Ectromelia virus Hantaan virus, Kilham rat virus,
Lymphocytic Choriomeningitis virus (LCMV), Mycoplasma
pulmonis (MP), Pneumonia virus of mice (PVM), Reo3 virus
and Sendai virus as screened by ELISA based methods using
commercially available kits from XpressBio Life Sciences
Products. The Institutional Animal Ethics Committee of the
ACTREC has approved the use of animals.Two hundred
micro liters of blood was collected directly from orbital
plexus of the mice without anesthesia whereas in rats, under
mild anesthesia by Ketamin injection at the dose rate of 10
mg/kg intramuscularly. Blood smears were prepared and
stained with Leishmans stain for Differential Leucocytes
Count (DLC).
Hematological parameters like RBC and WBC were carried
out using automated counter (Sysmex, model no. KX-21,
Japan). For RBCs, three parameters viz total RBC count, Hb
concentration and PCV percentage were measured whereas
for WBCs, total WBC count and their proportions were
measured by automated counter.
Results
Hematological parameters of mice and rat strains determined
in this study are presented in Table 1 and 2 respectively.
Average Hb values of all the mice and rat strains maintained at
ACTREC were found to be 14.5 and 15.6 gm/dl respectively.
The average RBC values of mice and rat strains were found
to be 8.6 and 8.8 millionos/cu.mm and average PCV values
were found to be 45.8 and 49 % respectively.
Table 1. Hematological data in mice strains
No Strain
Hb
gm/dl
RBC
10
6
/cu
mm
PCV
%
MCV
cu m
MCH
mmgm
MCHC
gm/dl
TLC
/cu mm
Differential Leucocytes
Count (DLC) %
N E L M
1 BALB/c
14.9
0.4
8.9
0.3
46
2.6
52
2.8
17
0.6
33
1.4
6680
964.7
24
8.9
1
0.5
74
8.8
1
0.8
2 C3H
14.5
0.5
7.9
0.7
45
3.0
49
3.4
17
0.5
32
1.3
7200
1500
41
5.3
1
0.9
57
4.8
1
0.6
3 C57BL/6
14.6
0.4
8.8
0.2
47
2.0
53
2.0
17
0.5
31
1.3
7418
2222
18
5.8
1
1.1
80
6.0
1
0.7
4 CD-1
13.2
0.9
7.8
0.5
42
2.7
54
2.3
17
0.6
31
1.0
7767
2790
18
5.2
3
1.7
78
5.4
1
0.7
5
Cr:ORL
SENCAR
14.8
0.3
9.0
0.2
49
2.0
55
2.4
17
0.5
30
1.0
6668
1109
23
8.5
1
1.0
75
8.3
1
0.5
6 DBA/2
14.5
0.4
8.8
0.2
45
2.0
51
1.7
17
0.5
32
1.2
7510
747.5
17
4.0
1
0.7
81
6.7
1
0.5
7 ICRC
14.5
0.7
8.7
0.4
45
2.7
51
2.0
17
0.4
32
1.7
6650
1844
24
5.7
1
0.7
74
5.7
1
0.6
8 S/RV/Cri
14.7
1.5
8.6
0.8
46
4.4
53
1.2
17
0.6
31
0.6
6950
1260
26
12.3
2
0.7
71
1.4
1
0.7
9
S/RV/Cri-
ba
14.6
0.5
8.8
0.3
47
3.5
53
2.6
17
0.4
31
1.4
6150
2063
57
9.7
1
0.7
40
9.3
2
0.8
Mean
14.5
0.5
8.6
0.4
45.8
1.9
52
1.8
17
0
31
0.9
6999
515.2
28
13.2
1
0.7
70
13.3
1
0.3
N- Neutrophils, E- Eosinophils, L- Lymphocytes, M- Monocytes
Vol -1, Issue - 1, Jan - Apr 2011 60
Average MCV values of mice and rat strains were found to be
52 and 53 cubic microns and average MCH values were found
to be 17 and 17 mmgm respectively. It was observed that
average MCHC values in mice and rat strains were 31- 32 gm/
dl respectively. Average TLC values of mice and rat strains
were found to be 6999 and 12096 per cubic mm respectively
The differential leucocyte counts were determined in rats
and mice and presented in Table 1 and 2. Average neutrophil
counts in mice and rat strains were found to be 28 and 16 %,
eosinophils were 1 and 1 %, lymphocytes were 70 and 82 %
and monocytes were found to be 1 and 1 % respectively.
Discussion
Hematological values are reported to be infuenced by variety
of parameter which includes site of sample collection, sex,
strain, age, stress level of animals, method of restraint, and
type of anesthesia (Thrall, 2004). Route of blood withdrawal
as well as analytical method and equipment used may result
in different values for some of the parameters (Lang, 1993).
Most of the hematological investigations include total count
and proportional values of RBC and WBCs. Type of food and
change in managemental practices are reported to infuence
the change in hematological parameters in the same strain
of rodents. For this reason, the present study was designed
to study hematological values of rodent strains housed at
ACTREC under standard housing conditions. Values obtained
in the present study were compared with the reported
literature. Hematological values can also help to detect the
population drift or analytical problems within a laboratory
(Giknis, 2008).
Hemoglobin amongst all strains of mice showed similar
values as reported by Aiso et al. (2005), Alter et al. (1974),
Charles River Laboratories (CRL) Technical Bulletin (1986),
Hariharan (1979), Harrison et al. (1978) and Hejtmancik et
al. (2002). However, Kawabe et al. (1993) reported that 17
% of hemoglobin as normal value in one of their experiments
on B
6
C
3
F1 mice whereas Merchant and Modi (2004) has
reported 8.9-10.6 gm % of Hb as normal values for mice in
their experiments. The CRL Technical bulletin (1986) has
even reported 8.2- 16.2 gm % Hb values in CD1 mice as
baseline values. These reports indicate that the range of Hb
values in case of mice strains may vary from 8.2-17 gm %.
Lower values shown here may be normal for one laboratory
but may be lower for another laboratory where normal values
for the mice strains are 17 gm % and vice versa..
Hemoglobin values in rats were found to be similar to that
reported by various investigators. Hemoglobin values
reported were 16.1 gm% (Aiso et al. 2005); 13.6% ( Alter
et al. 1974); 11-17gm% (Bhardwaj, 1992); 12.3 gm%
(Bhilegaonkar et al. 1995);

14.1-18.2 gm/dl (Charles River
Lab. Tech. Bulletin, 1982; 1984); 13.7- 17.6 gm% (Giknis
et al. 2008), 12.0-17.5 gm% (Hariharan ,1980); 14.0- 16.5
gm% (Hejtmancik et al. 2002); 9.19- 16.82 gm% (Lang,
1993), 13.6- 14.9 gm% (Lovell et al. 1981); 14.8-15.8 gm%
(Saibaba et al. 1995); 14.72 gm/dl (Stana et al. 2009); 14.3-
15.3 gm% (Walter , 1999) and 15.7 gm% (Waynforth, 1980).
On the contrary Jonkar and Till (1995) has reported 9.6 gm/
dl and Weber et al. (2002) has reported 8.2- 14 gm/dl of Hb
as normal values in their experiment using rats. This value
(8.2 mg/dl) is signifcantly low as compared to hemoglobin
values reported in the literature by others. This suggests that
there are differences in baseline values of Hb among different
laboratories and ranges from 8.2- 18.2 gm%.
Total Red Blood Cells (RBC) values in all strains of mice at
ACTREC were found to be varying between 8- 9 10
6
/cu mm,
which are comparable with the value reported by Aiso et al.
(2005); Charles River Lab. Tech. Bulletin (1986); Hariharan
(1979); Hejtmancik et al. (2002); Kawabe et. al. (1993)
and Merchant et al. (2004). Alter et al. (1974) has reported
6.89 10
6
/cu mm as RBC values in B6D2F1 mice which is
signifcantly low when compared to the values reported in
the literature. The review of the data indicates that the normal
RBC values in mice strains may vary from 6.89- 9 10
6
/cu mm.
Table 2. Hematological data in rat strains
No Strain
Hb gm/
dl
RBC
10
6
/cu
mm
PCV%
MCV
cu m
MCH
mmgm
MCHC
gm/dl
T.L.C.
/ml
Differential Leucocytes
Count (DLC) %
N E L M
1 S.D.
15.6
0.7
9.0
0.4
49
2.3
54
1.2
17
0.0
32
0.6
13800
2130
16
3.9
1
1.6
82
3.2
1
0.6
2
Wistar 15.8
0.3
8.0
0.2
51
1.9
55
1.9
17
0.4
31
0.9
12125
2733
18
5.7
1
0.9
79
6.0
2
0.5
3 F344
15.4
0.3
9.3
0.3
46
2.1
49
1.4
16
0.5
33
1.3
10363
1069.0
14
3.3
1
0.5
84
3.7
1
0.5
Mean
15.6
0.2
8.8
0.7
49
2.5
53
3.2
17
0.6
32
1.0
12096
1718.7
16
2.0
1
0
82
2.5
1
0.6
N- Neutrophils, E- Eosinophils, L- Lymphocytes, M- Monocytes.
Vol -1, Issue - 1, Jan - Apr 2011 61
The RBC values in all rat strains at ACTREC were found
to be in the range of 8.0- 9.3 10
6
/cu mm that are similar to
the values reported by Aiso et al. (2005); Bhardwaj (1992);
Giknis (2008); Hejtmancik et al.(2002); Jonkar and Till
(1995) and Waynforth (1980). However, Saibaba et al. (1995)
has reported 6.9 10
6
/cu mm of total RBC values as normal
value in Wistar rats. Lovell et al. (1981) and Walter et al.
(1999) has reported the RBC values ranging from 6.18- 8.47
10
6
/cu mm. Weber et al. (2002) has even reported 6.92- 11.21
10
6
/cu mm as normal values for RBCs in case of Wood rats.
Normal range of RBC values in case of rat strains are seen
varying from 6.1- 11.21 10
6
/cu mm.
Total Leucocyte Counts in all the strains of mice were found to
be in the range 6.2- 7.8 thousands/cu mm which is comparable
to the values reported by Charles River Lab. Tech. Bulletin
(1986); Hariharan (1979); Harrison (1978), Kawabe et al.
(1993) and Merchant and Modi (2004).
Total Leucocyte Counts in CRI rat strains was found to be
in the range of 10363- 13800/cu mm which is comparable
to the values reported by Bhardwaj (1992); Charles River
Laboratories Technical Bulletin (1982) and Hariharan (1980).
However, Alter et al. (1974) and Saibaba et al. (1995) have
reported 5000 and 5360/cu mm for Wistar and SD rats,
respectively as normal values. Even Lovell et al. (1981) has
also reported the TLC values ranging from 3.7- 7.6 thousands/
cu mm. This indicates that normal TLC values may ranges
from 3.7-13.8 thousand/ cu mm in rats.
Packed Cell Volume values in mice strains were found to
be varying between 42- 47 %. Hariharan (1979); Harrison
et al. (1978); Hejtmancik et al. (2002) as well as Kawabe
et. al. (1993) has reported similar values as normal in their
experiment. Aiso et al. (2005) has reported 50.2 % as PCV
values in BDF1 mice. The PCV values in mice ranges from
42-50% as reported in the literature.
Packed Cell Volume values in rats strains maintained in
ACTREC were found to be varying in the range of 46- 51
% which are comparable to the values reported by Aiso et
al. (2005); Giknis (2008); Hariharan (1980); Hejtmancik
et al. (2002); Lang (1993); NLAC News Letter (1992) and
Waynforth (1980). Weber et al. (2002) has reported PCV
values ranging from 26.9- 48.9 % whereas Alter et al. (1974)
has reported PCV values as 38.4 %. The PCV values in rats
varying in the range of 26.9- 51 % as reported in the literature.
Average Mean Corpuscular Volume (MCV) values in all mice
and rat strains were found to be varying in the range of 49- 55
cubic micron which matches with the values reported by Aiso
et al. (2005); Charles River Lab. Tech. Bulletin (1982); Giknis
(2008); Hariharan (1979); Hejtmancik et al. (2002); Jonkar
and Till (1995); Lang (1993); Merchant and Modi (2004) and
Walter (1999). Lovell et al. (1981) has reported MCV values
towards higher side in the range of 58- 67 cubic micron.
Mean Corpuscular Hemoglobin (MCH) values in all mice and
rat strains were found to be varying in the range of 16- 17
mmg which are comparable to the values reported by Aiso
et al. (2005); Alter et al. (1974); Charles River Lab. Tech.
Bulletin (1982); Giknis (2008) and Weber et al. (2002). Walter
(1999) and Lovell et al. (1981) has reported the MCH values
in the range of 18.0- 22.0 mmg which are towards higher side
as compared to all other references reported above. On the
contrary, Merchant and Modi (2004) have reported only 11.3-
12.5 mmg of MCH values in mice. The review of this index
shows that the MCH values in mice and rats may vary from
11.3- 22.0 mmg.
Mean Corpuscular Hemoglobin Concentration (MCHC)
values in mice strains were found to be varying in the range
of 30- 33 gm/dl which are comparable to the values reported
by the Charles River Lab. Tech. Bulletin (1986) where normal
range for their mice are shown varying from 31 to 39 gm/dl.
Hejtmancik et al. (2002) has reported 32- 33 gm/dl MCHC
values in case of B6C3F1 mice. These results and literature
survey indicates that the control values of MCHC in case of
mice may vary from 30- 39 gm/dl.
Mean Corpuscular Hemoglobin Concentration (MCHC)
values in rat strains were found to be varying in the range
of 31- 33 gm/dl which are comparable to the values reported
by the Charles River Laboratories Technical Bulletin (1984);
Lovell et al. (1981); Walter (1999) and Weber et al. (2002).
Alter et al. (1974) has reported 35 gm/dl of MCHC values for
their rats. Hejtmancik et al. ((2002) has reported 33.5- 35.2
gm/dl of MCHC values in case of F344 rats whereas Giknis
(2008) has reported MCHC values of CRL:WI(Han) rats in
the range of 32.9- 37.5 gm/dl. From the above literature, it
is observed that MCHC values are in the range of 31- 37.5
gm/dl.
Differential Leukocytes Counts determined in all mice and
rat strains were comparable to earlier reports by Bhardwaj
(1992), Charles River Laboratories Technical Bulletin (1982);
Charles River Laboratories Technical bulletin (1986); Giknis
(2008); Hariharan (1979)

and Lovell et al. (1981) except for
the strin S/RV/Cri-ba which shows reversal in values in case
of lymphocytes and neutrophils. The S/RV/Cri-ba is a hairless
mutant strain of mice originated from the Swiss strain of
mice at the Cancer Research Institute, Mumbai (Randelia and
Sanghvi, 1961). The reversal in lymphocytes and neutrophils
values of DLC in this strain need to be further examined and
characterized.
It is evident that hematological values of mice and rat strains in
ACTREC are comparable to the values reported in the literature.
Male and female animals did not show signifcant differences
in the hematological parameter studied. However, in some
cases the range of reported normal values in the literature
from laboratory to laboratory are so wide and this could be
due to some of the factors like either feed, managemental
practices, route of blood withdraw or equipments used for
testing. Only slight deviation in these values has been noticed
amongst the strains maintained in ACTREC except the strain
S/RV/Cri-ba where the neutrophils and lymphocyte values
are seen reversed. The varying levels of normal values of
the hematological parameters from laboratory to laboratory
highlight the importance of determination of normal base
line hematological and biochemical data for their own animal
strains.
Vol -1, Issue - 1, Jan - Apr 2011 62
References
Aiso S, Arito H, Nishizawa T, Nagano K, Yamamoto S,
Matsushima T (2005). Thirteen-week inhalation toxicity
of p-Dichlorobenzene in mice and rat. J . Occup. Health.
47: 249- 260.
Alter B, Kan Y, Nathan D (1974). Toxic effect of high-dose
Cyanate administration in rats. Blood. 43(1): 69- 77.
Bhardwaj K (1992). National Laboratory Animal Center
News Letter Rat, No. 2, Central Drug Research
Institute, Lucknow: 1-10.
Bhilegaonkar D, Deshpande B, Degloorkar N, Moregaonkar
S, Vadamudi V, Rajurkar S (1995). Hematobiochemical
Studies in sub-acute Benfuracarb Toxicity in Rats. Ind.
J. Vet. Pathol. 19(1): 15-18.
Charles River Laboratories Technical Bulletin for Wistar
Rats (1982). Baseline hematology and clinical chemistry
values for Wistar rats as a function of sex and age vol. 1,
No. 2, Wilmington, Massachusetts.
Charles River Laboratories Technical Bulletin for F-344 Rats
(1984). Baseline hematology and clinical chemistry
values for F-344 rats as a function of sex and age, vol.
3, No. 1, Wilmington, Massachusetts, USA.
Charles River Laboratories Technical Bulletin for CD-1
mice. Summer (1986). Baseline hematology and clinical
chemistry values for CD-1 mice as a function of sex and
age Wilmington, Massachusetts, USA.
CPCSEA Guidelines for Laboratory Animal Facility (2008).
II edn., Chennai. pp 12- 13.
Festing MFW (1979). In: Inbred Strains in Biomedical
Research, The Macmillan Press Ltd. London. pp 1-20.
Giknis MLA (2008). Clinical Laboratory Parameters for
Crl:WI(Han) rats. Charles River Laboratories, USA.
Green EL (1966). In: Biology of Laboratory Animals, 2nd
edn., The Blakeston Dit, New York. pp 352-353.
Hariharan S (1979). The Mouse- Care, breeding and
management. Laboratory Animals Information
Service Center News. National Institute of Nutrition,
Hyderabad No. 2: 14.
Hariharan S (1980). The Laboratory Rat, Laboratory
Animals Information Service Center News. National
Institute of Nutrition, Hyderabad, No. 3.
