STUDY ON CONCEPTION RATES IN SOWS BRED

WITH ARTIFICIAL INSEMINATION BY USING

CROSSBREED LWY DILUTED BOAR SEMEN
By
Dr. N.VAMSHI KRISHNA REDDY
B.V.Sc & A.H
RVM/14-13

THESIS SUBMITTED TO
P.V.NARSIMHA RAO TELANGANA VETERINARY
UNIVERSITY
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE AWARD OF THE DEGREE OF

MASTER OF VETERINARY SCIENCE

(VETERINARY GYNAECOLOGY AND OBSTETRICS)

DEPARTMENT OF VETERINARY GYNAECOLOGY AND OBSTETRICS

COLLEGE OF VETERINARY SCIENCE

RAJENDRANAGAR, HYDERABAD-500 030
P.V.NARSIMHA RAO TELANGANA VETERINARY
UNIVERSITY
HYDERABAD
JULY, 2016
 CERTIFICATE

Dr. N.VAMSHI KRISHNA REDDY, RVM/14-13 has satisfactorily prosecuted the

course of research and the thesis titled “STUDY ON CONCEPTION RATES IN

SOWS BRED WITH ARTIFICIAL INSEMINATION BY USING CROSSBREED

LWY DILUTED BOAR SEMEN” submitted is the result of original research work

and is of sufficiently high standard to warrant its presentation to the examinations. I also

certify that the thesis or part of thereof has not been previously submitted by his for a

degree of any University.

Date:

(K. MURALI MOHAN)

Major Advisor
 CERTIFICATE
This is to certify that the thesis entitled “STUDY ON CONCEPTION RATES
IN SOWS BRED WITH ARTIFICIAL INSEMINATION BY USING
CROSSBREED LWY DILUTED BOAR SEMEN” submitted in partial fulfilment of
the requirements for the degree of “MASTER OF VETERINARY SCIENCE” of the
P.V. Narsimha Rao Telangana Veterinary University, Hyderabad is a record of the
bonafide research work carried out by Dr. N. VAMSHI KRISHNA REDDY,
RVM/2014-13 under our guidance and supervision. The subject of the thesis has been
approved by the Student‟s Advisory Committee.
No part of the thesis has been submitted by the student for any other degree or
diploma. The published part has been fully acknowledged. All assistance and help
received during the course of investigations have been duly acknowledged by the author
of the thesis.

(K. MURALI MOHAN)

Chairman of the Advisory Committee

Thesis approved by the Student’s Advisory Committee

CHAIRMAN : Dr. K. MURALI MOHAN

Associate Professor
Principal
Animal husbandry polytechnic college
Siddipet, Medak. _________________

MEMBER : Dr. K. RAMCHANDRA REDDY

Associate Professor and Head
Department of Veterinary Gynaecology &
Obstetrics
College of Veterinary Science
Korutla, Karimanagar. _________________

MEMBER : Dr. C. Latha

Assistant Professor
Department of Veterinary Surgery and Radiology
Officer in charge and Head
Veterinary hospital, Warangal. _________________
 TABLE OF CONTENTS

Chapter Page
Title
No No
I 1. INTRODUCTION 1-4
II 2. REVIEW OF LITERATURE 5-26
2.1 Semen extenders 5
2.2 Semen evaluation 5
2.2.1 Motility 5
2.2.2 Concentration 7
2.3 Sperm concentration per insemination dose 8
2.4 Estrus 10
2.4.1 Weaning to estrus interval 10
2.4.2 Farrowing to estrus interval 11
2.4.3 Estrus synchronization 12
2.4.4 Estrus response rate 14
2.4.5 Interval between hormonal treatment and onset of estrus 15
2.4.6 Duration of estrus 16
2.4.7 Estrus intensity 17
2.5 Artificial insemination 18
2.5.1 Time of insemination 19
2.6 Conception rate 20
2.7 Pregnancy diagnosis 25
III MATERIALS AND METHODS 27-40
3.1 Selection of experimental animals 27
3.2 Experimental semen extenders preparation 29
3.3 Semen collection and evaluation 30
3.3.1 Estimation of motility and concentration 30
3.4 Semen processing and storage 32
3.5 Sterilization 32
3.6 Estrus 32
3.6.1 Natural and synchronized estrus 32
3.6.2 Estrus detection 33
3.6.3 Intensity of estrus 33
3.7 Experimental design 35
3.7.1 Group 1 35
3.7.2 Group 2 35
3.7.2.1 Sub group 2A 35
3.7.2.2 Sub group 2B 35
3.7.3 Group 3 36
3.7.3.1 Sub group 3A 36
3.7.3.2 Sub group 3 B 36
 3.8 Method of Artificial insemination 37
3.9 Pregnancy diagnosis 39
3.9.1 Non return rate 39
3.9.2 Ultrasound scanning 39
3.10 Statistical analysis 39
IV RESULTS 41-57
4.1 Estrus 41
4.1.1 Weaning to estrus interval 41
4.1.2 Interval between PGF2α treatment and estrus onset 41
4.1.3 Interval between farrowing to estrus 42
4.1.4 Duration of estrus 42
4.1.5 Estrus response rate 42
4.1.6 Estrus intensity 43
4.2 Conception rate 47
4.3 Pregnancy diagnosis 52
4.3.1 Non return rate 52
4.3.2 Ultrasound scanning 52
4.3.2.1 Fetal parameters 52
V DISCUSSION 58-63
5.1 Weaning to estrus interval 58
5.2 Farrowing to estrus interval 59
5.3 Synchronized estrus 59
5.3.1 Estrus response rate 59
5.3.2 Interval between hormone treatment and onset of estrus 60
5.3.3 Duration of estrus 60
5.3.4 Estrus intensity 61
5.4 Conception rate 61
5.5 Pregnancy diagnosis 62
5.6 Conclusion 63
VI SUMMARY 64-65
LITERATURE CITED 66-76
 LIST OF TABLES

PAGE
TABLE TITLE NO
NO

3.1 Body condition score index 28

3.2 Estrus intensity grading chart 33

4.1 Weaning to estrus interval and farrowing to estrus interval in natural 44

estrus

4.2 Correlation between weaning to estrus interval, farrowing to estrus 44

interval and duration of estrus with natural estrus

4.3 Comparison of estrus parameters between natural and synchronized 45

estrus in LWY cross bred or local pigs

4.4 Conception rate in LWY or local pigs with artificial insemination 49

using diluted semen with two different diluents.

4.5 Fetal parameters during transabdominal ultrasonography at different 54

stages of gestation
 LIST OF ILLUSTRATIONS

PAGE
Fig.No. TITLE
No.

3.1 Body condition score of the pigs 28

3.2 Stainless steel Dummy sow 31

3.3 Semen collection by gloved hand method 31

3.4A Mounting behaviour 34

3.4B Shoulder scratches 34

3.4C Swollen reddened vulva 34

3.4D Clear and sticky mucus 34

3.4E Lordosis 34

3.4F Riding test (standing reflex) 34

3.5 Equipments used for estrus synchronization and AI in swine 38

3.6 Inserting golden pig catheter at 30 º angle 38

3.7 Cervical Artificial Insemination method 40

3.8 Transabdominal ultrasonography 40

4.1 Estrus intensity grading in natural and synchronised estrus 46

Conception rate in LWY or Local pigs artificially inseminated to

4.2 50
natural and synchronized estrus
 Overall conception rate with AI using NBSE and PRIMXcell
4.3 50
extenders.

Comparison of conception rate in pigs artificially inseminated to

4.4 51
natural and synchronized heat with natural service.
Accuracy of pregnancy diagnosis with transabdominal
4.5 ultrsonography by using linear probe in relation to gestation period in 53
days.

4.6 Anechogenic smaller amniotic vesicles at 21 days of gestation 55

Anechogenic amniotic vesicle with maximum size at 30th day of

4.7 55
gestation

4.8 Echogenic embryo within the anechogenic amniotic vesicle (45 days) 56

An ultrasonographic measurment of CRL of fetus at 60 days of

4.9 56
gestation

An ultrasonographic measurement of BPD of a fetus at 60 days of

4.10 57
gestation

4.11 An ultrasonograhy of fetus at 78 days of gestation with ribs and spine 57

LIST OF SYMBOLS AND ABBREVIATIONS

˃ : Greater than

% : per cent

< : Lesser than

≤ : Less than or equal to

≥ : Greater than or equal to

µg : Microgram
0
C : Degree centigrade

A.I. : Artificial Insemination

AVD : Amniotic vesicle diameter

BCS : Body condition score

BTS : Beltsville thawing solution

BPD : Bipartial diameter

CAI : Cervical artificial insemination

CL : Corpus Luteum

Cm : Centimeter

CRL : Crown rump length

D : Days

DE : Duration of estrus

eCG : Equine chorionic gonadotropin

EOI : Estrus to ovulation interval

ERR : Estrus response rate

ES : Estrus synchronization

et al. : et alii (and other people)

Etc : et cet „er‟ a (and so on)

FEI : Farrowing to Estrus Interval

FR : Farrowing Rate

G : Grams

GEPS : Glucose sodium salt of EDTA-Potassium

sodium tartarate sodium citrate dehydrate
H : Hour

hCG : Human chorionic gonadotropin

HCl : Hydrochloric acid

i.e. : id est (that is)

i.m. : Intra muscular

ILFC : Instructional Livestock Farm Complex

IU : International Units

Kg : Kilogram

LWY : Large White Yorkshire

M : Months

MHz : MegaHertz

Min : Minutes

Ml : Milliliter

Mm : milli Molar

Mt : metric tones

NRCP : National research centre on pig

NRR : Non Return Rate

NSBE : Normal Boar Semen Extender

Ng : Nanogram

P : Probability
P4 : Progesterone

PD Pregnancy diagnosis

PGF2 α : Prostaglandin F2 alpha

PMSG : Preagnant mare serum gonadotropin

R : Correlation Co-efficient

RTU : Real time ultrasonography

SPSS : Statistical Package for Social Science

v/v : Volume per Volume

Vs : Against

w/v : Weight per volume

WEI : Weaning to Estrus Interval

 ACKNOWLEDGEMENTS

I praise the Almighty God for his abundant grace, mercy and blessings
showered on me in each and every step of my life and I am eternally indebted to him for
his kindness which enabled me to complete this work successfully.
With immense pleasure, I express my deepest sense of gratitude to major advisor
and Chairman of my advisory committee Dr. K. Muralimohan, Associate Professor,
Department of Veterinary Gynaecology & Obstetrics, Principal, Animal Husbandry
Polytechnic College, Siddipet for his kind nature and able guidance, constant
encouragement, meticulous attention to the details and sustained interest evinced
during present study. It is indeed my privilege for me to work under his unending
inspiration and indomitable spirit and I owe him a huge debt of gratitude for ever in my
life.
Diction fails to express my sincere thanks to Dr. K. Ramchandra Reddy,
Associate Professor and Head, Department of Veterinary Gynaecology & Obstetrics,
College of Veterinary Science, Korutla, Karimnagar and member of my advisory
committee, for his meticulous guidance andvaluable suggestions in planning and
execution of research work and in preparation of thesisandI am grateful to him for his
constant fomenting, punctilious and impeccable advice. Above all, his affectionate way
of dealing with things throughout the course of my study has helped me to consummate
the research work with a grand success. I take this opportunity to express my heartfelt
gratitude towards him. I had really a great pleasure and privilege to be associated with
him during this course of study.
I deem it my privilege to extol my sincere feelings of gratitude to Dr. C. Latha,
Assistant Professor, Department of Veterinary Surgery and Radiology, Officer in-
charge & Head, Veterinary Hospital, Warangal member of my advisory committee for
her moral support, valuable suggestions and constant encouragement during various
phases of my study.
My sincere are due to Dr. Eknath.B.Chakurkar, Prinicipal Scientist, ICAR
Central coastal research institute, Goa for providing the semen diluents and for his
generous help during the study.
I humbly place on record my sincere thanks to Dr. K. Chandrashekar Reddy,
Professor and University Head and Dr. K. G. Solmon Raju, Professor, Department of
Veterinary Gynaecology & Obstetrics, College of Veterinary Science, Hyderabad for
their constant encouragement and valuable suggestions rendered for preparing
manuscript of the thesis.
I am very much indebted to Dr. G. Arunakumari, Assistant Professor,
Department of Veterinary Gynaecology & Obstetrics, College of Veterinary Science,
Korutla, Karimnagar for her able guidance, generous help and transcendent suggestions
during my research work.
 I express my sincere thanks to Dr. L. Ramsingh and Dr. A. Sunil anand
Assistant Professors, Department of Veterinary Gynaecology & Obstetrics, College of
Veterinary Science, Rajendranagar, Hyderabad for their kind co-operation.
I express my special thanks to Dr. T. Raghunandan, Associate Dean, C.V.Sc,
Korutla, Department of Livestock Production and Managementand Dr. Sushma,
Assistant Professor, ILFC, C.V.Sc, Rajendranagar, Hyderabad, for permitting me to
carry out my research work at ILFC and for their kind help during period of study.

I am highly grateful to Dr. T. MadhavaRao, Professor and Head, TVCC,

College of Veterinary Science, Hyderabad, Dr. N. Nalinikumari, Associate Professor
and Head, Department of Animal Nutrition, C.V.sc, Korutla, Dr. M. Gnanaprakash,
Professor and Head, Poultry Research station, C.V.Sc, Hyderabad for their helping
hand, constant encouragement and providing necessary facilities to carry out my
research work.
There are no words in the available lexicon to express my gratitude and regards
to my beloved parents Mrs. Pushpalatha and Mr. Raghava Reddy, for their
inbounding love, Unflinching support andunconditional blessings. It is only due to
their instilling aspiration, I have reached this stage and this manuscript has taken its
form.