Harrison S, Burdeshaw J , Crosby R, Cusic A, Denine E
(1978). Hematology and Clinical Chemistry Reference
Values for C57BL/6 x DBA/2 F1 Mice. Cancer Res. 38:
2636-2639.
Hejtmancik M, Ryan M, Toft J, Persing R, Kurtz P, Chhabra
R (2002). Hematological Effects in F344 Rats and
B6C3F1 Mice during the 13-Week Gavage Toxicity
Study of Methylene Blue Trihydrate. Toxicol. Sci. 65:
126134.
Jonkar D, Till H (1995). Human Diets Cooked by
Microwave or Conventionally: Comparative sub-
chronic (13-wk) Toxicity Study in Rats. Fd. Chem.
Toxic. vol. 33, No. 4: 245-256.
Kawabe M, Tamano S, Shibata M, Hirose M, Fukushima
S, Ito N (1993). Subchronic toxicity study of methyl
hesperidine in mice. Toxicol Letters. 69: 37-44.
Lang P (1993). Hematology parameters for the Crl:CDBR
rats, Charles River Laboratories Technical Bulletin
for Crl: CDBR Rats. vol. 3, No. 1, Wilmington,
Massachusetts, USA.
Lovell D, Archer R, Riley J, Morgan R (1981). Variation in
haematological parameters among inbred strains of rat.
Lab. Anim. 15: 243-249.
Merchant M, Modi D (2004). Acute and chronic effects of
aspirin on hematological parameters and hepatic ferritin
expression in mice. Indian J Pharmacol. Vol 36, issue 4:
226-230.
Randelia H, Sanghvi L (1961). Bare, a new hairless mutant
in the mouse-genetics and histology. Genet. Res. 2:
283- 289.
Saibaba P, Urs J, Lokesh B, Suresh T (1995). Hematological
data of Indian desert Gerbil. Lab. Anim. India. vol. 4,
No. 2: 83-91.
Stana L, Trif A, Petrovici S, Stana L, Petcu M (2009).
Comparative Study Regarding the Potassium
Dichromate Infuence on the Resistance of Erythrocyte
Membrane in Female Rats in Accordance with the Dose
and Exposing Life Period. Lucrari Stiinifce Medicina
Veterinara. Vol. XLII (2): 286- 90.
Thrall MA (2004) Mammalian Hematologic: Laboratory
Animals and Miscellaneous Species. In: Veterinary
Hematology and Clinical Chemistry, Ed. DB Troy,
Lippincott Williams and Wilkins, Philadelphia. pp 211-
224.
Walter G (1999). Effect of Carbon Dioxide inhalation on
Hematology, Coagulation and Serum Clinical Chemistry
in Rats. Toxicol Pathol. Vol 27, no. 2: 217- 225.
Waynforth H (1980). Experimental and Surgical Techniques
in Rats, Academic Press, London: 240.
Weber D, Danielson K, Wright S, Foley J (2002).
Hematology and Serum Biochemistry Values of Dusky-
Footed Wood Rat (Neotoma Fuscipes). J. Wildlife
Diseases. 38 (3): 576 582.
Vol -1, Issue - 1, Jan - Apr 2011 63
Harikrishnan V.S. received his BVSc & AH from College of Veterinary and
Animal Sciences, Mannuthy, Kerala in 2003 and currently pursuing Master
of Laboratory Animal Science (FELASA-D accredited) from the University of
Copenhagen, Denmark partially supported by Laboratory Animals Limited,
London, UK and LASA, UK. Presently working as Scientist-B in the Division
of Laboratory Animal Science, Biomedical technology Wing, SCTIMST,
Thiruvananthapuram, Kerala. He is recipient of J apanese Laboratory Animal
Science Award in 2009.
Harikrishnan V.S.
Eradication of Pinworms (Syphacia spp.)
in a Laboratory Rodent Colony
Harikrishnan V.S, Ranaraj V.R, Sreeja K.R and Fernandez A.C.
Division of Laboratory Animal Science
Sree Chitra Tirunal Institute for Medical Sciences and Technology,
Biomedical Technology Wing, Trivandrum, Kerala, India
Dr. V.S. Harikrishnan, Scientist-B, Division of Laboratory Animal Science
Sree Chitra Tirunal Institute for Medical Sciences and Technology, Biomedical Technology Wing
Satelmond Palace, Poojappura PIN- 695012, Trivandrum, Kerala, India
Phone: +91 9447428597, Fax: 04712341814, email: harikrishnan@sctimst.ac.in
Abstract
Laboratory rodent colonies are commonly infested with pinworms namely Syphacia obvelata and Syphacia
muris, often results in clinical signs such as diarrhea and rectal prolapse in heavy infestation. The pinworm
infestation in rodents affects the experimental observations and hence it is important to eradicate them from the
colony. Division of Laboratory Animals at Sree Chitra Tirunal Institute for Medical Sciences and Technology
(SCTIMST) maintains rats and mice colonies. Pinworm ova were determined by perianal impression with
cellophane tape testing (CTT) under low power of a compound microscope. The mice and rats were divided
into 2 groups with 10 males and 10 females in each group. Group I was treated with piperazine (10.8 mg/ml of
drinking water) for a period of 28 days and group II was treated with piperazine (10.8 mg/ml) for a period of
14 days followed by ivermectin (0.01mg/ml) for 14 days. Cages and accessory equipments were disinfected
with activated gluteraldehyde, 2.45% w/v. All supplies including bedding and accessories were autoclaved
at 121
o
C temperature, 15 lb pressure for 20 minutes. The present study revealed that the parasites were
eradicated from those groups treated with combination drug (piperazine and ivermectin) therapy while the
group treated with piperazine alone could not demonstrate a complete eradication. The two drug (piperazine
and ivermectin) regime over a 4-week period, associated with appropriate sanitation programme was found to
be effective in eradication of pin worms in laboratory rodents.
key words : Syphacia muris, rodents, piperazine, ivermectin
Introduction
Candidate materials used for the fabrication of biomedical
devices such as heart valves, coronary stents, vascular
grafts, orthopaedic devices, cardiotomy reservoirs,
hydrocephalus shunts, blood pumps, oxygenators and blood
bags undergo a battery of toxicological tests like acute
systemic toxicity, hypersensitivity, hemocompatability,
pyrogen testing and osteocompatability in smaller laboratory
animal models. Laboratory rodents are an integral part of
biomedical toxicological studies. Conventional laboratory
rodent colonies are commonly infested with pinworms
namely Syphacia obvelata and Syphacia muris. Most of
the infestations are asymptomatic but complications like
decreased weight, behavioral changes and altered immune
response were often observed (Owen 1992; Baker 1998;
Huerkamp et al. 2000). Clinical signs associated with heavy
infestation are diarrhea, rectal prolapse, intussusceptions, and
fecal impaction (Harkness and Wagner, 1989; Lubcke, 1992;
Percy and Barthold 2001). The pinworms in rodents affect
the experimental observations and hence it is important to
eradicate them from the colony.
Vol -1, Issue - 1, Jan - Apr 2011 64
These parasites are extremely diffcult to eradicate, especially
from laboratory rodent colonies. Caesarian rederivation or
embryo transfer can be used to obtain parasite-free offspring
in order to provide breeders for a new colony. A number of
anthelminthics, including piperazine, ivermectin, trichlorfon,
fenbendazole, dichlorvos, thiabendazole and mebendazole,
have been used (Hoag 1961; Simmons et a1. 1965; Wagner
1970; MacArthur and Wood 1978; Baskerville et a1. 1988;
Flynn et a1. 1989; Coghlan et a1. 1993 ; LeBlanc et a1.
1993). The number and frequency of these reports indicate
a continuous search for a better therapy. In fact, only a
few treatment regimens are reported to be successful for
eradicating pinworms from large rodent breeding colonies.
This report describes an effective treatment protocol
using piperazine and ivermectin combination for pinworm
infestation in a medium sized rodent colony.
The present study was conducted in a medium sized
laboratory rodent colony, bred and maintained at the Division
of Laboratory Animals (DLAS), Biomedical Technology
Wing, Sree Chitra Tirunal Institute for Medical Science and
Technology, Thiruvananthapuram, Kerala, India. The animal
house is CPCSEA (Committee for the Purpose of Control
and Supervision of Experiments in Animals) registered and
the experimental protocols were approved and reviewed
by Institutional Animal Ethics Committee. The rat colony
consisted of two strains viz., Sctb: WI and Sctb: SD. Mouse
colony consisted of Sctb: Swiss and BALB/c.
The rodents were examined for pinworm ova by perianal
impression with cellophane tape as described by Dix et
al. (2004) under low power (X10) compound microscope
before the treatment. The sample size taken was as per the
recommendations of FELASA (Kraft et al. 1994; Nicklas
et al. 2002). Ten representative individuals from each strain
were sampled. The age group split up was 4 retired breeders
(>6 months), 4 young adults (10-14 weeks) and 2 weanlings
(6-8 weeks) (Table I). A 25 x 150 mm cellophane tape was
pressed against the area of pelt at peri-anal region and then
affxed to a microscopic slide. The entire slide was examined
thoroughly and systematically under X10 magnifcation to
detect ova of Syphacia spp. (Fig.1). Treated animals were tape
tested monthly after each treatment regime for four months
to determine the recurrence of infestation and effectiveness
of treatment.
Table I : Results of Cellophane Tape Test for Syphacia eggs
before treatment
Strain/
Stock
Treatment
Group I
Treatment
Group II
Males
(n=10)
Females
(n=10)
Males
(n=10)
Females
(n=10)
Sctb:WI 8 9 9 10
Sctb:SD 10 7 8 8
Sctb:Swiss 8 7 6 8
BALB/c 8 8 8 9
Treatment
A randomized block design was made for assessment of
treatment effectiveness in each stock/strain of animals. Each
strain/stock of animal was divided into two groups with equal
number of males and females. A total of 10 males and 10
females were selected from each strain/stock (Table 2).
Table 2 : Allocation of animals for treatment
Strain
Number of
animals
Treatment
Group I
Treatment
Group II
Males Females
Sctb:WI 10 10
Piperazine
(10.8 mg/
ml)
0-28 days
Piperazine
(10.8 mg/
ml)
0-14 days
Ivermectin
(0.01mg/
ml)
14-28 days
Sctb:SD 10 10
Sctb:
Swiss
10 10
BALB/c 10 10
Treatment group I was administered with piperazine alone
for 28 days at 10.8 mg piperazine (piperazine hexahydrate,
TTK Pharma) per ml of drinking water. Treatment group II
was administered with 10.8 mg piperazine per ml of drinking
water from day zero to day seven through drinking water. On
day seven, drinking bottles were washed and flled again with
piperazine and given until day 14. From day 14, 0.01 mg/ml
ivermectin solution (ivermectin tablets, Neomec, INTAS) in
drinking water was administered. On day 21, water bottles
were washed and flled again with ivermectin and given until
day 28.
Medicated water in each bottle lasted for one week, allowing
animals to get continuous treatment. Complete cage changing
was performed on day 28 for both the groups. Cages and
accessory equipments were disinfected with activated
gluteraldehyde, 2.45% w/v (Cidex, Johnson and Johnson). All
supplies including bedding, and accessories were completely
autoclaved in both the treatment groups.
Results
In the present study, treatment with two drugs (piperazine and
ivermectin) combination over a 4-week period, associated
with hygiene measures was chosen and tested in our animal
facility.
Table 3 : Results of Cellophane Tape Test for Syphacia eggs,
28 days after treatment
Strain/
Stock
Treatment Group I Treatment Group II
Males
(n=10)
Females
(n=10)
Males
(n=10)
Females
(n=10)
Sctb:WI 1 0 0 0
Sctb:SD 0 1 0 0
Sctb:Swiss 1 0 0 0
BALB/c 0 0 0 0
Eradication of Syphacia, Harikrishnan V.S.
Vol -1, Issue - 1, Jan - Apr 2011 65
Syphacia infestation in piperazine alone group (group I) was
not completely eradicated, which was evidenced by negligibly
small proportion of animals in the colony tested positive
although the number of ova found in these animals was very
low. It was observed that animals in group II with two drug
regimen is advantageous over group I animals (Table 3).
The present study showed that the oral administration of the
combination of piperazine and ivermectin, associated with
appropriate hygienic procedures, resulted in an inexpensive,
safe and highly effective treatment for mouse and rat
pinworms.
Fig.1 : Eggs of Syphacia muris

Discussion
Prior to the treatment, the colony showed 87% positive results
for Syphacia spp. The eggs (Fig.1) were identifed by the
descriptions given by Flynn (1989). The life cycles of these
parasites are direct. Gravid female worms migrate from
the caecum or the colon to the perianal region of the host,
deposit embryonated eggs on the skin, then dry up and die.
The eggs become infective within 6-24 h. The eggs hatch in
the alimentary tract of another rodent and the larvae continue
their way to the caecum where they moult through four stages
to sexually mature adults. After fertilization, the males die and
gets eliminated through faeces (Owen, 1992).
Only very few successful treatments were reported for the
eradication of pinworms from rodent colonies. The control of
Syphacia spp. infestation is diffcult as most anthelminthics
are only partially effective against adult worms and have
no impact on larvae or ova; and also the eggs embryonate
rapidly and can survive outside the host (Zenner, 1998).
Treatment failures reported were commonly associated with
reinfestation from eggs in the environment (Hoag, 1961).
Lipman et al. (1994) published a report on effective treatment
of S. obvelata in mice, using a combination of ivermectin
and piperazine. We followed the same protocol in infested
colonies of mice and rats but with modifcations in dosage of
piperazine (10.8 mg/ml) in drinking water when compared to
2.1 mg/ml of piperazine sulfate in drinking water by Lipman
et al. (1994). However, we could fnd that the availability of
piperazine hexahydrate as commercial medicine is ubiquitous
when compared to piperazine sulfate in India. The dosage of
2.1 mg/ml of piperazine sulfate in drinking water by Lipman et
al. 1994 could not be able to prevent recurrence of pin worm
when compared to a higher dosage of piparazine hexahydrate
(10.8 mg/ml in drinking water). An effective regimen
must be based on the combination of adequate hygiene
measures to remove parasite eggs from the environment, and
chemotherapy with effective drugs at appropriate treatment
intervals (Zenner, 1998).
The oral administration of piperazine citrate at a rate of
200-400 mg/kg for one week, followed by an interruption
for a week and another treatment during the third week, as
described by Hoag (1961), was used extensively. This protocol
reduces the number of oxyurids present in mice colonies, but
generally does not eradicate the parasites. Different treatment
regimes with piperazine alone have proved unsuccessful
in the eradication of pinworms (Lipman et a1. 1994) since
viable eggs were detected from 2 to 4 months at the end of
the treatment. Oral doses of ivermectin were effective against
adults, gravid females and immature worms in mice (Flynn et
al. 1985, Ostlind et a1. 1989). However, 3 weeks of treatment
with ivermectin in water was ineffective for the treatment of
a mouse colony infested with S. obvelata, since viable eggs
were detected soon after treatment (Lipman et a1. 1994).
Alternating the two regimens led to good results against S.
obvelata in mice (Lipman et a1. 1994), but was neither tested
in mice against S. muris nor in rats. The present treatment
regime resulted in eradication of Syphacia spp. in the rodent
colony. Results obtained after four months revealed that there
was no re -infestation with the present two drug treatment
regimen.
Acknowledgements
We express gratitude to the Director, and the Head, Bio
Medical Technology Wing, Sree Chitra Tirunal Institute for
Medical Sciences and Technology, Thiruvananthapuram,
Kerala, India for supporting this work.
References
Baker D (1998). Natural pathogens of laboratory mice,
rats and rabbits and their effects on research. Clin.
Microbiol. Rev.11:231- 266.
Baskerville M, Wood M, Newton CM (1988). Mebendazole
for worming mice: effectiveness and side effects. Lab.
Anim. 22:263-268.
Coghlan LG, Lee DR, Psencik B, Weiss D (1993). Practical
and effective eradication of pinworms (Syphacia muris)
in rats by use of fenbendazole. Lab. Anim. Sci. 43: 481-
487
Dix J, Astill J, Whelan G (2004). Assessment of methods
of destruction of syphacia muris eggs. Lab. Anim. 38:
11-16
Flynn BM, Brown PA, Eckstein JM, Strong D (1989).
Treatment of Syphacia obvelata in mice using
Ivermectin. Lab. Anim. Sci. 39: 461-463
Harkness JE, Wagner JE (1989). The biology and medicine
of rabbits and rodents. 3
rd
Edn. Philadelphia: Lea &
Febiger.
Vol -1, Issue - 1, Jan - Apr 2011 66
Hoag WC (1961). Oxyuriasis in laboratory mouse colonies.
Amer. J. Vet. Res. 22:150-153.
Huerkamp M, Bejamin K, Zitzow L, Pullium J, Lloyd J,
Thompson W, Webb S, Lehner N (2000). Fenbendazole
treatment without environmental decontamination
eradicates Syphacia muris from all rats in a large,
complex research institution. Contemp. topics. Lab.
Anim. 39:9-12
Kraft V, Deeny AA, Blanchet HM et al. (1994). Report
of the FELASA Working Group on Animal Health :
Recommendations for the health monitoring of mouse,
rat, hamster, guinea pig and rabbit breeding colonies.
Lab. Anim. 28: 1-12.
LeBlanc SA, Faith RE, Montgomery CA (1993). Use of
topical ivermectin treatment for Syphacia obvelata in
mice. Lab. Anim. Sci. 43: 526-528
Lipman NS, Dalton SD, Stuart AR, Arruda K (1994).
Eradication of pinworms (Syphacia obve1ata) from
a large mouse breeding colony by combination oral
anthelmintic therapy. Lab. Anim. Sci. 44: 517-520.