Special thanks to my seniors Dr. Vishal, Dr. Srilatha, Dr. Rajasri and
Dr. Anudeep Reddy for teaching patiently the basics of the work by their valuable
suggestions and for their assistance and encouragement throughout my research work.

I am extremely delighted to extend thanks to my colleagues Dr. M. Mahesh,

Dr. A. Priyanka, Dr. B. Priyanka and Dr. B. Gopi for their time sparing, kind
cooperation during my research workwho have been with me till the end of my work.

From inner core of my heart, I express my warmest thanks and attention to my

friends and juniors Raju, Anil, Rajesh, Yashashwini, Mallesh, Rajashekar Reddy,
Balakrishna, K. Mahendar Reddy, N. Anil Reddy, K.B.Ashok Reddy,
Chandrababu, Ravindranath Reddy, Shivanaresh, Jawahar, Rajith Reddy,
shashank Reddy, Venkat Reddy, Shashideep, Deepak Reddy, Sumanth and
Ramesh for their timely help rendered during the period of study.
I also express my heartfelt thanks to juniors Sai Hitesh, Sushanth, Aravind,
Raju, Sandeep, Santosh, Gopalakrishna, Phanindranath and Hemanth for their
timely help rendered during the period of study.
I express my deep sense of gratitude to the farmers Mr. Krishnamraju and Mr.
Anjaneyulu, Mahabubnagar for allowing me to continue the research work at their
private farm, kind cooperation, assistance and encouragement throughout my research
work.
 I am also happy to express my sincere gratitude to Mahesh, Mahendar and
Rambabu staff of ILFC. Moghulamma, Swaroopa, Anjaneyulu, Sriramulu and
Raju staff of Department of Veterinary Gynaecology and Obstetrics, College of
Veterinary Science, Rajendranagar, Hyderabad for their co-operation and help
rendered throughout the research period.

I deeply extend my thanks to Library staff of our college for their ever ready
help during my course of study.

I am thankful to P. V. Narsimha Rao Telangana Veterinary University,

Hyderabad for providing financial assistance in the form of stipend during my post
graduate study.

I place on record my apology and sincere thankfulness to the unmentioned

personalities, who have played a role in this study and preparation of this manuscript.
To wrap it up, thanks everyone for making my college life fun, enjoyable and
unforgettable.

Place: Hyderabad

Date: 30.07.2016 (N. VAMSHI KRISHNA REDDY)

 DECLARATION

I, N. VAMSHI KRISHNA REDDY, RVM/14-13 hereby declare that the

thesis entitled “STUDY ON CONCEPTION RATES IN SOWS BRED WITH

ARTIFICIAL INSEMINATION BY USING CROSSBREED LWY DILUTED BOAR

SEMEN” submitted to P.V. Narsimha Rao Telangana Veterinary University,

Hyderabad for the degree of MASTER OF VETERINARY SCIENCE is the

result of original research work done by me. It is further declared that the thesis

or any part thereof has not been published earlier in any manner.

Date: N. VAMSHI KRISHNA REDDY

Name of the author : N. VAMSHI KRISHNA REDDY

Title of the thesis : STUDY ON CONCEPTION RATES IN SOWS BRED

WITH ARTIFICIAL INSEMINATION BY USING

CROSSBREED LWY DILUTED BOAR SEMEN

Degree to which it

is submitted : MASTER OF VETERINARY SCIENCE

Faculty : VETERINARY SCIENCE

Discipline : VETERINARY GYNAECOLOGY AND OBSTETRICS

Chairman : DR. K. MURALIMOHAN

University : P.V.NARASIMHARAO TELANGANA VETERINARY

UNIVERSITY,HYDERABAD – 500 030

Year of submission : 2016

ABSTRACT

An experiment was designed to study the efficiency of two different boar semen
diluents-PRIMXcell a newly launched short term extender (IMV) and NBSE a short
term extender prepared by ICAR central costal research institute, Goa, India, on the
fertility of the LWY crossbreed boar diluted liquid semen at 15-18ºC with natural and
synchronized estrus.

Forty four non pregnant pigs (gilts or sows) were selected after screening the
animals with a transabdominal ultrasound for pregnancy. Three boars ageing 1-3 years
were selected and trained with a dummy sow for semen collection.

Twelve animals were kept as control (group 1), rest of the animals (n=12) were
observed for natural estrus and were inseminated (group 2) with NBSE (n=6) and
PRIMXcell (n=6) diluted semen. The other (n=20) animals (group 3) which could not
exhibit estrus within 14 days after weaning or gilts after attaining optimum body weight
were subjected for estrus synchronization with 175 µg of Cloprostenol (VETMATE)
intramuscularly.
 Semen was collected from the boars during early hours of the day and was
subjected for routine laboratory examinations usually mass motility, individual motility,
viability and concentration. The fresh undiluted semen with ≥75% motility, 80% normal
sperm morphology and 70% sperm viability was processed for dilution and utilised for
AI within 24 hours after the dilution.

The estrus response rate with natural estrus was 100% and with Cloprostenol
was 60% (12/20) and average time taken to exhibit estrus after hormonal treatment was
3.75 ± 0.44 days with an average estrus length of 42.00 ± 1.80 hours. However, only
25% animals were exhibited estrus behaviour with 2nd and 3rd grade estrus intensity and
35% animals showed 1st grade estrus intensity and remaining 40% animals could not
exhibit estrus.

Conception rate was 100% with both the diluents in natural estrus. Whereas, 33.
33% and 50%, respectively for PRIMXcell and NBSE diuted semen in synchronized
estrus. The overall conception rate with PRIMXcell was 66.66 % and with NBSE was
75%. The results indicated that the overall conception rate for both the diluents were
not significantly different. Conception rate with natural service and AI with natural
estrus was similar but AI with synchronized estrus was significantly lower.

Pregnancy diagnosis was done based on non return rates and also with
ultrasonography. Accuracy of non return rate was 80% whereas, in ultrasonography it
was 75% on 21st, 90% on 30th, 97% on 45th and 100% at 60th day of gestation.
 CHAPTER I

INTRODUCTION

The pig farming constitutes the livelihood of rural poor people belonging to the

lowest socio-economic status, especially tribal masses of India who rear pigs under

nomadic system (scavenging). The bulk of the pig population in India is of indigenous

type with poor growth rate and productivity. Pig production, among other species has a

higher potential to contribute to higher economic gain due to the facts: the pigs have

higher fecundity, higher feed conversion efficiency, early maturity, shorter generation

interval and relatively smaller space requirement. In India, 70% of the pig population is

being reared under traditional small holder, low-input demand driven production

system, except for limited number of semi-commercial pig farms in Kerala, Punjab and

Goa. Pig farming has the potential to have a positive impact on the livelihood of

millions of resource poor, under-privileged, landless and marginal farmers.

As per the XIX Livestock census (2012), the swine population of India is 10.29

million, with a decrease in population by 7.54% over the previous census. Porcine

population of Telangana state was 251 lakhs ranking 15th position in the country

(Census, 2012). According to Food and Agriculture Organisation of the United Nations

(2015), pork is the most consumed meat in the world and accounts for more than 35%

of the world‟s meat intake. Pork production in India is limited, representing only 7% of

the country‟s animal protein sources. Production is concentrated mainly in the north

eastern corner of the country and consists primarily of backyard and informal sector

producers. During 2012-13, India‟s domestic production of pork was 0.45 Mt with an

average meat yield of about 39 kg/animal, which is 55 % lower than the world average

of 79 kg/animal (FAOSTAT, 2009).

 Absence of sufficient number of breeder farmers throughout the country, is a

major constraint leading to lesser availability of quality pigs for farmers and market.

Therefore, genetic improvement of indigenous pigs through conventional and molecular

method must be undertaken on priority basis for production of superior germplasm.

Different strains of indigenous pigs from Indian states need to be identified,

characterized, documented, improved and conserved (in-situ and ex-situ). In view of the

importance of swine farming in terms of its contribution to rural poor and possible

potential for pig rearing in our country, large-scale application of Estrus

Synchronization (ES), Artificial Insemination (AI) technology under field conditions

and horizontal spread of superior germplasm needs to be taken up expeditiously.

Artificial Insemination technology holds enormous promise for genetic

improvement in farm animals and hence the use of AI for breeding of pigs has been

instrumental for facilitating global improvement in fertility, genetics, labour efficiency

and herd health. When compared with natural mating, artificial insemination is a very

useful tool to introduce superior genes into sow herds, with a minimal risk of disease

(Maes et al., 2008). By the year 2000, increases in AI use around the world had

occurred with several countries breeding nearly all pigs with AI (Weitze, 2000). Despite

the world scenario, AI in pig farming has not yet received acceptance due to lack of

awareness among the farmers especially in rural India. However, National Research

Centre on Pigs (NRCP), Rani, Guwahati a pioneer institute successfully introduced AI

technology at field level in Assam in 2009 under Institute Village Link Project (IVLP)

and also developed eight estrus synchronization protocols and thee protocols for treating

infertile sows.

Estrus synchronization enables farrowing synchronization, batch in-batch out of

piglets which suits the market demands, delayed onset of puberty and seasonal anestrus
in pigs can be avoided, exploitation of maximum number of farrowing rates and litter

size per sow per life time can be achieved by reducing the unproductive days of a pig.

One of the obstacles to the use of artificial insemination (AI) in swine is the

short storage life of boar spermatozoa, inseminations conducted are made with semen

that has been extended in the liquid state and used on the same day or stored at 15-20ºC

for 1-5 days (Johnson et al., 2000). Among mammalian species, boar spermatozoa are

extremely sensitive towards cold shock because of their peculiar plasma membrane

composition (White, 1993). The temperature of collection, storage, and the composition

of the storage medium are important for storage of liquid boar semen. It is found that

survival of cells is much greater after liquid than frozen storage of semen. The storage

media for liquid boar semen aim to prolong sperm survival, to provide energy to the

sperm cells, to buffer the pH of the suspension and to avoid the growth of bacteria (Vyt

et al., 2004). There are numerous commercial extenders for preservation of boar semen

in liquid state, NRCP developed a unique extender GEPS (Glucose sodium salt of

EDTA-Potassium sodium tartarate Sodium citrate dihydrate) which could preserve the

boar semen in liquid state for 7-8 days without losing the fertilizing capacity of the

spermatozoa and AI could be performed with satisfactory results. The farrowing rates

were 65% to 70% when the semen was used in the first 2 days after collection, but they

reduced to about 50% with 5 day-old semen (Johnson et al., 1988; Johnson, 1998). The

fertility of liquid boar semen decreases with duration of storage (Bennemann et al.,

2005) and is associated with a reduced ability of sperm to bind with oviductal

epithelium (Waberski et al., 2006).

A very few estrus synchronization protocols are practiced in swine unlike cattle.

There was limited information regarding synchronization protocols in pigs, although

there was information regarding fertility following Cervical Artificial Insemination

(CAI) with freshly diluted semen utilised within 24 h in pigs, there is a need to further

investigate the preservability of boar semen with an efficient diluent for utilising

superior germplasm. Hence this study was designed to compare the conception rate in

Large White Yorkshire (LWY) cross breed or local pigs bred by CAI using freshly

diluted and stored LWY cross breed boar semen at 15ºC-18ºC by using two different

extenders with natural and synchronized estrus. At the same time estrus response was

also evaluated to know the efficacy of ES protocol in pigs. Hence, the following

parameters were studied in this experiment.

1. To study the conception rates in pigs bred by AI.

2. To study the time interval between hormonal treatment and onset of estrus.

3. To study the estrus duration in natural and synchronized estrus.

4. To study the intensity of estrus symptoms.

5. To study the time interval between weaning to estrus.

6. To study the time interval between farrowing to estrus.

 CHAPTER II

REVIEW OF LITERATURE

2.1 SEMEN EXTENDERS

The storage media for liquid boar semen aim to prolong sperm survival, to

provide energy to the cells, to buffer the pH of the suspension and to avoid the growth

of bacteria (Vyt et al., 2004). Therefore, porcine semen extenders contain electrolytes to

maintain the osmotic pressure of the medium, glucose as energy source, buffering

systems to stabilize the pH of the extender and EDTA and antibiotics to prevent

bacterial overgrowth (Johnson et al., 2000). Commercial boar semen extenders vary in

composition and are classified as short-term (≤ 3 days) and long-term (≥ 3 days)

extenders based on how long they can preserve liquid semen without considerable loss

of its fertility. Short-term extenders are Beltsville liquid solution, Beltsville thawing

solution (BTS), Illinois variable temperature (IVT), Kiev, vital and long-term extenders

are Acromax, Androhep, Xcell, Zorlesco, Zorpvaetc (Gadea, 2003).

2.2 SEMEN EVALUATION

2.2.1 Motility

Motility of spermatozoa has always been considered a primary requirement to fertilize

eggs. Although the spermatozoa are brought to the fertilization site mainly by uterine

contractions (Langendijk et al., 2002), sperm motility is required for penetration of the

zona pellucida. Motility is known to be an important characteristic in predicting the

fertilizing potential of an ejaculate (Gadea, 2005). From the initial stages of AI

development until the present time, the assessment of the (motile) semen is the most

widely used test of semen quality. The percentage of total motile spermatozoa decreased
with storage time, without significant differences between extenders (Ambrogi et al.,

2006).

Flowers et al. (1997) reported that the minimum percentage of motile

spermatozoa for artificial insemination dose has been established to be at least of 60 %

because, under this threshold lower farrowing rates were experienced. Juonala et al.