Lubcke R, Hutcheson FAR, Barbezat DO (1992). Impaired
intestinal electrolyte transport in rats infested with the
common parasite Syphacia muris. Dig.Dis.Sci. 1:60.
MacArthur JA, Wood M (1978). Control of oxyurids in mice
using thiabendazole. Lab. Anim. 12: 141-143
Nicklas W, Baneux P, Boot R, Decelle T, Deeny AA ,
Fumanelli M, Illgen WB (2002). Recommendations for
the health monitoring of rodent and rabbit colonies in
breeding and experimental units. Lab. Anim. 36: 2042
Ostlind DA, Narcowicz MA, Mickle WC (1985). Effcacy
of ivermectin against Syphacia obvelata (Nematoda) in
mice. TournaI of Helminthology. 59: 257-261
Owen DC (1992). Parasites of Laboratory Animals,
Laboratory Animal Handbook No. 12. London: Royal
Society of Medicine Services Limited
Percy D, Barthold S (2001). Pathology of laboratory rodents
and rabbits. 2
nd
edn. Ames: Iowa state university press
pp 79-80
Simmons ML, Williams HE, Wright EB (1965). Therapeutic
values of the organic phosphate trichlorfon against
Syphacia obvelata in inbred mice. Lab. Anim. Care. 15:
382-385.
Wagner JE (1970). Control of mouse-pinworms, Syphacia
obvelata, utilizing dichlorvos. Lab. Anim. Care. 20: 39-
44
Zenner L (1998). Effective eradication of pinworms
Syphacia muris, Syphacia obvelata and Aspiculuris
tetraptera) from a rodent breeding colony by oral
anthelmintic therapy. Lab. Anim. 32: 337-342
Eradication of Syphacia, Harikrishnan V.S.
Vol -1, Issue - 1, Jan - Apr 2011 67
Shakthi Devan R.K. received MVSc, in Veterinary Pharmacology and Toxicology
from West Bengal University of Animal and Fishery Sciences, Kolkata in 2003. Cur-
rently working as Research Scientist at the Syngene International Limited, Ban-
galore. He is mainly involved in quality assurance program besides management
and maintenance of various species of laboratory animals, in addition to conduct-
ing experiments on animals. He has undergone training at NIEHS/NIH, Research
Triangle Park, NC, Virginia Tech, Blacksburg, VA and Bristol-Myers Squibb USA. He
has attended many workshop/conference at national and international level and
life member of several scientifc societies viz. LASA, AALAS, ISVPT and STOX. Nomi-
nated as AAALAC Ad-hoc Specialist in 2010
Shakthi Devan R. K.
Sentinel Monitoring Program: Quality Assurance in
Laboratory animal facility
Shakthi Devan R.K
1
, Parthiban.N
2
, Samarendra Narayanan
3
and Suresh Poosala
4
1, 2, 3
Syngene International Limited, Bangalore 560100, India
4
Bristol Myers Squibb India Pvt. Limited, Bangalore 560100, India
Abstract
Sentinel animal testing plays a pivotal role in management of any modern vivarium. It is an extremely
useful tool to assess the microbial status of the laboratory animals as part of the comprehensive animal
care program. Sentinels pick up the early infection if any, in the micro and/or macro environment, as these
animals are placed in a similar environment intentionally. Sentinels are representative animals, independent
of the research colonies and are maintained only for the purpose of screening the pathogenic organisms
at regular intervals. At times, as part of 3R's, we have on occasion used research animals to confrm our
sentinel results. Sentinel program is concerned with prompt detection of pathogens and the measures
to deal with them thereby ensuring the health status of the resident colony. With an efective sentinel
monitoring program preventive measures can be taken to enact either, treat, contain or eradicate the
pathogen. This review provides insights into the sentinel program with respect to disease surveillance of
laboratory animals and conveys why it is necessary for efective in laboratory animal program management.
Key Words: agents, sentinel program, health monitoring, test methods, serology, culture, polymerase
chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), immunofuoresence Assay (IFA)
Suresh Poosala, DVM, MS, Ph.D., Head, Veterinary Sciences & Toxicology, Bristol Myers Squibb India Pvt Limited
Biocon Park (BBRC), 1 & 2 Jigani Link Road, Bommasandra, Bangalore - 560099. India.
email: suresh.poosala@bms.com
Introduction
The use of laboratory rodents has become an indispensable
tool in modern biomedical research and has shown a steady
increase in various therapeutic areas of research. In recent
years, many institutions import animals from a commercial
vendor or breed them in their facility. Strains of genetically
engineered, immunocompromised animals now play a vital
role in research and various therapeutic diseases. Maintenance
of clean facilities becomes an even more challenging process
due to movement of animals between facilities on one hand
and the relatively unknown health status of newly arrived
animals on the other. Thus, though the animals may have
originated from barrier maintained facilities, there might still
be a risk of infection during transportation.
In any institution/organization, health monitoring is considered
an integral part of the quality assurance system, e.g. Good
Laboratory Practice (GLP), Association for Assessment
and Accreditation of Laboratory Animal Care International
(AAALAC), International Standards Organization (ISO)
(Nicklas et al. 2002 and for other regulatory purposes
(Weisbroth and Emily, 2000). Health monitoring is thus
Care and management
Vol -1, Issue - 1, Jan - Apr 2011 68
Sentinel monitoring programme, Shakthi Devan R.K.
an inescapable necessity and any institution which ignores
conducting a sound health monitoring plan does so at the
risk of losing all its valuable animals in the event of a disease
outbreak.
The outcome of any infection depends on multiple
factors relating to the characteristics of the pathogen, host
susceptibility, infection rate and mode of transmission.
However many a times before a pathogen elicits a response
in infected animals by way of evincing clinical signs, it
may harbour a low grade infection which may thus interfere
with the intended aim of the research. Systematic and
scheduled laboratory testing is the most effective way to
determine colony status and to prevent or detect infuences
on experiments (Nicklas, 2008). Therefore, it has been our
practice to deliberately subject certain susceptible strains
of animals to a challenging environment such as soiled
bedding, air or contact with animals of unknown health
status. Such animals have been called as sentinel animals, a
word derived from the French word sentinelle which means
watchtower. A sentinel animal is an animal known to be
susceptible to an infectious agent that is placed in the area
suspected of being contaminated (CCAC, 1993), and these
animals are intentionally used and then tested to see if it
became infected or developed antibodies to infectious agents.
The purpose of sentinel surveillance is to detect the
adventitious infections early (virus, bacteria and parasites)
among rodents/non rodents that could potentially interfere
with research. The aim of the sentinel surveillance is to obtain
timely information in a relatively inexpensive manner rather
than to derive precise estimates of prevalence or incidence in
the general population (CDCP, 2002).
General Considerations:
A decision should be made for type of agents to be
screened. Enlist the organisms of interest and may be
comprehensive or partial list based on the facilities
available.
Compile the prevalence of agents and the past incidence
of the facility if any.
Evaluate the reliability of the testing method (e.g.
ELISA, IFA, HAI and PCR etc.)
Frequency of the testing interval and cost of the testing
procedure.
Establishing a program:
Health monitoring program can be considered to comprise
of two complementary components: routine screening and
diagnostic evaluation. Routine screening involves periodical
sampling of a population to detect organisms based on
presumed morbidity and prevalence of the organisms
screened in the past. In contrast, the diagnostic evaluation of
the samples is retrospective and gives an idea for the cause
of the clinical disease or death in the animals. The organisms
may have invaded the population long before the initiation
of clinical signs and death of the animal. Hence, clinical
observation, complete morbidity and mortality records along
with the postmortem reports are required during the diagnostic
evaluation. The main goal for the health surveillance of the
colony is to detect the introduction of any agent and to act
quickly to control the spread within the colony, which will
mitigate the impact that disease has on the science.
Transmission routes
In many instances, the sentinel and the rest of the resident
animal population is exposed to infection via the same route.
Institutions that deal with frequent imports of animals and
other biological materials should establish and implement
an effective measure to reduce the risk of transmitting any
infectious agents to the laboratory animals and the staff. For
instance, Staphylococcus aureus is a common opportunistic
inhabitant of the skin, which can profoundly affect host
physiology (Baker, 1998a). Caging systems play an important
role in maintaining the integrity of the health status of the
animals and can minimize the exposure pattern of agents.
Nowadays, many institutions have started using individually
ventilated cages, biosafety cabinets for cage changes and
isolators to prevent cross contamination. Although disease
prevalence and outbreak management varied considerably
among the facilities, these variabilities create an environment
for possible cross contamination among the colonies (Carty,
2008). It is far better to prevent the introduction of pathogens
than to have to account for their presence when interpreting
experimental results (Baker, 1998b).
Animals
The transmission of any adventitious agent may occur from
rodents shipped from vendors and/or other institutions. In
order to avoid these transmissions, the packages for any
kind of animal transport should be designed to prevent the
animals escape, exclude the entry of microorganisms, allow
visual inspection of the animals without compromising
their microbiological status, and allow external disinfection
of the package on arrival at the receiving facility (Mahabir
et al. 2008). Animals may harbor any agent during transit
and adequate care should be taken at the time of receiving.
Improper disinfection of the exterior before unpacking can
lead to contamination (Reuter and Dysko, 2003). So, the
shipping crates needs to be decontaminated thoroughly before
placing them into the quarantine. However, even with these
extensive precautionary measures in place, there always
exists a potential risk (White et al. 1998) for introducing any
organism primarily of viral or bacterial origin.
Vol -1, Issue - 1, Jan - Apr 2011 69
Biological Materials
The use of biological materials such as cell lines, sera,
embryonic stem cells, and sperm derived from other animals
may result in the introduction of unwanted agents (Bhatt et al.
1986). If any of the afore mentioned materials are imported into
the facility, they should be procured from institutions/vendors
with necessary testing certifcate ensuring that the biological
materials are free from pathogens and/or need to be screened
before use. A quality assurance program should confrm the
status of these materials from time to time. Routine diagnostic
tests like Mouse/Rat/Hamster antibody production test (MAP,
RAP, and HAP) have been used extensively to identify
the presence of infectious virus in biological specimens
(Weisbroth et al. 1998). These traditional tests have now been
effectively replaced by novel PCR diagnostic methods. Some
of the murine viruses like lymphocytic choriomeningitis virus
(LCMV) (Bhatt et al. 1986) minute virus of mice (MVM),
mouse adeno virus, kilham rat virus (KRV), Toolans H-1,
mouse hepatitis virus (MHV), and reovirus3 have been
detected in biological materials (Nicklas et al.1993). The most
frequent contaminant is lactate dehydrogenase-elevating virus
(LDV) (Nicklas et al. 1993) because this virus causes long
lasting viremia in mice without any evidence of clinical signs.
The use of parvovirus-infected rodents and contaminated cell
lines can affect immunology, transplantation and oncology
research (Besselsen et al. 2008).
Personnel
Personal hygiene and periodical monitoring of PPEs
(personal protective equipment) for their quality and
integrity of the materials are essential. Personnel may act
as effective carriers of infection from contaminated to non-
contaminated units (La Regina et al. 1992). Organisms may
enter the facility if any breach in the PPE procedure and/or
traffc pattern of the personnel occurs. Organisms of human
origin like Staphylococcus aureus or Klebsiella pneumoniae
are occasionally responsible for research complications,
particularly in immunocompromised animals (Nicklas et
al. 1993) because humans act as a mechanical or biological
carrier for the transmission. An established traffc pattern
and continuous education to the personnel can minimize the
level of cross contamination into the facility. In addition,
management should ensure the facility personnel coming
into contact with laboratory animals have no access to other
animals of unknown microbiological status.
Vermin Control
The great concerns of preventing major infections through
adventitious agents are achieved by pest control program.
Facilities should have rigorous monitoring procedure to
prevent the entry of fying and crawling insects and wild
rodents as they can carry unknown agents. Hence, the animal
diets, bedding and waste materials may attract these external
vermins. It is, therefore necessary to maintain the facility
without any cracks, crevices and small holes especially in the
walls, roofs and perimeter because these can allow entry of
wild rodents into the facility.
Other Factors
The most important factors are equipment and materials used
for animal experiments because these materials are considered
as potential source of contamination as animals are often in
contact with them. In many instances, the equipment may be
shared with other laboratories within the facility increasing the
possibility of contamination. An established system should be
followed for transportation of materials within the facility and
also for decontamination procedure by appropriate method.
It is always necessary to use sterilized items (autoclaving/
irradiation) in the facility and experimental materials can be
disinfected with suitable agents. Drinking water is unlikely
to be a source of infection, if it can be treated with a suitable
method, such as (hyperchlorination/ozonization/ultra
violet/reverse osmosis fltration) to eliminate the infectious
microorganisms.
Sample Size for sentinel testing
The necessary sample size entirely depends on the size of
animal population, the prevalence rate of eventual infection
and the frequency of the testing period. Ten animals should
be monitored to detect at least one positive animal if the
suspected prevalence rate of an infection is 30% (confdence
level: 95%) (Nicklas et al. 2002; Shek, 2008). There is
always a heightened potential risk of introducing agents into
the facility. For instance, frequent introduction of animals
from multiple sources and personnel movement all can lead
increased chances of infectious outbreaks. Therefore, the
frequency of screening should be optimized based on the
practice, type of research and physical nature of the facility.
Several reports revealed the statistical consideration for the
size of the samples (Selwyn and Shek, 1994; Clifford, 2001).
Sampling techniques and frequency
Once the types of screening methods and kind of organisms
have been determined, the number and frequency of the
samples needs to be decided for consistency. The frequency
of sampling is driven by the historical rate of contaminations
with extraneous agents that compromise the microbial status
of the colony (Selwyn and Shek, 1994). Many biological
considerations that affect the sampling frequency, particularly
for serologic estimation requires minimum 14 to 21 days
necessary from the time of infection until the antibody titer
raises in the serum of the infected animal. Depending upon the
rate and method of transmission, additional time is required
Vol -1, Issue - 1, Jan - Apr 2011 70
for the animal to show a detectable antibody titer and to reach
maximum morbidity. Considering all variables, at least three
to six weeks may be required from the introduction of the
agent and to develop suffcient enough serologic evidence of
the organism in animals.
Sentinel Program
Sentinel animals are placed to monitor the population being
surveyed and these animals are indirectly exposed to the
infectious agents by using soiled bedding. These animals are
independent of the research colonies and placed only for the
testing purpose to evaluate the pathogens if any. The selection
of strain, stock, age and sex are important. For instance, the
C57BL/6 mice are not susceptible to MPV in comparison to
white mice.
Generally female animals of the same species are preferred
as sentinels because they fght less than males when group
housed. Age and sex of the sentinels can be selected based
on the type of research adopted by the institution. In general,
outbred stocks have high vigor, robust immune response to
a larger pool of antigens and involves relatively low cost.
Institutions using immunocompromised animals such as Nude
or SCID (severe combined immunodefciency) mice may
use heterozygotes as sentinels. In breeding facilities, retired
breeders may also be screened because these animals were
exposed to infectious agents for longer duration and presumed
that these animals are likely to have seroconverted during
their tenure. Hence, seroconversion occurs consistently in
young mice exposed high doses of mouse parvo virus (MPV1)
equivalent to those shed by acutely infected mice (Besselsen
et al. 2008). Nevertheless, the duration of sentinel exposure
can be decided based on the characteristics of the organisms.
The sentinels should be selected from any commercial
vendor provided all the health reports and/or from the In-
house breeding facilities of known pathogen free status. If
the animals have been procured from an outside vendor, they
should be quarantined and screened before releasing them into
the facility for use to avoid introducing any infectious agents.
Sentinels used can be either contact sentinels, in which case
they share housing with the animals being monitored, or as
indirect sentinels, in this case animals are exposed through
transfer of contaminated bedding and/or air from animals
selected for monitoring. Contact sentinels are preferable
when assessing the health status of small number of animals
(e.g., import animals in quarantine), whereas indirect
exposure methods are desirable when monitoring large
colonies (Lipman and Homberger, 2003). Suffcient period of
association is allowed for transmission of infectious agents
and presumed increase in serologic titers or development of
disease. It may be desirable to select a sentinel that has higher
levels of exposure and which is therefore more likely to show
evidence of a pathogen if it is present than to directly survey
the population itself (Halliday J o et al. 2007). In addition,
any animal found sick, moribund or dead during the course
of time are submitted for a detailed examination so that the
occurring infections can be detected at a very early stage.
Generally, sentinel cages are placed on the lower tier of the rack
to maximize the fomite transmission through air particulate
movement. Transmission of infections by soiled bedding
was shown experimentally for fecally excreted pathogens:
sialodacryoadenitis virus (La Regina et al. 1992), mouse
hepatitis virus (Smith et al. 2007), kilham rat virus (Ueno et
al. 1996 and Smith et al. 2007) and Helicobacter hepaticus
readily transmitted to sentinel mice via contaminated bedding
(Livingston, 1998). Scheduled transfer of soiled bedding to
the sentinel cage is mandated from rest of the housing cages
of the population being monitored. The soiled bedding of
adequate quantity can be transferred to the sentinel cages
by using a cup to avoid any cross contamination during the
practice. It is preferable to use equal amount of soiled and
clean bedding mix together to provide comfort to the sentinels.
Exposing sentinel animals to dirty bedding enhances the
sentinels chances of becoming infected and could reduce the
time required to detect some pathogenic agents (Thigpen et
al.1989). The sentinel program can be practiced in quarantine,
breeding and experimental unit and cages should be identifed
by using bright label with all the necessary information.
Testing Methodologies
It is necessary to evaluate the testing strategy for sensitive
and specifc diagnostic approaches to accurately identify the
pathogen in the infected colonies. The most common method
to assess the presence of viral agents is to examine serum for
the presence of specifc antibody. Most of the viral antibodies
are detected using ELISA method. Exceptions would include
mouse thymic virus, for which the test recommended is
indirect immunofuorescence assay (IFA), and lactate
dehydrogenase-elevating virus which can be detected by
supplementing a test to detect elevation of LDH in the serum.