(1998) reported that a subjective total motility of > 45 % in a 7 day stored semen (at 16-

20°C) should be the requirement for use in artificial insemination.

Britt et al. (1999) observed that ejaculates with motility ≤ 60 % fertilize fewer

eggs. Johnson et al. (2000) reported that a minimum of 60 % motility is essential to

attain optimal fertility. The high % of progressive motility indicates their plasma

membrane integrity and excellent metabolism. Althouse et al. (2002) reported that a

minimum of ≥ 70 % gross motility is essential for use of unextended fresh boar semen

for artificial insemination.

Vyt et al. (2004) reported that with extenders like Mulberry III, AndrohepTM,

Acromax, Kobidil+ and BTS, the average motility scores of the five extenders during a

period of 7 days significantly decreased with preservation period (p < 0.001). Decrease

in motility over time was lowest in Mulberry III, i.e. from 4.9 at day 1 to 3.7 at day 7.

Gadea et al. (2004) reported that farrowing rate was < 85 % with 74.09 ± 0.63

motility and forward progressive motility (FPM) 3.24 ± 0.03 and ˃ 85 % with 75.31 ±

0.63 motility and FPM 3.20 ± 0.03, respectively. Whereas, the total number of piglets

born showed a significant positive correlation with motility.

Zhou et al. (2004) evaluated sperm motility of Harbin white boar semen stored

at 20ºC in Kiev, BTS, ZO, ZO-BSA and ZO+PVA extenders for different time and

reported that there was no significant difference among the 5 extenders during the first 3
days of storage. Thereafter, sperm motility declined in all extenders but more rapidly in

the Kiev, BTS and ZO-BSA. A motility of more than 40% was maintained for 7, 9, 10

and 12 days in Kiev, BTS, ZO-BSA, ZO and ZO+PVA respectively. Whereas with

ZO+PVA extender after cooling down to 20°C, 15°C and 5°C was 91.3%, 89.6% and

75.6%, respectively.

Kaeoket et al. (2010) reported that the % of progressive sperm motility after

dilution on day 2 with Merck-III, Androstar®Plus, Modena, X-Cell, Dofu gold, Vitasem

LD and Duragen was 82.0 ± 3.4, 83.0 ± 6.5, 89.0 ± 5.5, 72.0 ± 8.4, 86.0 ± 10.8, 85.0 ±

10.6 and 87.0 ± 9.7, respectively with a significant difference of (p < 0.01).

Mapeka et al. (2012) reported that the total sperm motility of semen stored at

25°C was higher with BTS and Kobidil+ extended semen when compared with the egg

yolk citrate diluent. Sperm progressive motility was higher in the BTS group, whereas

lower in Kobidil+ and non-extended groups.

Sa et al. (2013) recorded the effect of commercial semen extenders Androhep+,

BTS, ModenaTM, Seminark, Vitasem LD on sperm motility (%) of Korean Native boar

semen stored at 17℃ for 96 h and reported that all extenders were maintained a high

level (80%) of sperm motility thoughout the storage period. They observed no

significant difference (p > 0.05) in sperm motility among the extenders. Patra et al.

(2016) reported that the progressive sperm motility (%) of Large Black, Ghungroo,

Hampshire and Hampshire Ghungroo cross was 66.3, 80.7, 76.5 and 85.4, respectively.

2.2.2 Concentration

Strzezek et al. (1995) reported that the concentration of the ejaculates from

Landrace, York, Duroc and Chester were 286 ± 8.072 x 106, 211 ± 22.07 x 106, 315 ±

47.43 x 106 and 363 ± 50.16 x106 spermatozoa/ml, respectively.

 Cerolini et al. (2001) reported that the boar sperm mean concentration was 100-

300 million/ml. Hence, boar semen was 5-10 times less concentrated when compared

with bull semen and was approximately equal to stallion semen. The variation limits are

very large, between 10 million and 1 billion spermatozoa/ml in boar semen.

Kommisrud et al. (2002) recorded that the sperm concentration of the boar

ejaculates from Duroc, Landrace, Duroc/Landrace and Yorkshire were 133 ± 49x106, 86

± 32x106, 100 ± 21x106 and 117 ± 35x106 spermatozoa/ml, respectively.

Turba et al. (2007) stated boar semen as being a dense ejaculate which contained

between 0.151 and 0.400 billion spermatozoa/ml. Youngquist and Threlfall (2007)

reported that the fully diluted porcine semen should contain a final sperm concentration

of 25-80 million sperm cells per ml. Hafez and Hafez (2008) reported that, the

spermatozoa concentration of boar semen was 200-300 million/ml and 30-60 billion

sperms per ejaculate. Whereas, Cupps (2009) reported that sperm concentration in boar

was 25-350 × 106 spermatozoa/ml.

Patra et al. (2016) evaluated the ejaculates obtained from different breeds: Large

Black, Ghungroo, Hampshire, Hampshire Ghungroo cross and reported concentration

(millions/ml) was 197.09 ± 39.44, 259.28 ± 21.88, 535.63 ± 61.36 and 141.32 ± 14.77

and total sperm per ejaculate (billion) was 32.49, 48.96, 50.18 and 24.65, respectively.

2.3 SPERM CONCENTRATION PER INSEMINATION DOSE

Willmen et al. (1991) observed a significant reduction of fertility from 90.2% to

58.3%, when the number of spermatozoa in the inseminate was reduced from 2 to

0.5×109 spermatzoa/ml. Colenbrander et al. (1993) reported that in AI of swine several

dose regimens were applied, which were ranged from 1.5x109 to 6.0x109 spermatozoa

per intra-cervical insemination dose. Soede et al. (1995) found that a good fertility was
obtained when inseminations containing 3×109 spermatozoa were administered 24 h

prior to ovulation.

Nissen et al. (1995) reported good fertility when sows were inseminated within

28 h before ovulation by using 2×109 spermatozoa/insemination. Xu et al. (1998)

observed that the range in mean litter size was between 10.2 to 11.5 and 9.1 to 10.1 in

pigs, respectively when 3×109 and 2×109 spermatozoa cells were used.

Krueger et al. (1999) reported that the lower doses tended to reduce litter size

and % of normal embryos, but could not affect the conception rates. Singleton (2001)

reported that the number of viable sperm per dose ranges from 2.5 to 4 billion motile

cells and volume ranges from 70 to 100 ml. When estrus detection and insemination

programmes were optimal, acceptable reproductive performance can be achieved with

only 1.5 to 2 billion sperm per dose. Behan and Watson (2006) reported that, acceptable

fertilization rates (88.2%) were achieved with the insemination dose of 80 ml which

contain 1×109 sperm cells with appropriate estrus detection.

Alm et al. (2006) observed a significant decrease in fertility parameters like non

return rate (NRR). The NRR % was 75.8 % and 84.0 % with a dose of 2 and 3 billion

spermatozoa, respectively. Garcia et al. (2007) reported that insemination with 1.25×109

sperm resulted in a lesser (P < 0.02) farrowing rate when compared to 2.5×109 sperm.

Pelland et al. (2008) reported that acceptable farrowing rate and litter size were

obtained when inseminations were done close to ovulation time by CAI with 1×109

sperm per dose. Whereas, Cupps (2009) reported that the dose of sperm to be

inseminated was 5×109 in 50 ml volume. Stancic et al. (2010) reported that the

farrowing rates (%) were 73.3, 66.7 and 50% and average litter size was 10.77, 10.35

and 10.54 with diluted sperm dose 50 ml in volume and spermatozoa number per dose

4, 2 and 1 x109 sperms with intra cervical insemination, respectively.

 Sa et al. (2013) reported that in practice, artificial insemination with fresh liquid

semen is performed immediately after dilution or after keeping for a day at 15-20℃,

using a dose of 3 billion spermatozoa with a volume of 80-100 ml. Apic et al. (2015)

reported that the farrowing rates with at volumes 50 ml with 2×109 and 4×109 were 66.7

and 73.3% and at 100 ml volume with 2×109 and 4×109 were 76.7 and 83.3%,

respectively with intra cervical insemination.

2.4 ESTRUS

The first estrus typically occured within 5 to 7 months of age in gilts that have

received boar stimulation to induce puberty. In mature sows, estrus usually occurs

within 3 to 7 days post weaning. Removal of the suckling stimulus for the sow at

weaning, causes an immediate decrease of prolactin secretion followed by an increase

of gonadotropin secretion, follicular growth and estradiol-17β concentrations that

culminates in the induction of estrus, a preovulatory GnRH/LH surge and ovulation. For

sows, the weaning to estrus interval should be approximately 4 to 5 days. As this

interval extends beyond 4 or 5 days, the actual heat period shortens and conception rate

and litter size decreases (Steverink et al., 1999).

2.4.1 Weaning to estrus interval (WEI)

Weaning typically resumes follicular development and estrus occured within 3

to 8 days after weaning (Youngquist and Threlfall, 2007; Hafez and Hafez, 2008). The

occurrence of post weaning estrus, was influenced by many factors including lactation

length, parity and season. Seasonal delays in WEI are expected in late summer and early

fall (seasonal infertility).

Weitze et al. (1994) observed a decrease of 10 h for duration of estrus (DE) and

7 h for estrus to ovulation interval (EOI) for each day that weaning to estrus interval
increased from 3 to 5 days and then an increase of 6 h for DE and 5 h for Estrus to

Ovulation Interval (EOI) as weaning-to-estrus interval increased from 5 to 6 days.

Mabry et al. (1996) reported that the lactation length exerted a significant

quadratic effect on weaning-to-first-service interval. The weaning-to-first-service

interval appeared to be minimized at a lactation length of 22–27 days. Weaning-to-first-

service interval was significantly increased when lactation length was either less than 22

days or greater than 27 days.

Kemp and Soede (1996) reported that a consistent decrease of 8 h for DE and 5

h for estrus to ovulation interval for each day that weaning-to-estrus interval increased

from 3 to 6 days. Steverink et al. (1999) found a decrease of 5h for DE for each day that

weaning-to-estrus interval increased from 4 to 6 days after analyzing several months of

DE records data based on twice daily detection of estrus in 55 farms.

Tantasuparuk et al. (2000) reported that reproductive performance of sows

showing estrus between 6 and 12 days after weaning was lower than that of sows

showing estrus earlier or later after weaning. Anil et al. (2004) reported that a higher

total born per litter was associated with a WEI of 5 days. Whereas, Youngquist and

Threlfall (2007) reported that sows with shorter WEI tend to have an increased litter

size. Cavalcante-Neto et al. (2008) reported that an increase of size in the following

litter with an increase in the weaning to estrus interval.

2.4.2 FARROWING TO ESTRUS INTERVAL (FEI)

This is the period between farrowing and onset of estrus. It includes lactation

period of the sow and weaning to estrus interval.

Correa et al. (2014) reported an increase of litter size as Farrowing to Service

Interval (FSI) increased. In part, this was expected because lactation length and
Weaning to Service Interval, the two components of farrowing to service interval,

increased litter size as well.

2.4.3 Estrus Synchronization

A seasonal reduction of sow reproductive performance during the summer and

early fall months is a common occurrence (Love et al., 1993). Weaning-to-estrus

interval is typically increased during this period, which tends to reduce DE and EOI.

Lindloff et al. (1976) reported that estrous cycle length was significantly

reduced by 2.0-2.1 days when 6 mg Prostaglandin F2α-tromethamine salt was infused

intrauterine (iu) on day 13th of the estrous cycle in the cycling miniature Gottingen

strain of sow.

Connor et al. (1976) observed that the estrous cycle length was unaffected with

20 mg PGF2α administered on day 9, or on days 9th and 10th. However, when 20 mg

PGF2α was injected on day 12th, estrus occurred an average of 1.7 days earlier when

compared with control animals.

Guthrie and Polge (1976b) reported that the estrus was synchronized in LWY

gilts by inducing accessory CL with 1500 IU of PMSG and 750 IU of hCG to delay

estrus and ovulation and by injecting 0.5 or 1mg of PGF2α analogues to regress the

accessory CL at a predetermined time.

Guthrie and Polge (1978) reported that the pregnant gilts (days12-40 of gestation

period) which were administered with 0.5 and 1.0 mg of Cloprostenol intramuscularly

at an interval of 24 h, induced a fertile estrus. Pressing et al. (1987) reported that

pregnancy was terminated with 500 µg of Cloprostenol and gilts returned to estrus with

normal fertility within 4 to 6 days.

 Estill et al. (1993) observed that the pig was susceptible to the luteolytic effects

of 12.5 mg PGF2α (Dinoprost), if repeated injections were given from Day 5th to 10th

for every 12 h estrus was induced after day 12th. They also concluded that PGF2α

analogue induced estrus which was accompanied by normal follicular development and

ovulation, thus resulted in better conception.

Foxcroft (2001) reported that synchronization of estrus in gilts could be obtained

with PGF2α administration on day 12th of the estrous cycle by which estrus was

advanced by 5 days. Estienne and Harper (2002) reported that the gilts could be

synchronized by feeding Alternogest for 14-18 d and followed by administration of

PG-600 (400 IU of PMSG and 200 IU of hCG) intramuscularly.

Kuge et al. (2006) reported that the length of estrous cycle was reduced

significantly for 12.5 ± 0.25 days with 1.5 mg Cloprostenol administered through

vaginal vestibule when compared with intramuscular route. Davis (2008) reported that

the estrus was induced in gilts provided with 15 mg of alternogest (Regumate®) for 14-

18 days. Noguchi et al. (2010) reported that the estrus was induced in cycling gilts

(Landrace x LWY) and the induced pseudo pregnant gilts (Estrodiol propionate 20 mg)

when treated with 15 mg of Dinoprost twice with a 24 hour interval on day 10th & 11th

and on day 36th & 37th, respectively.