Haemagglutination Inhibition (HAI) test also be used for viral
screening, however ELISA and IFA are more sensitive than
HAI and are commonly used as primary tests for most of the
viral agents and mycoplasma.
Nowadays, polymerase chain reaction (PCR) technique is
used to detect many of the pathogens and this test doesnt
require the host to have a completely functional immune
system to enable detection. The PCR technique is the reliable
method for the detection of Helicobacter spp. (Fox et al.
1994; Pritchettt-Corning et al. 2009) CAR bacillus (Baker,
2003) and Pneumocystis spp. (Pritchettt-Corning et al. 2009)
in laboratory animals. In addition, novel multiplex diagnostic
system that employs fuorescent microbeads coated with
Vol -1, Issue - 1, Jan - Apr 2011 71
Table 1 : List of infective organisms screened in laboratory animals
Agents Rat Mouse Hamster Guinea Pig Rabbit
Mouse corona virus / Mouse hepatitis virus (MHV)
Sendai virus
Rat corona virus / Sialodacryoadenitis virus (RCV/SDAV)
Kilham rat virus (KRV)
Minute virus of mouse / Mouse parvo virus (MVM/MPV)
Theilers murine encephalomyelitis virus (GDVII)
Rota Virus (EDIM/MRV)
Pneumonia Virus of Mice (PVM)
Rat parvo virus (RPV)
Reovirus 3
Lymphocytic choriomeningities Virus /Arena virus (LCMV)
Lactate dehydrogenase-elevating virus (LDV)
Ectromelia virus/Mouse pox
Adenovirus (MAV 1 & MAV 2)
Polyoma virus
Hantaan virus
Toolans virus (H1)
Mouse thymic virus (MTLV)
Cytomegalovirus (CMV)
Hemorrhagic disease virus
Myxoma Virus
Mycoplasma pulmonis
Corynebacterium kutscheri
Streptococcus pneumoniae
Klebsiella pneumonia
Cilia-associated respiratory bacillus (CARB)
Pasteurella pneumotropica
Pasteurella multocida
Pasteurellaceae
Bordetella bronchoseptica
Pseudomonas sp
Staphylococcus sp
hemolytic Streptococci sp (group B)
Clostridium piliforme
Chlamydia psittaci
Streptococcus moniliformis
Salmonella sp
Citrobacter rodentium
Yersinia pseudotuberculosis
Helicobacter sp
Encephalitozoon cuniculi
Pneumocystis carinii
Syphacia muris
Syphacia obvelata
Aspiculuris tetraptera
Other endoparasites
Dermatophytes
Ectoparasites
Vol -1, Issue - 1, Jan - Apr 2011 72
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Vol -1, Issue - 1, Jan - Apr 2011 74
purifed antigens is useful or simultaneous serodetection of
various infectious agents (Khan et al. 2005).
Samples preferred to be collected for screening of bacteria
are taken from upper respiratory tract (nasopharynx/trachea)
and intestinal tract (caecal contents or feces). Selective
media should be used to distinguish the pathogenic bacteria
from the non-pathogenic. There are some bacteria including
mycoplasma which can be detected by using ELISA and PCR
method. Regardless of the method adopted by the institution,
an established standard operating procedure should be
implemented to obtain consistent testing results.
In addition, pelt examination from the animals often provides
the evidence for the presence of any ectoparasites. Skin
scrapings can be taken from lesions if any and the same can
be screened for mite infestation. Tape test or direct fecal
foatation techniques are used to detect the presence of
pinworms of Syphacia spp. because they deposit their ova in
the perineum of the host. In contrast, Aspiculuris tetraptera
pinworms deposit their egg within the large intestine of the
host and caecal examination is most commonly used to detect
the ova and adult worms. Similarly, caecal examination is
used for other endoparasites (e.g. tape worms).
A detailed list of orgonisms and type of samples used for
assays are given in Annexure-1.
Documentation of results
Detailed results pertaining to sentinels should be reviewed
and fled based on functional area. Besides, the data can be
incorporated as part of the experimental data. The test results
should include all the relevant informations like test method,
source of the samples (isolator, barrier and conventional),
profle of organisms (partial/ comprehensive/full). Test
results should be archived annually for further traceability and
this can be served as historical data for the facility. A list of
organisms provided (Table 1) and one can decide the profle
of the organisms to be screened based on their nature of work.
Response to the positive resultsIt is always advisable to
confrm the positive results by another laboratory and/or
by repeated testing by using other reliable methods such as
IFA, PCR, culture or histopathology exists for the pathogen.
Serology provides an indirect measure of exposure to an
agent, whereas PCR directly detects the presence of the agent
and histopathology detects pathological changes induced
by the microbe (Livingston and Riley, 2003). Generally, the
eradication involves standard approaches (e.g. depopulation,
rederivation, antimicrobial therapy, stop breeding, test and
cull) (Clifford and Watson, 2008; Shek, 2008). An action
plan pertaining to any emerging infectious agents should
be in place to guide during confrmed positive result of any
agent. For any contamination, animal care personnel and
research staff work together and discuss about the impact on
the research of the positives and discuss the possible action
plans for elimination of the pathogen, decontamination of the
facilities and evaluation of the colony.
Conclusions
The objective of this review is to provide an insight into
the role of sentinel animals in detecting the adventitious
microbial infections early among rodents and non rodents
used in biomedical research. The effective health monitoring
systems rely on early detection of unwanted organisms
(Clarke and Perdue, 2004). The use of sentinel animals
for disease monitoring is relatively inexpensive when no
animal from hygienic unit to be examined are available for
microbiological examination (e.g. immunocompromised
and transgenic animals). Animal health and welfare,
not to mention protection of scientists from the adverse
effects of infectious disease on their research animals, are
unequivocally institutional and governmental responsibilities
(Barthold, 1998). The biomedical research community and
laboratory animal professionals should take a proactive role
in understanding the need of animal health surveillance and
implementation of sentinels programs in their facility with the
available resources. Further more, disease free animals can
provide clean status of the facility, animal health assurance to
the researchers and in turn ensure meaningful research data.
References
Baker DG (1998a). Future Directions in Rodent Pathogen
Control. ILAR J.39 (4): 312-315.
Baker DG (1998b). Natural Pathogens of Laboratory Mice,
Rats, and Rabbits and Their Effects on Research. Clin.
Microbiol. Rev. 11(2) : 231-266.
Baker DG (2003). Natural Pathogens of Laboratory Animals.
Washington ASM Press.
Barthold SW (1998). Opportunistic Infections in research
Rodents: The Challenges Are Great and the Hour Is Late.
ILAR J. 39 (4) : 316-321.
Besselsen DG, Franklin CL, Livingston RS, Riley LK (2008).
Lurking in the shadows: Emerging Rodent Infectious
Diseases. ILAR J. 49 (3): 277-290.
Besselsen DG, Myers EL, Franklin CL, Krote SW, Wagner
AM, Henderson KS, Weigler BJ (2008). Transmission
Probabilities of Mouse Parvovirus 1 to Sentinel Mice
Chronically Exposed to Serial Dilutions of Contaminated
Bedding. Comp. Med. 58(2): 140-144.
Bhatt PN, Jacoby RO, Barthold SW (1986). Contamination
of transplantable murine tumors with lymphocytic
choriomeningitis virus. Lab. Anim. Sci. 36(2) : 136-139.
Vol -1, Issue - 1, Jan - Apr 2011 75
Carty AJ (2008). Opportunistic infections of Mice and Rats:
Jacoby and Lindsey Revisited. ILAR J. 49(3) : 272-276.
CCAC (1993). Guide to the care and use of experimental
animals. Volume1, 2nd edition.
CDCP (2002). Centers for Disease Control and Prevention.
Manual for the surveillance of vaccine-preventable
diseases, 3rd edition.
Clarke CL, Perdue KA (2004). Detection and Clearance
of Syphacia obvelata Infection in Swiss Webster and
Athymic Nude Mice. Contemp. Top. Lab. Anim. Sci.
43(3) : 9-13.
Clifford CB (2001). Samples, sample selection, and statistics:
Living with uncertainty. Lab. Anim. (NY). 30:26-31.
Clifford CB, Watson J (2008). Old enemies, Still with Us
after All These Years. ILAR J. 49(3) : 291-302.
Companion Guide to Infectious Diseases of Mice and Rats
(1991). National Research Council National Academy
Press.
Fox JG, Dewhirst FE, Tully JG, Paster BJ, Yan L, Taylor
NS, Collins JR MJ, Gorelick PL, Ward JM (1994).
Helicobacter hepaticus spp. Novel Microaerophilic
Bacterium Isolated from Livers and Intestinal Mucosal
Scrapings from Mice. Am. Soc. Microbiol. 32(5) : 1238-
1245.
Halliday Jo EB, Meredith AL, Knobel DL, Shaw DJ,
Bronsvoort BM de C, Cleaveland S (2007). A framework
for evaluating animals as sentinels for infectious disease
surveillance. J. R. Soc. Interface. 4 : 973-984.
Khan IH, Kendall LV, Ziman M, Wong S, Mendoza S,
Fahey J, Grieffey SM, Barthold SW, Luciw PA (2005).
Simultaneous serodetection of 10 highly prevalent mouse
Infectious Pathogens in a Single Reaction by Multiplex
Analysis. Clin. Diag. Lab. Immunol. 12(14) : 513-519.
La Regina M, Woods L, Klender P, Gaertner DJ, Paturzo
FX (1992). Transmission of sialodacryoadenitis virus
(SDAV) from infected rats to rats and mice through
handling, close contact and soiled bedding. Lab. Anim.
Sci. 42 : 344-346.
Lipman NS, Homberger FR (2003). Rodent Quality
Assurance Testing: Use of Sentinel Animal Systems.
Lab. Anim. 32(5) : 36-43.
Livingston RS, Riley LK (2003). Diagnostic Testing of
Mouse and Rat Colonies Infectious Agents. Lab. Anim.
32(5) : 44-50.
Livingston RS, Riley LK, Besch-Williford CL, Hook Jr.
RR, Franklin CL (1998). Transmission of Helicobacter
hepaticus Infection to Sentinel Mice by Contaminated
Bedding. Lab. Anim. Sci. 48(3) : 291-293.
Mahabir E, Bauer B, Schmidt J (2008). Rodent and Germplasm
Traffcking: Risks of Microbial Contamination in a High-
Tech Biomedical World. ILAR J. 49 (3) : 347-355.
Nicklas W (1993). Possible routes of contamination of
laboratory rodents kept in research facilities. Scand. J.
Lab. Anim. Sci. 20 : 53-60.
Nicklas W (2008). International harmonization of Health
Monitoring. ILAR J. 49(3) : 338-346.
Nicklas W, Baneux P, Boot R, Decelle T, Deeny AA,
Fumanelli M, Illegen-wilcke B (2002). FELASA:
Recommendations for the health monitoring of rodent
and rabbit colonies in breeding and experimental units.
Lab. Anim. 36, 20-42.
Pritchettt-Corning KR, Cosentino J, Clifford CB (2009).
Contemporary prevalence of infectious agents in
laboratory mice and rats. Lab. Anim. 43:165-173.
Reuter JD, Dysko RC (2003). Quality assurance/surveillance
monitoring programs for rodent colonies,. In J . D. Reuter
and M. A. Suckow (ed.), Laboratory animal medicine
and management. International Veterinary Information
Service, Ithaca, N.Y. pp. 113.
Selwyn MR, Shek WR (1994). Sample sizes and frequency
of testing for health monitoring in barrier rooms and
isolators. Contemp. Top. Lab. Anim. 33:56-60.
Shek WR (2008). Role of Housing Modalities on Management
and Surveillance Strategies for Adventitious Agents of
Rodents. ILAR J. 49(3) : 316-325.
Smith PC, Nucifera M, Reuter JD, Compton SR (2007).
Reliability of Soiled Bedding Transfer for Detection of
Mouse Parvovirus and Mouse Hepatitis Virus. Comp.
Med. 57(1) : 90-96.
Thigpen JE, Lebetkin EH, Dawes ML, Amyx HL, Caviness
GF (1989). The Use of Dirty Bedding for Detection of
Murine Pathogens in Sentinel Mice. Lab. Anim. Sci.
39(4) : 324-327.
Ueno.Y, Sugiyama.F, Yagami.K (1996). Detection and In
vivo transmission of rat orphan parvovirus (ROPV). Lab.
Anim. 30 : 114-119.
Weisbroth S, Emily P (2000). Global Harmonization of
Laboratory Rodent Health Surveillance Standards. Lab.
Anim. 29(6) : 43-47.
Weisbroth SH, Peters R, Riley LK, Shek W (1998).
Microbiological Assessment of Laboratory Rats and
Mice. ILAR J. 39 (4): 272-290.
White JW, Anderson LC, Geisfeild J, Martin D (1998). Current
Strategies for Controlling/Eliminating Opportunistic
Microorganisms. ILAR J. 39 (4) : 291-305.
Vol -1, Issue - 1, Jan - Apr 2011 76
Sai Tummala is AVMA Licensed veterinarian, and a Diplomate of the American College
of Laboratory Animal Medicine. He is also a Certifed Professional IACUC Administrator
and an Adhoc specialist for the AAALAC International. Has signifcant experience in
managing animal facilities with special emphasis to sentinel programs, staff training,
animal model development, bio-containment, facility design, operational effciency and
lean practices. Experience in both academia and pharmaceutical preclinical research.
Has the knowledge of GLP and FDA regulations pertinent to discovery and preclinical
drug development studies for small molecules and biologics. Currently working as
Director at Biological Resource Center, National J ewish Helath, Denver, USA.
Sai T.
An overview of the key concepts required for managing
a laboratory animal (rodent) facility
Managing laboratory animal facility, Sai T.
Sai Tummala DVM,MS,DACLAM,CPIA, Director, Biological Resource Center
National J ewish Health, 1400 J ackson Street, D104, Denver, CO 80206, USA.
Phone: 303-398-1432, Fax:303-270-2172, email: tummalas@njhealth.org
Abstract
The use of laboratory animals primarily rodents (mice) has increased over the past 20 years especially with
the availability of transgenic and mutant strains. This increased use of mice along with novel research models
with unique needs, has contributed to the evolution of the modern vivariums, decontamination equipment,
imaging cores, ultra clean barriers, decontamination of feed and water etc. In addition, the industry has
also made signifcant improvement in operating animal facilities. Automation of cage processing is just one
example that signifes the sophistication which is an ongoing process. This sophistication and availability of
better options not only improved animal welfare and ergonomics, but also effciency in operating the modern
animal facilities. As the animal facility operation involves several components, it could be challenging for
an entry level manager to be knowledgeable about the best options that suits the needs of the facility. This
article provides an overview of the most popular practices for the crucial aspects of animal care involving
housing, animal health monitoring, feed and watering systems in addition to quality aspects. To provide a
complete review of the laboratory animal facility operations and management would exceed the scope of this
article. However, the topics discussed here are worthy of note when assessing the essential care and welfare
of laboratory animals
Key words : laboratory animals, rodents, facility, management, feed and water quality, health
monitoring, decontamination.
Introduction
Laboratory mice constitute more than 90 percent of all the
animals used in research. Over the years the professional
feld has made signifcant progress in understanding the
physiology, pathology and quality of care for the valuable
strains of research mice. Here, we discuss broadly the key
factors that could impact the micro and macro environments
in a rodent facility. The daily operation in the facility involves
humane care for lab animals, protecting the animals from
pathogens, to keep them well fed and watered, and to maintain
their genetic integrity, in addition facilitating the ongoing
research activities in the facility. One should never forget
that the pathogen protection can never be achieved without
mandatory adherence to pathogen protection standards of all
activities related to animal care at the institution.
Animal health monitoring and
pathogen protection plan
There is no universal agreement on the desired health status
of mice used in research. Scientists and laboratory animal
professionals are in agreement and feel the need to avoid
Sai T.
Biological Resource Center, National Jewish Health, Denver, Colorado, USA
Vol -1, Issue - 1, Jan - Apr 2011 77
microbial contaminants that could impair the performance of
mice in research either directly by causing clinical disease or
indirectly by causing physical or physiological changes that
could alter or confound data. Although there is widespread,
if not universal, interest in excluding the pathogenic murine
viruses from research facilities, there is no consensus on
the importance of excluding organisms such as pinworms,
Helicobacter, Pneumocystis murina, Staphylococcus and
Pseudomonas. To complicate matters, some of the scientifc
evidence and animal models requiring certain agents for
expression of desired pathology/phenotype etc. is undeniable
and emphasizes the reality for lack of universal exclusion
list of agents. There are many examples where a desired
phenotype of a mouse model was lost following rederivation
in to an ultra-clean barrier facility. For example, in most
mouse models of infammatory bowl disease (IBD), diabetes,
immunology and enterocolitis etc. requires a complex
enteric fora and sterile conditions may negatively affect the
desired phenotype. In addition, the absorption and toxicity
of the investigational new drug compounds can be affected
by altered gut fora and or pathogens. Recent publications
support the role of normal fora and their impact on immune
Animal Housing
Summary of common types of mouse caging
System and description Advantages Disadvantages
Conventional cages and shelving
Boxes sit on, or ft into, open metal
shelves.
Boxes have wire lids that contain
bins for feed and water bottles; lids
may or may not have covers.
Air circulation in cages is determined
by room circulation and by airfow
caused by the thermal heat load of
the mice (Reeb et al. 1997).
Is least expensive.
Allows easy access to mice.
Allows cage changing and any
work with mice on open tables.
Even with flter covers, provides
lowest level of pathogen
protection.