Diaz et al. (2011) tested the hypothesis that acute inhibition of P4 by treatment

with epostane (EPO; 3beta HSD inhibitor) in CL lacking luteolytic capacity (Day 9 CL)

will allow PGF to induce responses associated with luteolysis. EPO dramatically

decreased production of P4 by luteal tissue (ng/mg tissue) by 90% and 95% in EPO and

PGF+EPO groups, respectively, when compared to control (p < 0.01). Low production

of PGF by luteal tissue was found in Control, PGF, and EPO groups; however,
treatment with PGF+EPO dramatically increased (˃ 82%) luteal PGF production and

induced luteolytic response in CL lacking luteolytic capacity.

Stancic et al. (2014) reported that the estrus was induced in gilts with two

separate i.m injections of luteolytic preparation like 5 mg Dinoprost (Dynolitic®,

Phizer) at interval of 11 days. They also reported that, estrus was induced in cycling

gilts with a single injection of 1000 IU of eCG (Folligon®, Intervet) and also by feeding

20 mg Alternogest Regumate® (Roussel Uclaf, Bernburg, Germany) for 18 days.

2.4.4 Estrus response rate

Guthrie and Polge (1976b) analysed luteal regression, based on plasma

progesterone concentrations and the occurrence of estrus were 54, 83 and 89 % of the

gilts with one injection of 0.5 mg of Cloprostenol, two injections of 0.5 mg of

Cloprostenol with an interval of 24 h and injections of 1.0 and 0.5 mg prostaglandins,

with an interval of 24 h, respectively. Guthrie and Polge (1978) observed that the LWY

gilts after insemination were aborted by administering Cloprostenol on 8, 12, 20, 26, 32

and 40th days of gestation. Almost 87% of the gilts in the groups D12, D20, D26, D32

and D40 exhibited a synchronized estrus 4 to 7 days after the administration of first

injection. Gilts in group D12 had fewer CL per animal than the animals in other groups.

Jochie et al. (1983) reported that 84 % of gilts were synchronized within 10 days

after treatment with 2 mg of Alfaprostol after 21 days of lactation. Treatment of

seasonal anestrus with Alfaprostol shortened effectively the interval to heat (5.9 vs 17.4

days, in gilts, 5.6 vs 9.7 days in sows and greatly increased the number of animals in

heat (81 vs 47% in gilts, 83 vs 62% in sows). Pena et al. (2001) reported that treatment

with 37.5 µg of D-Cloprostenol intrmuscularly at weaning during summer increased

27.93% (P < 0.001) of sows in estrus within 7 days after treatment.

 Estienne and Harper (2002) reported that the % of gilts exhibiting estrus < 7 d

after P.G. 600 injection was greater (88.9%) for the group, fed with alternogest for 18 d

than for the 14 d treated (63.2%). Breen et al. (2005) reported that the short-term boar

exposure before PG600 for gilts between 160 to 180 d of age can be used to improve the

estrus induction response.

Davis (2008) reported that 85% or more cycling gilts have expressed estrus

within 4 to 10 days after last treatment with altrenogest at the dose rate of 15 mg daily

for 14 days. Chakurkar (2009) reported that 85 and 70% of estrus was exhibited in

LWY and local pigs when treated with 7.5 mg of Dinoprost 55 days post farrowing.

Noguchi et al. (2010) reported that the standing estrus was exhibited by 80 and

83% of cycling and pseudo pregnant animals, respectively after treatment with 15 mg of

Dinoprost. Konch et al. (2012) reported that gilts were exhibited 100% estrus response

when treated with 5ml of Iliren.

Stancic et al. (2014) reported that 90, 40 and 65% of the gilts exhibited estrus

which were treated with 20 mg of Altrenogest for 18 days, two i.m injections of 5 mg of

Dinoprost (Dynolitic®) and single injection of 1,000 IU eCG (Folligon), respectively.

2.4.5 Interval between hormonal treatment and onset of estrus

Kraeling et al. (1975) reported that the gilts were treated with 5 mg of estradiol

benzoate from day 10th to 15th of the estrous cycle and then 10 mg of PGF2α was

administered on day 20th. The estrus was observed 5 ± 0.7 days after administration of

PGF2α.

Moeljono et al. (1976) reported that estrus was exhibited on an average of

88.0+13.5 h after administration of 20 mg of PGF2α twice daily with 12 h interval on

day 17th of estrous cycle in bilaterally hysterectomised gilts.

 Guthrie and Polge (1978) reported that estrus was induced 4-7 days after

treatment with two i.m. injections with 24 h apart by of 1.0 and 0.5 mg Cloprostenol in

gilts between 12 and 40 days of gestation.

Estill et al. (1993) reported that the mean inter estrus interval was reduced to

13.3 ± 0.5 days in gilts treated with PGF2α when compared with 19.8 ± 0.6 days for

control gilts. The serum progesterone levels declined below 1 ng/ml by Day 10.5 in

PGF2α treated gilts when compared to Day 17.5 in control animals.

Chakurkar (2009) reported that the average time taken by LWY and local pigs

when treated with 10 and 7.5 mg PGF2α was 87.50, 60.30 h and 92.40, 78.0 h

respectively. Noguchi et al. (2010) reported that 100% estrus was exhibited with

induced pseudo pregnant gilts 5.9 ± 0.5 days after the first administration of PGF2α.

Konch et al. (2012) successfully synchronized cyclic Hampshire gilts with 5ml

of Iliren (Intervet) with single and double dose. All the gilts were exhibited estrus

within 4-6 days with a mean value of 5.00 ± 0.26 days following administration of

single dose of PGF2α and 4-8 days with a mean value of 5.33 ± 0.61 days after the

second dose of PGF2α.

Stancic et al. (2014) reported that the average interval from the end of treatment

to onset of estrus was 4.2, 5.5 and 5.3 days with eCG, PGF2α and Alternogest,

respectively.

2.4.6 Duration of estrus

The average length of estrus varies due to parity, season, weaning-to-estrus

interval and farm management. Moeljono et al. (1976) observed an average duration of

estrus was 66.0+16.4 h with 20 mg of PGF2α twice daily with 12 h interval on day 17th

of estrous cycle in bilaterally hysterectomized gilts. Weitz et al. (1994) reported that,
most of the sows returned to estrus after an average lactational periods of 16 to 22 days

within 3-8 days and in these sows, duration of estrus was usually 56 h. Steverink et al.

(1997) observed that the average duration of estrus was 40 and 48 h in gilts and sows,

respectively which ranged between 12-88 h.

Soede and Kemp (1997) reported that the duration of estrus usually lasts for 45-

65 h and ovulation occurred 65% of the way through estrus. Roberts (2004) reported

that the duration of estrus was 1 to 4 days with an average of 2-3 days or 60 h. Hafez

and Hafez (2008) reported that the pubertal estrus period was usually shorter (47 h) than

the later ones (56 h). Gilts usually have a shorter period of estrus when compared to

sows.