With flter covers, poor
circulation can affect intra-cage
temperatures, ammonia levels,
humidity.
Requires more frequent cage
changing than other systems.
Microisolator cages and conventional shelving:
Similar to conventional systems,
except individual boxes have covers
containing high effciency particulate
air (HEPA) flters.
Is a cost-effective, fexible
way to provide higher level
of pathogen protection than
elsewhere in a mouse room.
To maintain higher level of
pathogen protection, cage
changing and any work with mice
must be done in HEPA-fltered
hood.
Air circulation may be poor,
usually worse than conventional
cages and shelving.
May require more frequent cage
changing.
Ventilated, HEPA-fltered caging systems, with heating, ventilation and cooling (HVAC) functionality:
Boxes ft snugly into closed, forced
ventilated shelving system.
Provides highest possible
pathogen protection for the mice
and allergen protection for the
technicians.
Provides good air exchange.
Cages remain dry, which may
reduce cage change frequency
and labor expense.
Requires capital expense.
Cage changing and any work with
mice must be done in HEPA
fltered hoods.
Ventilation can result in drafts
within the cages, which might
stress some mice.
Portable ventilated caging systems:
Similar to stationary ventilated units,
but
boxes ft into stand alone units on
wheels, and
incoming air is fltered room air;
exhaust is fltered back into the
room.
Provides fexible solution for
pathogen protection.
Works well for light mouse loads.
Cages remain dry, which may
reduce cage change frequency
and labor expense.
Cages can be pushed together
for foor space economy with
out inhibiting air circulation for
animals.
Numerous portable racks can
increase the heat load in the
room. An option is to modify rack
ventilation so it is exhausted
outside the room.
Vol -1, Issue - 1, Jan - Apr 2011 78
function. These fndings will only complicate the validity of
research data published from different facilities across the
globe as one can anticipate variability in the gut fora among
research facilities.
Given these complications, unique scenarios and research
types, it will be up to the facility to establish suitable standards
for their research needs and should develop an animal health
program that meets their standards. The four essential
components for any program should be (1) The exclusion
agent list (2) Preventive measures (3) Monitoring procedures
(4) Containment and eradication procedures (in the event of
an outbreak). Stringent standard operating procedures (SOPs)
for each of these components are vital and all vivarium staff
including management staff and research personnel should be
trained on the SOPs.
In the event of an out break, appropriate notifcation and
communication to researchers at academic institutions, to
customers by animal breeders or suppliers and to clients by
contract research organizations is critical to save valuable
time and resources.
Once the exclusion list is defned for the program appropriate
preventive measures should be considered for protecting the
animals. In reality, these measures should be envisioned at
the facility design stage, and use of multiple strategies should
be considered. These aspects can be logically delineated by
concentrating on the macro and micro environments and also
port of entry and port of exit (with respect to animals, staff,
supplies and reagents). Macro environment components with
respect to pathogen protection includes attention to separation
of clean and dirty supplies and traffc, and air quality from
HVAC system. Micro environment components include,
quality of feed, water, bedding, cage processing and cage
changing techniques. The port of entry components should
address the quality of animals and appropriate quarantine
and or rederivation measures, disinfection of supplies at
receiving dock and or in to barrier facilities, proper garbing
of staff entering the facility and proper screening of reagents
especially biologicals prior to injecting the mice in facility.
Rodent health monitoring
As most of the rodent viral agents do not produce clinical
signs, serological monitoring for the presence of antibodies
for the detection of exposure to an agent is the most commonly
Animal Housing
Advantages and disadvantages of different types of bedding for mice
Type of bedding Advantages Disadvantages
Hardwood
shavings
Are cost effective.
Are physiologically inert;
do not elevate cytochrome
p450. (Weichbrod et al.
1988)
May be dusty and irritating.
Is not as absorbent as other materials.
May be from an environment in which
chemicals were used.
Softwood shavings
(cedar, pine)
Are cost effective. Same as hard wood shavings, plus
Cedar and pine can be dusty and irritating.
Cedar, in particular, elevates hepatic p450
enzyme systems (Wade et al. 1968).
Fractions or pellets
of corn cob
Are non irritating.
Are physiologically inert; do
not elevate p450 system.
Is moderately expensive.
Is not good for nesting; additional nesting
material may be required.
May be prone to mold.
Is less absorbent than other bedding.
Recycled paper,
cellulose chips
Provides superior
absorbency and ammonia
control.
Is moderately expensive.
Can be dusty.
Fibers may be irritating to certain strains (e.g.,
nude or SKH-1) that have no eye lashes
(White, 2007).
Cotton based
material
Is highly absorbent.
Is good for ammonia control
when shredded.
Is expensive.
Is effective against odors only if shredded.
Fibers may entangle infant limbs and cut off
circulation.
Paddy husk Inexpensive, readily
available.
Is a good absorbent.
Very light and could be dusty.
Potential for pesticide residues depending on
source.
Not well studied compared to other materials.
Vol -1, Issue - 1, Jan - Apr 2011 79
used method. As one cannot monitor each and every animal,
using targeted sentinel animals typically per rack is most
commonly used method. Sentinels are of two types: Dirty
bedding sentinels are exposed to dirty bedding from research
animals at every cage change (a small amount of dirty bedding
collected from the colony animals cumulatively transferred
to sentinel cage). Contact sentinels are other type where the
sentinel animal lives with in the same cage/pen of the research
animal and is in direct contact with the colony animals. The
contact sentinel system is used in limited settings for example
quarantine facilities, etc.
Although use of sentinel animals has been the most pragmatic
and so far the widely acceptable approach for disease
surveillance, this system has several major caveats and
concerns. 1) The choice of sentinel animals is very crucial,
they should be not only clean but also immuno-competent for
the agents that are surveyed. 2) The dirty bedding exposure
system is ineffective in transmitting the agents to sentinels.
This is because either the agent is not shed into bedding (e.g.
Sendai virus) or the agent is shed intermittently (e.g. Mouse
Parvo Virus-MPV) and or the agent may be unstable in the
environment and loose effcacy to mount immune response in
sentinels (e.g. Helicobacter, Mouse Hepatitis Virus-MHV). 3)
In case of the contact sentinels, unwanted mating and fghting
with the colony animals are obvious issues (castrated animals
are available to eliminate mating issues but still fghting are
a problem). Contact sentinels, if rotated among several cages
they may actually spread the agent in case of an outbreak. 4)
Regardless of the type of sentinels, it takes at least 4 weeks
exposure to mount effective immune response for antibody
detection. Hence, the sentinel method is not indicative of the
current health status but the status 4 to 12 weeks prior (based
on frequency of sentinel screening). One pragmatic approach
that is being used to overcome the ineffciencies in sentinel
system is to use the littermate or adult female (mother) after
weaning for diagnostics. Typically in quarantine settings
when the concern of pathogen entry is high, we can set up
the breeding with in quarantine and only the weaned litter is
allowed in facility with negative results from full necropsy
of the mother. Of course, this scenario may not be possible
in all settings but is effective wherever possible. Hence, the
facilities using sentinel health monitoring system should
critically evaluate serology data of any positives with
respect to the pathogenesis of the agent to logically arrive
at the current status. This comprehensive understanding is
critical for development of best effective containment and
remediation plan. In addition, the sensitivity and specifcity of
the assays used should be factored in for an in-depth analysis
of the prevalence and incidence of any agent detected.
Cage changing, feeding and watering
of animals
Cage changing is the most common and routine operation
that could pose a threat to disease prevention. Ideally the cage
changing should be performed using microisolation technique
in a biosafety cabinet or in a cage change station with hepa
fltered laminar fow. The best practice would be to use
forceps to transfer mice and precautions should be taken not
to touch the mice or bedding (even with gloved hands). Using
hands for collecting sentinel bedding can facilitate transfer
of agents from cage to cage as the disinfectants used to spray
the gloves are ineffective due to lack of suffcient contact
time in between cages. Hence, using forceps that is dipped
in a disinfectant is the most ideal method. In addition, use of
forceps also prevents the spread of pheromones (minimizing
Bruce and Whitten effects) between cages there by improving
breeding performance. Use of autoclaved cages and bedding
for cage change outs is gaining popularity and could become
the norm in the near future.
Types of diets and quality control
aspects
Mouse diets are broadly classifed in to types based on
ingredients (natural, purifed, or chemically defned) and the
formula (open or closed).
Natural
The most common type of diet from natural ingredients
contain agricultural products and by-products such as whole
grains, mill by-products, high-protein meals (animal and
vegetable), yeast and mined or processed mineral sources
(NRC,1995).
Purifed and Chemically defned diets:
Purifed diets also known as semi-synthetic diets contain
ingredients that represent single nutrients. For example
casein, starch, dextrins, sucrose, glucose, specifc oils,
cellulose vitamins and minerals. Purifed diets are expensive
than natural ingredient diets, but are useful and an absolute
requirement when the exact composition of a diet must be
controlled. In addition, nutritional and toxicological studies
may require purifed diets to eliminate the variability in
results from the low concentration of contaminants and
immunogens. Recently, the concern of phyto-estrogens
from natural ingredient diets and its effects on reproductive
and physiological research is being debated. In addition, the
concern of Bisphenol A contaminant in diet is also being
investigated. Purifed diets could help to reduce such concerns
and variability in studies where ever required.
A chemically defned diet is a type of purifed diet in which
individual amino acids are used in place of a protein source
and specifc fatty acids are used in place of oils. Chemically
defned diets are very expensive are used mainly in amino
acid and fatty acid metabolism studies.
Formula types Open and fxed:
Open formula diets can be made of either natural ingredients
or purifed ingredients, however the formula is fxed i.e.,
the macronutrient source is consistent and it is not variable.
Manufacturers openly publish the exact proportions of
ingredients.
Formula types Closed and variable:
Closed formula diets are always made of natural ingredients
and the proportions/formula is proprietary. Manufacturers
publish the ingredients but not specifc concentrations or
Vol -1, Issue - 1, Jan - Apr 2011 80
formulations. The source of ingredients and proximate analysis
of protein, fat and ash content may be found on these types of
diets. It is important to keep in mind that the macronutrient
source can be variable according to considerations such as a
market price. The protein source is especially liable to vary
in the closed formula diet. For example milk protein may
be substituted for fsh meal. Knapka (1997) has argued that
the varying ingredient inclusion rates in variable formula
diets poses a considerable risk of experimental variability.
Researchers that are concerned about the possible effects of
varying dietary nutrients should use purifed fxed formula
diets or have the relevant nutrients assayed independently
by commercial laboratories. The biology of mouse can
be affected by varying the source of protein. For example
varying the protein source, not the amount, could double the
inducible tumor incidence (Guo et al. 2004). In addition, the
gut micro fora composition and its effect on immunology,
obesity etc. is gaining importance. Variability in diets could
alter the gut micro fora that eventually could lead to altered
research results.
Type of feed based on Physical Form
The most common physical form of feed is pellets and
extrusions (collets). The manufacturing process for both these
forms start the same way: raw food material is ground, sifted
and mixed with vitamin and mineral supplements into a meal.
Pellets:
For pelleting, the meal is mixed with steam raising the
temperature to 65-80 C and gelatinizes the starches, thus
binding the diet ingredients together. Heat also reduces the
microbiological load by about 2 folds in magnitude. The meal
is forced through a die and cut to specifc length. The resulting
pellets are dried so that moisture content typically is about
12%, a level at which free water content is too low to support
growth of microbes. Hence, pelleted feed when protected
from moisture can remain on a shelf for about 6 months
before nutritive loss or growth of mold becomes a concern.
Purifed diets because of the ingredients, for example
simple carbohydrates and casein, are very sensitive to the
temperatures during pelleting process. Hence, to minimize
loss of purifed ingredients, the meal is mixed with water,
pelletized, and then dried at a low temperature (less than 60
C) in a vacuum. Purifed pelleted diets generally have a shelf
life of about 4 months when refrigerated or frozen.
Extrusions:
For extrusions, the meal is ground fner than for pelleting.
Meal is mixed with steam and hot water to a temperature of
80-95 C and extruded under pressure (about 35 atmospheres)
to temperatures of 150C. As steam is trapped within food
during extrusion, the extruded pellets (collets) become honey
combed. The collets are dried by hot air to a moisture content
of 8-12%. Due to the high temperature for this process, the
loss of vitamins is greater, at the same time the mold and
bacterial spores are also destroyed there by facilitating longer
shelf lives than pelleted diets.
Quality control
Decontamination of feed:
The risk of contamination by insects and microbiological
organism is unavoidable in feed manufacture, packaging,
storing and shipping. Anecdotal evidence and personal
communications from facility directors, it has been shown that
mouse parvo out breaks are controlled by switching the feed
to irradiated diet. The scientifc reasoning and data to explain
the mechanism is not available although some studies are
under way. From practical point of view, the ingredients (for
example corn) get stored in silos that are infested with wild
rodents. The parvo virus that could get into the ingredients,
if survived (not known) the manufacturing process could
cause random positivity in the facilities. Irradiating the diets
could eliminate the active infection that cannot be spread to
sentinels. This reasoning seems unlikely but at least points to
the steps and complications in the manufacturing and storage
conditions that could lead to contamination.
The two most common methods used for decontamination of
feed are autoclaving and irradiation. Pasteurization has been
used historically, as this process only eliminates vegetative
bacteria and some spores, this is currently not a popular
method.
Autoclaving:
Autoclaving is the most common method of sterilizing
mouse feed. The process involves sterilizing with steam at a
specifc temperature and pressure for a specifc length of time.
Typically, this process is carried out on site using double-door
autoclaves facilitating the pass through of sterilized feed into
barrier facility. Manufactures produce feed specifcally to be
autoclaved and mark the feed bags accordingly.
Nutrients that are susceptible to damage by heat, moisture, and
oxygen are especially affected by autoclaving. Browning
reactions among amino acids, especially methionine, cysteine
and lysine and between amino acids and carbohydrates, alters
the structure of the amino acids and can substantially diminish
protein bioavailability.
Manufacturers supplement the feeds to be autoclaved with
increased protein content and supplemental amino acids.
Heat labile vitamins (B1, B2, B12, B6 and Pantothenate)
are particularly sensitive to autoclaving; modest losses of
vitamins A, D3 and folate also occur. Manufacturers generally
add additional vitamins to diets that will be autoclaved.
Because of this reason potential toxicity can result if mice
are fed autoclavable feed that is not autoclaved. Autoclaving
also affects the hardness of the pelleted diets which should be
monitored. Clumping of diets specifcally the ones that had
coatings is also another problem evidenced from autoclaving.
Irradiation:
Irradiation is the process of exposing feed to radiation for
the purpose of destroying microorganisms. The radioactivity
damages the genetic material (DNA) that prevents replication.
Vol -1, Issue - 1, Jan - Apr 2011 81
There is no transfer of radioactivity to the irradiated diet during
the irradiation process. This process requires specialized
facilities.
Gamma irradiation is the most commonly used form of
irradiation for diet decontamination. Irradiation at doses
less than 10kGy (radicidation or radurization) is equivalent
to pasteurization. A minimum dose of 21kGy is suffcient
to kill most bacteria, molds and fungi, and is considered a
sterilizing dose (however clostridium and bacillus spores may
require above 30 kGy). Doses at 30kGy may be necessary to
inactivate some viruses (Baldelli, 1967). The sensitivity of
many pathogens to irradiation is provided by the world health
organization (WHO, 1999).
Most mouse diets are irradiated at doses of 20-25kGy which
does not have an effect of protein bioavailability (Eggum,
1979; Ford, 1979) and loss of vitamins are less than 20 percent
(Ford 1979; Isler and Brubacher, 1999). The most concerning
factor in irradiated diets is the free radical induced oxidation
of fats and production of peroxides. The peroxide values
increased 6- 8 fold in a high fat irradiated diet at 25kGy (Ford,
1979). The increase was reduced to 3-4 folds by irradiating
under vacuum (peroxide level continued to increase during
storage).

Provision of Water
Quality of water
Water provided for mice should be free of microbial
contamination. Although there are no set guidelines for safe
water, the common treatment methods used are autoclaving,
acidifcation, hyper-chlorination, reverse osmosis and ultra
violet light exposure.
There is no set standard for the type of water with respect to
laboratory mice. Tap water has wide spread variations, such
as mineral content, general hardness, presence of fuoride.
The generally accepted notion is that if water is good enough
for the community, it is good enough for the laboratory mice.
Water delivery systems
Type of system Advantages Disadvantages
Automatic Individual cages will never run
low.
No sterilization of water
bottles and lids is required.
It requires a capital expense.
System must be maintained to
prevent buildup of mold and
bacteria.
Some mice may need training, to
get used to the system.
It is diffcult to determine water
usage for specifc cages.
Leakage in some systems may
cause mice to drown if cages fll
with water.
Cages must be checked daily for
evidence of leaks.
Glass or plastic bottles in
different confgurations:
Rubber stopper with
sipper tube.
Solid stopper or solid lid
with gasket; hole in bottle
to provide water.
Metal lid with gasket;
sipper hole in the middle
of the cap.
Water usage per cage can be
easily monitored.
Sterilization of bottles and
Lids is easy. High pathogen
protection.
Delivery of medications and
or reagents is easy.
Individual bottles and lids must go
through sterilization process.
Technicians must deal with
individual bottles and check for
air bubbles in sipper tubes (labor
intensive).
Bottles and cages must be
checked daily for any leaks.
Plastic bags/pouches with
water:
Plastic bag punctured with
a sipper stem and encased
in a mouse proof holder.
Water can be stored long
term, treated or untreated.
Disposable bags eliminate
labor and expense to wash
and process bottles.
Bags can be sterilized
separately.
Good for emergency planning.
Capital expense.
Maintenance costs for the
machine and materials in case of
disposable bags.

Vol -1, Issue - 1, Jan - Apr 2011 82
This notion does not recognize the variability in specifc
research studies. The best strategy is to use consistent source
of water and consistent treatment method. The GLP studies
and toxicity studies should pay special attention to the quality
of water as the contaminants could potentially affect the
results. In addition, micro fora of the gut could be altered with
quality of water, an issue that is gaining popularity. Regardless
of the source and treatment method, the delivery mechanism,
for example, bottle, auto water, pouches etc. should be cleaned
and sterilized for maintaining overall quality of the program.
Conclusions
Although we talked about several aspects, this certainly is
not comprehensive information and each topic can be a book
chapter by itself and the reader is recommended to explore
additional resources for a thorough understanding as needed.
In addition to these aspects there are several other areas for
example facility design, traffc patterns, cage wash setup,
decontamination procedures, animal procurement, quarantine
and rederivation facilities, handling-identifcation and basic
procedures, security and disaster planning etc. which also
are an integral component of any animal care program. From
this, it should be understood that animal facility management
is complex and requires knowledge and expertise in several
aspects, eventually facilitating valuable research studies in
physiologically normal animals handled in a humane way.
Acknowledgements
The Jackson Laboratory Handbook on genetically standardized
mice Sixth edition by Kevin Flurkey and J oanne M. Currer
(eds), has been the primary source for the information, which
is also added as a reference. I also extend my deepest gratitude
to Dr. S.G Ramachandra for helping me with the review,
format and editing of the content.
References
Eggum BO (1979). Effect of irradiation on protein and amino
acids in laboratory rodent diet, Decontamination of
animal feeds by irradiation. International Atomic Energy
Agency, Vienna. pp. 55-67.
Ford DJ (1979). Observations on the infuence of irradiation
on fat and vitamin A in dry laboratory cat diets,
Decontamination of animal feeds by irradiation.
International Atomic Energy Agency, Vienna. pp. 77-81.
Guo JY, Li X, Browning JD Jr, Rottinghaus GE, Lubahn
DB, Constantinou A, Bennink M, MacDonald RS
(2004). Dietary soy isofavones and estrone protect
ovariectomized ERalphaKO and wild-type mice from
carcinogen induced colon cancer. J Nutr. 134:179-182.
Isler D, Brubacher D (1999). Effect of sterilization on
vitamin retention in laboratory animal feed. Proceeding
of the International XII ICLAS and VII FELASA Joint
Meeting. Tur-Mari JA, Orellana-Mureiena JA (eds).
London, Laboratory Animals Ltd. pp. 242-248.
Kevin Flurkey and Joanne M. Currer (eds) (2010):
The Jackson Laboratory Handbook on genetically
standardized mice Sixth edition (Primary Source)
Knapka JJ (1997). Natural-ingredient diets: managing the
variation in dietary nutrient concentrations. Lab. Anim.
26:40-42.
National Research Council (1995). Nutrient Requirements of
Laboratory Animals, Fourth Revised Edition. National
Academy Press, Washington, DC.
Reeb CK, J ones RB, Bearg DW, Bedigian H, Paigen B
(1997). Impact of room ventilation rates on mouse cage
ventilation and microenvironment. Contemp. Top. Anim.
Sci. 36:74-79.
Wade AE, Holl J E, Hilliard CC, Molton E, Greene FE
(1968). Alteration of drug metabolism in rat and mice by
an environment of cedar wood. Pharmacology. 1:317-
328.
Weichbrod RH, Cisar CF, Miller J G, Simmonds RC, Alvares
AP, Ueng TH (1988). Effects of cage beddings on
microsomal oxidative enzymes in rat liver. Lab. Anim.
Sci. 38:296-298.
White, WJ (2007). Management and Design: Breeding
Facilities, in The Mouse in Biomedical Research,
Vol.III, Normative Biology, Husbandry, and Models.
2
nd
Edition. Fox J G et al. (eds). American College of
Laboratory Animal Medicine Series; Academic Press,
Elsvier, Burlington, MA.
Vol -1, Issue - 1, Jan - Apr 2011 83
Dr. Jessica N. Keen received her MS degree in Biological Sciences in 2002 from
the University of South Alabama in Mobile, AL. She received her DVM degree in
2006 from Auburn University College of Veterinary Medicine in Auburn, AL. After
graduating from veterinary school, Dr. Keen worked for the USDA-APHIS-VS as a
Veterinary Medical Ofcer and Provisional Designated Scrapie Epidemiologist for
New York. In 2008, Dr. Keen entered a two year residency program in laboratory
animal medicine at the University of Rochester Medical Center in Rochester, NY. Dr.
Keen serving as a facility veterinarian at Marshall BioResources since the comple-
tion of her residency in June 2010.
Keen J.
Production Facility of Purpose-bred Beagle dogs
Abstract
The dog, Canis lupus familiaris, has been domesticated for several thousand years and has been used as a
research animal since the early 1600s (Fox et al. 2002). Dogs are often docile, friendly animals, especially
when they are well-socialized. For a production facility of purpose-bred dogs, it is important to provide high-
quality, well-adjusted, healthy animals that can readily transition to a variety of different research environ-
ments. Animal welfare is the cornerstone of an effective program and benefts all parties involved. The goal
of this paper is to provide the reader with a general view of the practices and animal care as it relates to a high
volume, purpose-bred beagle production facility in the United States.
Key words : beagle dog, production, management
Dr. Keen J, Marshall BioResources, 5800 Lake Bluf Road, North Rose, New York, USA
Phone : 315-587-2295, Fax: 315-587-2109, email : infous@marshallbio.com
Introduction
The domestic dog, Canis lupus familiaris, is a mammal of the
order Carnivora, family Canidae and thought to be a descendent
from the grey wolf (Fox et al. 2002). Dogs have been widely
used in biomedical research due to their docile nature, biologic
features and reasonable size. Dogs, particularly beagles,
have been involved in research that includes cardiovascular
pharmacology, alternative drug delivery systems, drug safety
assessment, dental and periodontal disease research and
surgery, radiation oncology and skeletal physiology (Fox
et al. 2002). Much of this research has benefted not only
humans, but dogs as well, thereby advancing therapeutic and
diagnostic techniques for veterinary medicine (NRC, 1994).
Husbandry
Housing Facilities: Marshall BioResources follows the federal
regulations as set forth in the Animal Welfare Regulations
(AWR, 2008).

Facilities that house dogs are designed and
constructed in a manner such that they are structurally sound,
protect animals from injury, contain them securely and restrict
other animals from entering (AWR, 2008; ILAR, 1996).
According to the AWR, the housing facilities at Marshall
BioResources are considered to be sheltered housing facilities
(AWR, 2008). This type of housing protects the animals from
the elements and temperature extremes at all times (AWR,
2008). All buildings housing dogs are enclosed, insulated,
heated and ventilated. Each one is composed of four walls,
a roof and a foor. The fooring within each building may be
composed of either earth or concrete depending on the year
the structure was built. Those structures with earth fooring
have asphalt walkways between each row of pens. A thick
layer of sand is spread underneath the row of pens in order to
trap waste and odors. Those structures with coated concrete
fooring have a fushing system to remove waste and odors.
Translucent panels are located along the length of the top of
two walls to allow natural light to enter. Adjustable, solid
panels are located along the bottom of the walls so that they
may be opened or closed in order to maintain the interior
temperature, humidity and air quality as appropriate.

Keen J and Jasmin B.
Marshall BioResources, New York, USA
Vol -1, Issue - 1, Jan - Apr 2011 84
Beagle dog production facility, Keen J.
Environmental Control:
Dogs are able to tolerate a reasonable range of environmental
conditions as long as they are provided appropriate food,
water, shelter and time to acclimate to their environments.
Facilities housing dogs are suffciently heated and cooled
as necessary to protect animals from extreme temperatures
(AWR, 2008). The ambient temperature is not allowed to fall
below 45 F (7.2 C) or rise above 85 F (29.5 C) for more
than four consecutive hours when dogs are present (AWR,
2008). Our facilities are adequately ventilated to minimize
odor levels and provide for the health and well-being of the
dogs. Auxiliary ventilation is employed as necessary by using
fans, opening moveable side panels, or air-conditioning as
appropriate for the building. Translucent panels and artifcial
lighting is provided so that the dogs are maintained on a
normal diurnal light cycle. The lighting is maintained at a
suffcient level to allow for adequate housekeeping practices
and daily observation of the dogs (AWR, 2008).
Primary Enclosures:
Each building consists of several rows of individual pens
that are suspended above the ground for housing dogs. The
interior surface of each pen is composed of materials that are
impervious to water and easily sanitizable. The fooring within
each pen consists of expanded metal in a diamond-shaped
pattern. We feel this design offers a higher level of sanitation
in the primary animal enclosure because waste is allowed to
fall through to the ground surface beneath each pen. Any
solid waste that does not fall through the fooring is removed
daily from the pens and as needed to provide the dogs with a
cleaner primary enclosure. Most animal behaviorists agree
that approaching a dog from above represents a threatening
stance. This suspended cage design allows the dogs to be at a
similar level to the staff which may have an added beneft of
reducing any perceived threat by the dogs.
Dogs at Marshall BioResources are housed in socially
compatible groups as appropriate. Each pen is constructed
of suffcient size and strength to accommodate the number
of animals within it. All dogs within the enclosure are able
to move about freely, stand, sit and lie in a normal position
(AWR, 2008). The minimum interior height of the primary
enclosure is required to be at least six inches higher than the
head of the tallest dog standing in a normal position within
the enclosure. The minimum foor space per dog is calculated
according AWR standards (AWR, 2008). In order to provide
additional foor space and increased enrichment, our newer
cage designs employ a tiered system. This system includes
a ramp to a second level where the dogs may walk up and
down as they like. In addition to social housing and daily
human interaction, dogs are also provided various toys as
additional forms of enrichment. All toys are constructed of
sturdy materials that are easily sanitizable, appropriately sized
for the age of the dogs and replaced as needed.
Sanitation:
Proper cleaning should promote the health and well-being of
the animals. Marshall BioResources follows the requirements
as set forth in the AWRs (AWR, 2008). All primary enclosures
are cleaned daily to remove waste that has not fallen through
the pen fooring. The primary enclosures are sanitized at least
once every two weeks using appropriate methods (AWR,
2008). Waste that accumulates under the pens is removed
at timely intervals to minimize the attraction of pests and
odors. A dynamic pest control system is also in place year-
round in order to minimize pests. Dogs are removed from
the primary enclosure prior to sanitizing. Modifcations to the
pen sanitizing schedule may be necessary for whelping and
nursing dogs so as to minimize any unnecessary disturbances.
However, cleanliness of the primary enclosure during these
critical times is of utmost importance for the health of the
neonates (NRC, 1994).
Dietary Requirements
Dogs should be fed a high-quality canine diet that is
nutritionally balanced, free of contaminates, appropriate for
the age of the animal, and is available in suffcient amounts
to maintain normal body condition and weight (AWR,
2008). Feed must be provided at least once daily, unless
otherwise approved by the Institutions Animal Care and Use
Committee (IACUC) or the equivalent, or for health reasons
as approved by the attending veterinarian or his/her designee.
At Marshall BioResources, dogs are provided ad libitum
access to feed. Large quantities of feed are delivered in bulk
trucks and pumped into numerous dry feed storage silos. The
silos are connected to buildings via plastic pipes with moving
internal augers which deliver the feed to drop tubes extending
into each cage. Each silo will last approximately 2-4 weeks
depending on the size and number of buildings being supplied.
Each load of feed is accompanied by a guaranteed analysis.
Annual testing by an independent lab is conducted along with
a triennial vendor audit of the feed plant.
The energy requirements for breeding and nursing dogs are
different from the standard maintenance diet. Premium diets
with 30% protein and 20% fat are used for active breeders.
If potable water is not provided ad libitum to dogs then it
should be offered not less than twice per day for at least one
hour each time (AWR, 2008). Municipal water is provided
ad libitum to all dogs in our facility via an automated water
system with lixit style water sippers in each cage. The water
is tested by the municipality and quarterly by the New York
State Department of Health (NYSDOH). Watering devices
Vol -1, Issue - 1, Jan - Apr 2011 85
are checked daily and fushed weekly to ensure they are
working properly.
Behavior and Handling
Social Behavior:
The social unit of the dog is the pack. Within the pack there
is a dominant social hierarchy structure. Often, dogs will
accept humans as their dominant individual provided they
receive appropriate positive reinforcement (petting, feeding,
gentle handling, etc.) and minimal negative experiences.
Staring directly into a dogs eyes or leaning over them may
be perceived as threatening and should be avoided, especially
in cases where the dogs fnd themselves in a new, unfamiliar
environment. Dogs should be housed in socially compatible
groups and have positive daily interaction with staff. Personnel
are better suited to work with dogs if they understand the
dogs behavior, social structure and importance of positive
reinforcement.
Handling:
Proper handling and restraint of the dog is necessary to ensure
the safety of the animals and personnel involved. Handling
and restraint should be performed in a manner that does not
cause distress, physical harm or discomfort to the dogs. Small
to medium-sized dogs, such as the beagle, can be effectively
carried by placing a hand under the chest and abdomen,
holding the dog close to your body and restraining the head if
needed. Dogs can also be trained to walk on a leash if desired.
Health Care
Immunization:
Marshall BioResources dogs are vaccinated for protection
against common canine diseases including the following:
canine distemper, parvovirus, adenovirus type 2,
parainfuenza, leptospirosis, bordetella, canine papilloma type
1 and 2, and rabies. Immunization of the pups is initiated at
appropriate intervals. Immunization may be tailored based on
customer request. Any preventive medicine program should
be based on potential risk of exposure to endemic pathogens
and regulatory requirements in ones jurisdiction.
Physical Examination:
Routine physical examinations are part of a comprehensive
health maintenance program for Marshall BioResources
dogs. The normal canine body temperature range is between
99.5 -102.5 F (37.5 C - 39.2 C). The heart rate averages
approximately 70-180 beats per minute. The normal
respiratory rate is 20-40 breaths per minute (Fox et al. 2002).
Basic normative biological parameters for dogs such as blood
chemistry, hematology, and coagulation profles are well
published. The reader may refer any standard veterinary text
book. Establishing in-house normative values may also be
desirable (Field and Jackson, 2007).
Routine preventive anthelmintics are administered to
control parasites. Regular fecal and blood sample testing
from different age groups monitor the effectiveness of the
anthelmintic program and profles the health status of the
colony. Marshall BioResources dogs are free from helminth
parasites, heartworm and brucellosis.
At approximately 6 months of age, blood is drawn from all
beagles and analyzed for complete blood count (CBC) on
an Advia hematology analyzer. The most recent CBC and
differential values are printed on the individual beagle history
that accompanies each dog to our customers. Diagnostic data
are used to monitor the colony and individual dog health.
Common Diseases
The most common clinical disease entities seen in our animals
are loose stools in post weaning age pups and upper respiratory
signs in juveniles. Diarrhea does not correlate with a specifc
enteric pathogen, and weaning stress has been determined to be
the single most important factor in its occurrence. The upper
respiratory syndrome is a typical kennel cough (Bordetella
bronchiseptica) presentation with increased lung sounds,
mucoid nasal discharge and mild depression. Signs may be
mild and self-limiting, but sometimes requires symptomatic
Vol -1, Issue - 1, Jan - Apr 2011 86
treatment with oral antibiotics for rapid resolution and to
decrease the likelihood of potential opportunistic secondary
bacterial pneumonia.
Reproduction
Breeders go through extensive diagnostic, temperament and
pedigree screenings prior to acceptance in the breeder pool.
A full-time, on-site genetic analyst is charged with genetic
monitoring. A proprietary computer program was designed
and provides a list of studs to be mated to each female in
estrus based on quality statistics and optimal out-breeding.
Breeders are kept on a high quality diet to ensure optimal
condition. Dogs are mated beginning with their frst estrus
and continued for each subsequent estrus. Estrus is detected
visually. Breeding females are group-housed. Each female
is brought to a stud when she is ready to mate. Females are
mated to the stud at least two times and three times if possible.
Each mating is recorded.
Dams are moved into the whelping building and singly-
housed approximately two weeks prior to parturition.
Bedding and heat lamps are provided in each pen. A smaller
partition is located within the pen to provide a nesting area.
Technicians are on duty twenty-four hours, seven days each
week to monitor whelping dams and new-born pups. All
newborns are examined for any defects and navels are dipped
in iodine. Newborn pups are then weighed and monitored to
ensure successful nursing. Dams must be observed for any
mistreatment of pups.
Birth weights of the pups are important statistics for
evaluating breeders prior to their next estrus. Unique events
such as illness, treatment or injury are written on the pups
individual card. In these cases, the pup receives a microchip
or an ear tag for identifcation. According to the USDA
regulations, all weaned dogs must be individually identifed
by means of an appropriate dog tag or permanent tattoo
(AWR, 2008). Animal numbers must not be reused within 5
years (AWR, 2008). Weaned puppies, approximately 8 weeks
of age, are tattooed in the right ear with a unique seven digit
number. Identifcation numbers are never reused at any of
our facilities.
Euthanasia
Humane euthanasia is performed according to the American
Veterinary Medical Association (AVMA) Guidelines on
Euthanasia (AVMA, 2007). Euthanasia is performed by
intravenous administration of barbiturate euthanasia solution.
If intracardiac euthanasia is required, the animal must be
heavily sedated, anesthetized, or comatose (AVMA, 2007).
Conclusions
The domestic dog has been a valuable research model since
the early 1600s. With appropriate socialization and positive
human interaction, dogs readily accept a wide variety of
manipulations that may be encountered in a research setting.
Animal production involves several aspects, but most
importantly, animal welfare is of the utmost concern. Proper
husbandry practices throughout all life stages will ensure the
health and well-being of the animals. A structured preventive
health care program along with daily observation of all dogs
for any signs of illness or injury improves the quality of the
animals available for research. The information provided
here is only a cursory review of the current practices and
animal care as it relates to a purpose-bred beagle production
facility. The reader is encouraged to seek out additional texts
and guidelines on more specifc topics, as appropriate, in
order to better understand the beagle as a research subject.
References
Animal Welfare Regulations (2008). 9 CFR 1-4.
American Veterinary Medical Association (AVMA)
Guidelines on Euthanasia (2007).
Field G, Jackson TA (2007). The Laboratory Canine. Boca
Raton (FL): Taylor and Francis Group.
Fox JG, Anderson LC, Loew FM, Quimby FW ( 2002).
Laboratory Animal Medicine 2nd Edition. San Diego
(CA): Academic Press.
Institute for Laboratory Animal Research (1996). Guide
for the care and use of laboratory animals. Washington
(DC): National Academies Press.
National Research Council (1994). Laboratory Animal
Management: Dogs. Washington (DC): National
Academy Press.
Beagle dog production facility, Keen J.
Vol -1, Issue - 1, Jan - Apr 2011 87
Satish T Panchal received BVSc and MVSc (Pathology) from Anand Agricultural
University, Anand, Gujarat. He is extensively involved in the feld of laboratory
animal medicine, toxicology and pathology since 12 years. He worked in various
organizations viz. Jai Research Foundation, Torrent Research Center and Alembic
Research Centre prior to joining Sun Pharma. Currently, working as group leader at
the Department of Biological Research Toxicology, Sun Pharma Advanced Research
Co. Ltd., Vadodara.
Satish T. P.
Environmental enrichment for laboratory animals
Abstract
Laboratory animals (Rat, Mice, Rabbit, Guinea pig and Dog) are extensively used in pre-clinical
safety and effcacy research over several decades. Their use has led to signifcant contribution in
drug discovery and development which ultimately has helped in fnding cure for several human
diseases. Hence, it is necessary to care the laboratory animals with the highest standards to ensure
the scientifc data obtained from these animals is not miss represented. Presently followed housing
in research setup provides little for their behavioral and physiological needs, which compromises
the welfare of the animals and can affect the validity of research results if stress has affected the
animals. Environmental enrichment (EE) is the alteration of an animal's housing environment to
create an opportunity to express species-typical behavior, which will reduce abnormal behaviors.
The EE will also enhance animal welfare. In this review, various types of EE of laboratory species is
discussed with an aim to integrate them into routine laboratory animal husbandry.
Keywords: environmental enrichment, welfare, stress, animal behavior
Satish P, Department of Toxicology, Sun Pharma Advanced Research Co. Ltd.
907/4, GIDC Makarpura, Vadodara 390010, Gujarat
Phone : 09825319421, email : satish.panchal@sunphrama.com
Introduction
Environmental enrichment is defned by Beaver (1989) as
additions to an animals environment with which it can
interact. Laboratory animals spend most of their lives in their
cage so any positive change in the environment of home cage
can lead to improvement in overall enrichment and welfare
of animals. Hence, EE is a key component of animal welfare
since it infuences the animals overall well-being, reduces
stress, provides opportunities for activity, and encourages
appropriate behaviors (Sparling et al. 2009). Several aspects
of rodent enrichment have received a signifcant amount of
attention during the last few years, which has lead to great
advances in developing a benefcial enrichment program
(Galef, 1999; Hutchinson et al. 2005). Restriction of
opportunities for physical activity in the currently used
standard cages for housing laboratory animals often results
in inactive and obese animals, which are more prone to stress
and anxiety. Exercise benefts in improving animal health by
positively affecting animal welfare, hence system which can
enhance animals physical activity would be benefcial form
of EE. Physical activity can offset the deleterious effects of
a sedentary life combined with overeating and may prevent
stress-induced immune suppression and consequent disease.
Animals under experimental condition should have freedom
from hunger, thirst, discomfort, pain, injury, disease, fear and
distress and should have freedom to express normal behavior.
This can be accomplished only by providing ready access
to feed, water, appropriate environment, prevention or rapid
diagnosis and treatment, suffcient space and facilities and
company of animals own kind (Moraska and Fleshner, 2001).
Satish T.P.
Department of Toxicology, Sun Pharma Advanced Research Co. Ltd.
Vadodara 390010, Gujarat, India
Vol -1, Issue - 1, Jan - Apr 2011 88
Humans are also part of social environment of laboratory
animals and handling the animals is a very important aspect
of the daily care and use of animals. It is also benefcial to
train animals to become used to routine care and management
practices. Most of the animal models are social animals
in nature and get benefted from the living association of
same species or humans. Welfare of animals is improved by
interactions among animals. Animals gain variety of experience
during the growing phase which usually determines the social
behavior of animals and hence conditions of animal holding
in a breeding colony will affect on the animals future well-
being. Vaious aspects of enrichments viz. social environment,
nutritional environment and sensory environment of various
laboratory species are described below.
Enrichment for rodents
Enough considerations should be given for environmental
factors like lighting, heating, ventilation and caging to fulfll
social needs of rodents (rat and mice) used in research,
teaching, or testing. In the case of individually housed
animals, daily observation gives other form of social contact
for the animal and will also facilitate easy handling in that
the animal becomes familiar to the human presence. Group
housing in mice (especially for breeders) will be successful
if the social arrangement is limited to one male with more
than two females and monitored for pecking order. Female
mice from the same litter can be group housed (Wolfensohn
and Lloyd, 1998). However, this may not be always possible
for male mice as they often exhibit increasing aggression and
dominance behavior (Brain, 1992), interestingly male rats
unlike male mice easily get used to group housing situations
(Barnett, 1975). Several evidences indicate that individually
housed mice may have a poor immune system and developed
tumors faster when singly housed (Schwartz et al. 1974; Riley,
1981). Therefore, when feasible, rodents should be housed
socially in compatible groups. Hierarchy and relationship
between dominant and submissive individuals should be
given consideration while group housing mice.
Depending upon the involvement of strain, subordinate
mice can be killed in group housing, hence it is necessary to
routinely observe for signs of incompatibility and separate
the mice in question. For deciding the degree of socialization,
strain specifc variations in behavior and vendor should also
be evaluated (Haemisch & Grtner, 1994). Wistar rats housed
in group of three or less revealed reduced stereotypic behavior
compared to individually-housed animals (Hurst et al. 1997;
1998). Individually housed rats which can smell, see and hear
nearby housed animals also seem to be benefted. In some
studies individually housed animals had abnormal levels
of cholesterol, triglyceride and glucose (Perez et al. 1997).
Rodents get highly aggravated to make use of food item as
enrichment. In the wild, rodents spend a major portion of time
searching for food whereas under laboratory condition food is
mostly provided ad libitum and is easily available to animals.
Food items scattered in the cage gives animal an opportunity
to forage and prevents boredom as in nature. It has been found
that rats preferred earned food though free food was available
(Carder and Berkowitz, 1970). Rodents are nocturnal and
sleep during the day. Also, albino rodents (unpigmented eyes)
are susceptible to retinal degeneration if exposed to higher
light intensities than normally recommended in animal rooms.
The light intensity experienced by the rodents is reduced
by the presence of wire bar lids, food, water bottles, and
microisolator tops. It can also be further decreased by use of
reduced lighting, with timer-controlled supplemental lighting
when people are working in the room; by use of opaque cages;
by not housing animals on the top shelf near the light source;
and by providing nesting material or a nest box so the rodents
can move away from the light (Mortell, 2001).
Enrichment for dogs
In case of dog, benefts of socialization with other dogs
and care takers have been demonstrated experimentally
(Hubrecht, 1993). Abnormal behavior such as fearfulness,
aggression due to fear, hyperactivity, immobility and timidity
are observed in case pups are not adequately socialized during
the sensitive period of their life i.e. 4-14 weeks of age (Scott
et al. 1967). Such pups suffer from isolation syndrome later
in life and do not make good research subject due to the
distress experienced by them while performing common
laboratory procedures. Physiological parameters monitoring
of such animals may fall outside normal limits (Vanderlip et
al. 1985). Socially-housed dogs interact with kennel-mates
and spend more time sniffng the pen, probably as a result
of the increased diversity of scents (Hubrecht et al. 1992),
and considerable reduction in stereotypic circling is evident,
as compared to individually-housed animals. Individually
housed dogs vocalize more, sleep less and exhibit more
stereotypy than group-housed dogs (Hetts et al. 1992), and it
was concluded that social isolation might well be even more
harmful than space limitations. The maximization of visual
contact with other dogs, pair housing, periods of exercise
are all considered benefcial. In addition, it also seems that
proximity with humans, by means of grooming, handling
and petting while performing any procedures, are important
aspects of human interactions in improving the wellbeing
of laboratory dogs. In many kennels, cages are very simple
without any complexity, and in some countries it is still legal
to house dogs in small cage. It is doubtful that such small
cage will provide dogs physiological and psychological
needs. Housing system should allow the dog to maneuver or
masticate safe objects, and provide opportunities for human
and dog interaction (Hubrecht, 1993). Pens/cages should
be divided into sleeping and exercise areas which provide
complexity, allows the dog to defecate/urinate away from
its resting area (Fox, 1986). Solid partitions between cages
provide seclusion and prevent injuries, but can detach the dog
from its surrounding environment. A good pen design should
allow the dog to fulfll its inquisitiveness about events taking
place outside its home cage.
A provision of platform can be made at a height that allows
the dog to see over the partitions while lying down (Hubrecht,
1993). Sometimes, dogs have to be housed individually for
research or quarantine purposes, in which case greater care
should be taken to provide extra human interaction time and
an enriched environment. Animal welfare regulation requires
that dogs over three months old (except lactating and nursing
females with pups) must be given exercise regularly if they
Environmental enrichment, Satish P.
Vol -1, Issue - 1, Jan - Apr 2011 89
are housed individually in an area that is less than two times
required foor space. Group housed dogs do not require
exercise provided total foor space equals the sum of required
spaces for the dogs if housed individually. Forced exercise
programs are not considered ethical. If group of dogs are
released together for exercise, group should remain as stable
as possible because acceptance of new dog in group varies
and group may attack new member. It is also necessary to
frequently observe the pack to ensure safety of the group.
Interestingly, presence of one or more persons in exercise area
helps achieving positive social interaction. Indoor-outdoor
pens are an option in some cases and offering dogs access to
the outside is an enriching experience for them. Dogs are very
motivated by toys or chews that have an appetizing aroma
or taste; therefore, it receives a lot of attention. Such items
increase their active period and decrease destructive behavior.
Enrichment for rabbits
Social organizations of the rabbit are infuenced by how they
are housed in experimental settings. It is reasonable to suggest
that they should be housed in groups since wild rabbits live
in groups containing at least one other rabbit of the same
sex (Cowan, 1987), except matured males (Gunn & Morton,
1995). Irrespective of type of housing used for rabbits,
occasionally they should be provided exercise and social
interaction which may allow them to have control over their
immediate environment (Batchelor, 1991). Floor pen housing
allows easier way to achieve contacts with others, wherever
cages are used, group housing of compatible animals in a
large cage is recommended. Socialization has been reported to
reduce abnormal physiological and psychological behavior. If
group housing is followed, cage furniture should be designed
in such a way that animals have some form of refuge. Group
housing also facilitates normal behavior, such as reciprocal
grooming and chasing, jumping, rearing but groups should
not be composed of more than 6-8 mature animals since
monitoring turn out to be diffcult (RSPCA, 1993). The cages
should be large enough to allow rabbits to sit on hind legs
and lie down with full stretch (Gunn & Morton, 1995) and
should have visual contact with other rabbits. If they are
grouped at younger age they tend to get adjusted to the social
group better whereas older animals, particularly males have
tendency to fght and intimidate younger ones (Davys, 1994).
Since they are social in nature, they mix well at an early age;
there may be problems with removal or replacement of adults
in an established group (Love and Hammond, 1991). As
enrichment, rabbit should be provided with woodchip litter
or shredded paper or straw bedding. Rabbits prefer straw or
shredded paper and avoid sawdust or wood shavings (Turner
et al. 1992). Hay should be provided for foraging and building
nest and nest boxes for females under reproduction, designed
in such a way that it makes impossible for delivered females
to see each other and trigger infanticide behavior. Shelves may
be provided to offer comfortable resting and refuge places.
Rabbits generally spend about 20% of the time gnawing hard
objects like wood sticks (Stauffacher, 1992). Rabbits get
easily frightened and react to environmental noises, hence
care takers and experimenters should work calmly and quietly
in rabbit rooms. For enrichment and supply roughage, rabbits
may be supplied variety of food treats.
Enrichment of G.pigs
Guinea pig is a social animal and hence should be always
housed in group unless there is a veterinary or experimental
requirement. To avoid aggression and stress, group should
be as stable as possible with adequate space for housing and
nursing. Females can be kept in groups and also males (till
four months age) whereas housing in pair is not recommended
for boar above four months of age. To avoid the social tension
due to presence of several males, a male should be housed with
3-4 females and pups should be removed at two - three weeks
to prevent overcrowding. Adult animals can be housed in the
unisex group provided that male groups have no olfactory
or visual contact with females. Smell of female urine turns
the friendliest males into enemies and will not tolerate each
others presence. Exchanging males for breeding purposes are
easily accepted by females without any aggression similarly
strange female can also be easily introduced without any
aggression (Raje & Stewart, 2000) as long as none of them
is nursing. Cage design should be such that they have place
of refuge to improve their welfare. Place of refuge is utilized
by them for sleeping together and for pregnant females to
deliver (Gray, 1988). They do well on pelleted diet but for
enrichment, fresh hay can be supplemented and animal
express great pleasure in eating and burrowing inside the hay
(Sutherland and Festing, 1987). To prevent G. pigs developing
overgrown front teeth, they must be engaged in regular
gnawing behavior by providing hard food pellets, carrots and
softwood sticks (Scharman, 1991). Guinea pigs get easily
frightened and react to environmental noises and hence care
takers and experimenters should work calmly and quietly in
Guinea pig rooms.
Summary
Any modifcation in the environment of the laboratory animal
that enhances its psychological and physiological well being
by providing stimuli, meets animals species-specifc needs.
Fulflling animals need is not equal to going back to nature/
wild or to bring wild behavior in laboratory but is to bring
crucial features of wild environment into the laboratory e.g.
foraging. Social needs of animal can be met by housing them
with conspecifcs and by providing good care takers whereas
physical needs can be fulflled with the provision to rest,
explore, groom, dig, nest or hide. Other needs of animals like
eating, foraging, light, temperature, humidity, defection and
urination should also be appropriately met. Several researches
conducted in past supports that EE in the form of nesting
material and foraging behavior and physical activity or
exercise are effective in enhancing the welfare of laboratory
animals. More research is still required in establishing
feasible and cost effective EE methods which does not affect
the research and at the same time improves animal welfare.
This review presents a fair scientifc evaluation of important
topic like this and it will be helpful in developing enrichment
programs and in planning the studies that produce positive
effects on laboratory animals welfare and research outcomes.
Vol -1, Issue - 1, Jan - Apr 2011 90
References
Barnett, S (1975). The Rat. University of Chicago Press,
Chicago.
Batchelor GR (1991). Group housing on foor pens and
environmental enrichment in sandy lop rabbits (I).
Anim. Techno. 42, 109-120.
Beaver BV (1989). Environmental enrichment for
laboratory animals. ILAR News. 31(2): 5-11.
Brain PF (1992). Understanding the behaviours of feral
species may facilitate design of optimal living
conditions for common laboratory rodents. Anim.
Techno. 43: 99-105.
Carder B, Berkowitz K (1970). Rats preference for earned
food in comparison with free food. Sci. 169:1273-1274
Davys JS (1994). The foor pens for laboratory animals a
mixed blessing? Anim. Techno. 45 : 95-100.
Fox MW (1986). Laboratory Animal Husbandry: Ethology,
Welfare, and Experimental Variables. State University
of New York Press: Albany, NY. pp. 1-98.
Galef BG Jr. (1999). Environmental enrichment for
laboratory rodents: animal welfare and the methods of
science. J. Appl. Anim. Welf. Sci. 2(4):267-280.
Gray G (1988). Guinea pigs. Humane Innovations and
Alternatives in Animal Experimentation 2. 48-49.
Gunn D, Morton DB (1995). Inventory of the behaviour of
New Zealand White rabbits in laboratory cages. Appl.
Anim. Behav. Sci. 45: 277 292.
Haemisch A, Grtner K (1994). The cage design affects
intermale aggression insmall groups of male
laboratory mice: strain specifc consequences on social
organization, and endocrine activations in two inbred
strains (DBA/2J and DBA/J). J. Exp. Anim. Sci. 36 :
101-116.
Hetts S, Clark JD, Calpin JP, Arnold CE, Mateo JM (1992).
Infuence of housing conditions on beagle behaviour.
Appl. Anim. Behav. Sci. 34:137-155.
Hubrecht RC, Serpell JA, Poole TB (1992). Correlates of
pen size and housing conditions on the behaviour of
kennelled dogs. Appl. Anim. Behav. Sci. 34:365-383.
Hubrecht RC (1993). A comparison of social and
environmental enrichment methods for laboratory
housed dogs. Appl. Anim. Behav. Sci. 37: 345-361.
Hurst JL, Barnard CJ, Nevison CM, West CD (1997).
Housing and welfare in laboratory rats: welfare
implications of isolation and social contact among
caged males. Anim. Welfare. 6: 329-47.
Hurst JL, Barnard CJ, Nevison CM, West CD (1998).
Housing and welfare in laboratory rats: welfare
implications of isolation and social contact among
caged males. Anim. Welfare 7: 121-36.
Hutchinson E, Avery A, Vandewoude S (2005).
Environmental enrichment for laboratory rodents. ILAR
J. 46(2):148-161.
Love JA, Hammond K (1991). Group-Housing Rabbits. Lab.
Anim. 20 (8): 37 -43.
Moraska A, Fleshner M (2001). Voluntary physical activity
prevents stress-induced behavioural depression and
anti-KLH antibody suppression. Am. J.Phy.Reg.
Integra. Comp. Physiol. 281: 484-489.
Mortell N (2001). Practical environmental enrichment for
rats and mice: the results of a survey. Anim. Technol.
52: 1-17.
Perez C, Canal J R, Domimgues E, Campillo J E, Guillen
M, Torres MD (1997). Individual housing infuences
certain biochemical parameters in the rat. Lab. Anim.
31: 357-61.
Raje SS, Stewart KL (2000). Group housing female guinea
pigs. Lab. Anim. 29(8) : 31-32.
Riley V (1981). Psychoneuroendocrine infuences on
immunocompetence and neoplasia. Sci. 212: 1100-1109.
Scharmann W (1991). Improved housing of mice, rats and
guinea pigs: A contribution to the refnement of animal
experiments. ATLA, 19: 108-114.
Schwartz R, Sackler A, Weltman A (1974). Adrenal
relationships and aggressiveness in isolated female
mice. Experientia. 30: 199-200.
Scott JP, Shepard JH, Werboff J(1967). Inhibitory training
of dog: Effects of age at training in basenjis and
shetland sheepdogs. J. Psychol. 66(2):237-252.
Sparling JE, Mahoney M, Baker S, Bielajew C (2009). The
effects of gestational and postpartum environmental
enrichment on the mother rat: A preliminary
investigation. Behav. Brain Res. 208(1):213-23.
Stauffacher M (1992). Group housing and enrichment cages
for breeding, fattening and laboratory rabbits. Anim.
Welfare. 1: 105-125
Sutherland SD, Festing MFW (1987). The Guinea-pig. In
The UFAW Handbook on the Care and Management
of Laboratory Animals, Sixth Edition Poole TB (ed),
Churchill Livingstone, New York, NY. pp 393-410.
Turner RJ , Selby J I, Held SDE, Howells KJ , Eveleigh J R,
Wootton RJ (1992). Preferred substrates for penned
laboratory rabbits. Anim. Technol. 43: 185-192.
Vanderlip SL, Vanderlip JE, Myles S (1985). A socializing
program for laboratory-raised canines. Part 2: The
puppy socialization schedule. Lab. Anim. 14(2):27-32.
Wolfensohn S, Lloyd M(1998). Handbook of laboratory
animal management and welfare. 2nd ed. Blackwell
Science. Oxford, United Kingdom.
Environmental enrichment, Satish P.
Vol -1, Issue - 1, Jan - Apr 2011 91
Honours and Awards bestowed on LASA members
The following members of LASA have won international honours and awards for
their signifcant contribution in the feld of laboratory animal science. The LASA
would like to congratulate these members for their signifcant achievement.
Dr. Suresh Poosala is heading the Veterinary Sciences and Toxicology
programs at Bristol-Myers Squibb India Ltd., Bangalore. He has been
an AAALAC ad hoc visitor and a specialist since 2001. Recently, he has
been elected as a Council member to the Pacifc Rim Section of AAALAC
International. Dr. Suresh is the frst and the only council member from
India for AAALAC International.
Dr. Rahul Thorat, working as Scientist C at the Laboratory Animal
Facility, Advanced Centre for Treatment, Research and Education
in Cancer (ACTREC) Tata Memorial Centre (TMC), Mumbai has been
bestowed with International Award of the Japanese Association for
Laboratory Animal Science (JALAS) as excellent young researcher for the
year 2010 for his abstract entitled Establishment of breeding stock of
rodent strains from cryo-preserved 8-cell to morula stage embryos using
controlled rate freezing.
Mrs. Rosa J. Samuel is currently working at the Central Animal Facility,
Indian Institute of Science, Bangalore. She has been bestowed with Young
Investigator Award at the 4th Asian Federation of Laboratory Animal
Science Associations (AFLAS) Congress 2010 which was held at Taipei,
Taiwan from November 9-11, 2010 for the abstract entitled An alternate
and rapid method for detection of bacterial agents in laboratory animal
facilities".
Vol -1, Issue - 1, Jan - Apr 2011 v
1. Aims and scope
The J ournal of Laboratory Animal Science (JLAS) is
the offcial journal of The Laboratory Animal Scientists
Association (LASA), India, and published biannually in
English. The journal publishes original research articles,
case reports, brief communications, letters to the editor,
and review articles on all aspects of the use of animals in
biomedical and veterinary research as well as in testing and
evaluation of the toxicity of agrochemicals, pesticides and
other compounds.
The JLAS publishes a diverse range of papers dealing
with both use of animals and their management in any
research, consistent with the aims of the Laboratory Animal
Scientists Association. The journal considers manuscripts
on facility design, environmental enrichment, laboratory
animal care and nutrition, pain management, genetics,
health monitoring and diagnostics, transgenic animals,
new animal models of disease, novel methods of effcacy
testing for vaccines, pharmaceuticals and delivery systems,
regulations/guidelines pertaining to the husbandry and use
of animals, education and training, personnel and facility
management, and animal welfare for publication.
2. Editorial policy
Peer review
All contributions are reviewed by independent referees
and the Editorial Board reserves the right to make the fnal
decision on acceptance or rejection of a manuscript.
Ethical approval and other ethical considerations
All research articles submitted for publication must be
approved by the Institutional Animal Ethics Committee
(IAEC). Articles will only be published if the experimental
procedures employed conform to the accepted principles
of animal usage in biomedical research. Housing
conditions shall be mentioned with standard management
procedures and type of the facility used with environmental
conditions. Wherever possible, mention the standard
international nomenclature for laboratory rodents. Give a
history on the source of animals if purchased or if in-house
bred, mention whether in-bred or out-bred or the type of
breeding followed.
A sentence mentioning the date of approval of the IAEC
for small laboratory animals and the Committee for the
Purpose of Control and Supervision of Experiments on
Animals (CPCSEA) in case of large animals should appear
in each manuscript, and the corresponding author must
make the approval certifcate available if requested by the
Editorial Board.
Permissions
All previously published material must be accompanied by
the written consent to reproduction of the copyright holder.
An acknowledgement of permission should be included
at appropriate place in the paper, and a full reference to
the original place of publication should be included in the
reference list.
Copyright
The authors of the manuscripts accepted for publication
will be required to assign copyright to JLAS and a form for
this purpose will accompany the proofs.
Manuscript Review and Status
The Editor reviews all submissions and makes an initial
determination regarding suitability for publication. Before
being sent for peer review, newly submitted manuscripts
are screened to ensure that the text, fgures (charts, graphs,
images), and tables comply with the criteria described
in the Guidelines for Authors. All manuscripts undergo
thorough peer review, typically by three reviewers with
relevant experience. Selection of the panel of reviewers
ultimately is the prerogative of the Editor.
3. Types of articles
Review Articles
Review Articles are generally invited by the Editorial
board and the Editor may be contacted by prospective
authors to verify the journals interest and suitability of the
manuscript. These articles (should not exceed 5500 words,
cited references to be limited to about 75 in number) are
expected to appraise and discuss current developments
in the relevant feld. They should be well focused and
organized.
Original Articles
An article describing original or substantially incremental
research that falls within the Aims and Scope of the journal
is considered for publication under this category. These
articles should be up to 4500 words and must include an
Abstract, Introduction, Material and Methods, Results,
Discussion, Acknowledgements and References.
Short Reports
Technical notes and preliminary communications
with adequate methodological details and conclusions
are accepted under this section. Case reports or case
studies with signifcant and original observations will be
considered for publication. These reports should be less
than 1500 words, including an Abstract of less than 200
words, and no more than two fgures or tables.
JOURNAL OF LABORATORY ANIMAL SCIENCE
(Ofcial Journal of the Laboratory Animal Scientists Association)
About the Journal - Mission Statement
The mission of the Journal of Laboratory Animal Science (JLAS) is to disseminate high-quality, peer-reviewed information that
expands biomedical knowledge and promotes human and animal health through the study of laboratory animal diseases, animal
models of disease, basic biological mechanisms related to disease in humans and animals, and information on animal biology,
technology, facility operations and management.
Guidelines for Authors
Vol -1, Issue - 1, Jan - Apr 2011 vi
Letters to the Editor
Letters to the Editor will be considered for publication but
only on issues related to the scientifc or ethical content of
the journal, and authors will be permitted the opportunity to
publish a reply to any letters.
4. How to submit a manuscript
All submissions must be in English and of good grammatical
standards. The manuscripts should be submitted as single
word fle via email to editor.jlas@gmail.com Manuscripts
should be prepared in accordance with the guidelines below.
5. How to prepare a manuscript
Formatting
Manuscripts must be submitted using double line-spaced,
unjustifed and line numbered text throughout, with
headings and subheadings in bold case. The line numbering
of text must start at the abstract, must continue throughout
the entire text and must not re-start at the top of each page.
Manuscripts must be prepared in a font and size suitable for
reading at 100% zooming. Authors are recommended to
use Times New Roman (font size : 12).
Title page
The frst page should contain the full title of the manuscript,
a short title, the initials and last names of all the authors and
their affliations, the department(s) and the institution(s)
where the work was carried out, and the name, postal
and email addresses and telephone and fax numbers of
the author responsible for all communications about the
manuscript and proofs.
The title should be concise, informative and must not be
unnecessarily punctuated. The short title should be no more
than six words long.
Abstract
An unstructured abstract of no more than 250 words (or no
more than 200 words for Short Reports) that sequentially
summarizes the background, rationale, methods, results and
conclusions of the work and a list of up to fve key words
must accompany all Review Articles, Original Articles,
and Short Reports. Letters to the Editor do not require an
abstract.
Body
Use coded or nonproprietary language throughout the
manuscript. Cite the proprietary, brand, or vendor name
associated with an assay, instrument, machine, service,
or compound only in Materials and Methods. Defne all
nonstandard abbreviations and acronyms at frst use. Limit
the number of novel abbreviations used. Refer to the list of
Standard Abbreviations for abbreviations that can be used
without defnition.
Introduction
Provide the rationale and supporting background for
the submitted article and its importance and relevance.
Extensive reviews of the existing literature are
inappropriate for research reports and case studies/
reports.
Describe the animals, husbandry, tests, equipment,
procedures, reagents, and services used in detail with
citation of published references.
Include statistical methods where relevant and
attribute (name of software program used) or reference
them appropriately. In addition, provide the P value
used to defne statistical signifcance.
Include a statement regarding Institutional Animal
Ethics Committee approval (or equivalent) for
procedures and protocols involving animals.
Insert callouts (in parentheses) for all Figures and
Tables, which are numbered in order of their mention
in the text.
Follow correct nomenclature for laboratory animals,
genes, genetic markers, alleles, mutations, and
microbes. Biological names of organisms and names
of genes must be italicized.
Wherever possible, use International System of Units
base and derived units for numerical data.
Results
Accompany statements of differences between groups
with appropriate statistics.
Summarize selected data from Figures and Tables
in the Results section; do not merely repeat all
information presented in graphics.
Save interpretation of data for the Discussion section.
Discussion
Begin the Discussion with a brief summary of the key
fndings.
Limit discussion of study fndings to those that have
been presented in the Results.
Address any limitations of the study and directions for
potential future research.
Tables
Tables must be prepared using the Table feature of the word
processor. Tables should not duplicate information given
in the text, should be numbered in the order in which they
are mentioned in the text, and should be given a brief title.
Each Table should appear on a separate page at the end of
the manuscript as part of the text fle. Vertical rules and/or
background shading should not be used. The legend of a
table should be concise and enable the reader to understand
the data without excessive reference to the text. The
Table should be designed in such a way so as to convey
the information without extensive footnotes. Authors
are encouraged to convert Tables into Figures whenever
possible and appropriate.
Figures
All fgures should be numbered in the order in which they
are mentioned in the text. All fgures must be accompanied
by a fgure legend. If fgures are supplied in separate fles,
the fgure legends must all be listed at the end of the main
text fle.
Line drawings should be produced electronically and clearly
labeled. Graphs may be supplied as Excel spreadsheets
(one per sheet). Other line drawings should be supplied in a
suitable vector graphic fle format (e.g. .eps)
All photographic images should be submitted in camera-
ready form (i.e. with all extraneous areas removed), and
where necessary, magnifcation should be shown using a
scale marker.
Vol -1, Issue - 1, Jan - Apr 2011 vii
Digital fgure guidelines
To ensure the highest quality reproduction in the journal,
original digital fgures are preferred. When creating and
submitting digital fles, please follow the guidelines below.
Formats
For publication, use TIFF, EPS or postscript (ps) fles in
PC or MacIntosh format, preferably from PhotoShop or
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fles must be converted to postscript (ps) format.
Resolution and fgure quality
Figure fles must be submitted at an appropriate
resolution for print publication:
Color, 300 d.p.i. minimum, convert all color fles into
CMYK mode
Grayscale, 600 d.p.i. minimum for black & white
photographs
Line art, 1200 d.p.i. minimum for graphs and
illustrations
References
Only essential references should be included. Authors
are responsible for verifying them against the original
source material. Automatic numbering should be avoided.
References should include the names and initials of up to
six authors. If there are more than six authors, only the
frst three should be named, followed by et al. Publications
for which no author is apparent may be attributed to the
organization from which they originate. Simply omit the
name of the author for anonymous journal articles avoid
using Anonymous. Punctuation in references should be
kept to a minimum, as shown in the following examples:
Some basic rules applicable to all formats indexed by
author name(s):
All citation entries are listed in alphabetical order based on
the frst authors last name
If the same author(s) are cited for more than one paper
having the same order of authors names, the papers should
be listed in chronological sequence by year of publication.
References in the text are given as the author(s) and year,
i.e. (Evans, 1961; Smith and Jones, 1990) or Evans (1961).
Papers with more than two authors are cited as et al. i.e
J ones et al. (1989).
References in the text within the same parentheses are given
in chronological order. The fnal list of references should be
alphabetical.
The format in the reference list is as follows: author(s)
name(s) and initials, year of publication in parentheses,
full title of article, journal title as abbreviated in the World
List of Scientifc Periodicals, volume number and page
numbers.
Evans B, Jones T, Smith R (1990). The role of
estrogen in mouse courtship behavior changes as
mice age. J. Physiol. 62(6):1130-1142.
References by the same frst author and published in
the same year should be labeled a, b, c etc within the
text (e.g. Smith, 1992a) and listed sequentially in the
reference list. For example:
Evans B (1970a). Physiological effects of estrogen
on mouse courtship behavior. J. Physiol. 40(2):140-
145.
Evans B (1970b). Physiological effects of estrogen
analogs: Insincere courtship behavior in female mice.
J. Physiol. 40(8):1240-1247.
Journal Article: Single author
Evans B (1970). Physiological effects of estrogen on
mouse courtship behavior. J. Physiol. 40(2):140-145.
Journal: Two authors
Jones T, Evans B (1989). The role of whisker length
in mouse nose-twitch courtship behavior. J. Physiol.
61(3):113-118.
Journal: Multiple authors
Evans B, Jones T, Smith R (1990). The role of
estrogen in mouse courtship behavior changes as
mice age. J. Physiol. 2(6):1130-1142.
References to book articles should be mentioned as follows:
author(s) name(s) and initials, date of publication
in parentheses, title of chapter or article, full title
of book, edition, name(s) of editor(s) if relevant,
publisher, place of publication and pages referred to:
e.g. Robin C (1991). Calcium in plants eaten by
horses. In: Dietary Calcium, 2nd edn., Ed: J. Chalk,
Blackwell Scientifc, London. pp 195-201.
It is the responsibility of the authors to ensure that all
reference details are accurate.
Acknowledgments
Recognize (with their permission) and acknowledge people
and institutions whose contributions of funding, technical
assistance, reagents, data collection and analysis, and other
services that do not meet the criteria for authorship.
Abbreviations
Symbols and abbreviations should be those currently in use.
Authors should not create new abbreviations and acronyms.
References to animal strains should be in accordance with
current nomenclature. For reference, use The International
Index of Laboratory Animals, 6
th
edn. (Festing M, 1993);
the Mouse Genome database and the Rat Genome Database.
Units
All measurements should be expressed in SI units.
6. Proofs and e-prints
Proofs will be sent by email to the designated corresponding
author as a PDF fle attachment and should be corrected
and returned promptly; corrections should be kept to a
minimum. A PDF e-print of each published article will be
supplied free of charge to the author for correspondence;
hardcopy off-prints may be ordered from the publisher.
Vol -1, Issue - 1, Jan - Apr 2011 viii
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Dr. Syed S.Y.H. Qadri
Secretary
H.No. 12-13-483/A/1
Lane Opp. Mahalakshmi Super Market
Nagarjuna Nagar, St. No. 14, Tarnaka
Secunderabad-560017, Andhra Pradesh
Phone (Of) : 040-27008921
(Res.) : 040-65280225
Mobile : 9849518510
E-mail : lasaindia@yahoo.co.in
Web : www.lasaindia.org
Laboratory Animal Scientists Association
(Registered Society under Andhra Pradesh Society Registration Act, 2001)
Registration number 415 of 2004
LASA is a national organization dedicated to advancing laboratory animal science by pro-
moting the ethical care and use of laboratory animals in biomedical and veterinary research.
LASA is a registered member of the International Council for Laboratory Animal Science
(ICLAS), Barcelona, Spain and Asian Federation of Laboratory Animal Science Associations,
Japan. LASA has also been recognized by FELASA (Federation of European Laboratory Ani-
mal Science Associations.
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Chicken Embryo As A Diabetic Animal Model

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