Noguchi et al. (2010) reported that the duration of estrus was 43.5+13.3h and

50.4+13.8 h in PGF2α administered and PGF2α treated pseudo pregnant animals,

respectively. Konch et al. (2012) reported that the mean duration of estrus was

48.33 ± 1. 20, 48.67 ± 0.99 and 46.67 ± 1.76 h with single, double dose of Illerin and in

control cycling gilts, respectively.

2.4.7 Estrus intensity

Accurate detection of onset of estrus is critically important for the proper timing

of AI. Heat detection is most successful if females are subjected to a back-pressure test

in the presence of a boar once every 12 h or more frequently; snout to snout contact

with the boar is best, particularly for gilts.

Roberts (2004) reported that the 50% of estrus sows were showed immobilizing

reflex by pressure of hands on the back of sow and nearly 100% sows showed the saw

horse stance in the presence of the boar.

 Youngquist and Threlfall (2007) reported that lordosis or standing heat, usually

is identified by signs such as swelling and reddening of the vulva, vocalization, boar

seeking behaviour, ear popping and standing for back pressure.

Ramakrishnan et al. (2013) observed that the intensity score of estrus in gilts

and weaned sows along with boar was 2.8 ± 0.2 indicating that, the presence of boar in

the pen enhanced the intensity of estrus and probably the reproductive performance due

to the fact that high intensity of estrus helps in easy detection of heat and timely mating

and hence may be advantageous to the farmer.

2.5 ARTIFICIAL INSEMINATION

Artificial insemination was first attempted as a practical procedure in Russia

during the early 1900‟s (Foote, 2002) and has been used in pigs since the 1930‟s.

However, it is only in the past few decades that wide commercial application in the pig

industry has taken place, owing to standardised procedures being established. More than

99% of inseminations conducted worldwide are made with liquid-stored semen at 15-

20oC for 0-5 days with 85% of all inseminations were carried out on the day of

collection or on the following day for meat production market.

For successful AI or any controlled breeding system, the critical factors

controlling successful conception and maximum litter size are the proper timing of

insemination and the proper placement of semen. Sperm fertilizing capacity after boar

semen storage was usually assessed by artificial insemination (Johnson et al., 1988;

Johnson, 1998). However, a concern is that use of AI doses older than 12–24 h

following extension of the semen may lead to fertility losses, particularly in terms of

litter size (Waberski et al., 1994) and (Christensen et al., 2004). The reduction in

fertility of stored semen is a gradual process (beginning at ejaculation), regardless of the

extender used.
 Roca et al. (2006) recommended that at least 2.5x109 sperm cells for CAI with

semen extended in a liquid state for optimal results. However inseminating procedures

allowing semen delivery into the uterus body (intra uterine insemination, IUI) and the

proximal uterine horn (deep uterine insemination, DUI) are commercially available for

pigs which require low number of sperm (1billion) with acceptable fertility rates.

Krikwood (2006) reported that currently 3x109 spermatozoa in 80-100 ml

extender were deposited in the cervix. The large number is necessary because most of

the sperm will be lost due to backflow of semen as well as entrapment and death in

cervix and uterus.

Vazquez et al. (2008) stated that, to achieve systematic high fertility rates

among pig farms, deposition of 3x109 spermatozoa in a large volume (80-100 ml), two

or three times during the estrus period is recommended.

2.5.1 Time of insemination

Artificial insemination in gilts was done at 12 and 24 h after first standing heat

was noticed whereas, in sows at 24 and 36 h after first standing heat was observed as

the ovulation in pigs occur at 2/3rd of the estrus.

Waberski et al. (1994) stated that acceptable fertilization rates (≥ 80 %) were

achieved with inseminations (2 to 3×109 spermatozoa) done within 24 h prior to

ovulation in gilts. Rozeboom et al. (1997) reported that inseminating late in estrus or

just after the end of estrus could actually decrease the farrowing rate of gilts and

second-parity sows and also decrease the number of total and live-born piglets in all

pregnancies.
 Nissen et al. (1997) reported that wider insemination-to-ovulation interval range

of 28 h pre to 4 h post ovulation resulted in optimal fertilization rates, farrowing rates

and litter sizes in sows.

Garcia et al. (2007) observed farrowing rates for sows receiving semen 6 h prior

to the predicted time of ovulation were greater than those of sows receiving semen 24 h

prior to the predicted time of ovulation (85% versus 61%).

Roca et al. (2011) reported that, highest fertility could be achieved when liquid-

stored semen is placed in the reproductive tract within 12-24 h before ovulation,

regardless of sperm number delivered.

2.6 CONCEPTION RATE

Conception and farrowing rates after artificial insemination in swine depend on

the time of insemination relative to ovulation (Soede and Kemp, 1997). The best

conception rates and litter sizes were achieved when the female is inseminated

anywhere from 24 h before to 4 h after ovulation (Waberski et al., 1994), with the

ovulation occurring anywhere from 10-85 h after the onset of estrus (Soede and Kemp,

1997). As a general rule the farrowing rates were 65 to 70 % when the semen was used

in the first 2 days after collection, but they reduced to about 50 % with 5 day-old semen

(Johnson et al., 1988; Johnson, 1998).

Guthrie and Polge (1976b) reported that approximately 80% of the gilts with

PGF2α-synchonized estrus had embryos 4-7 days or 24-30 days after insemination.

Guthrie and Polge (1978) reported that 85% of fertility was observed with Cloprostenol

induced abortion and synchronized estrus.

Machaty et al. (1992) reported that inseminations with BTS-diluted semen

containing 5×109 spermatozoa and used on both D 0 and D 4 achieved a farrowing rate
significantly higher than that of MK-diluted semen (74.5% vs. 54.2%; P < 0.05). Sows

and gilts inseminated with BTS semen had a greater total number of piglets born alive

per litter when compared to MK semen (9.5 vs. 8.9; P < 0.05).

Alexopoulos et al. (1996) reported that the farrowing rate in relation to the

number of spermatozoa per insemination dose 1x109, 3x109 and 5x109 were 80.0, 82.5

and 87.5%, respectively in sows inseminated with BTS diluted semen within 0-24 h

after semen collection. Steverink et al. (1999) reported that the double inseminations on

an average improve farrowing rates from 80.8 to 85.1% for sows and from 81.2 to

88.2% for gilts.

Kuster and Althouse (1999) reported that the gilts inseminated with the

Androhep B diluted semen showed a decrease (P < 0.001) in farrowing rate when

compared with gilts inseminated with the X-CELL semen when stored for 5 to 6 d prior

to use. Gilts inseminated with Androhep B extended semen produced smaller litters

when semen was stored for 4 to 5 d (p < 0.05). No differences were detected in the litter

size or farrowing rate for gilts bred with semen stored for 2 to 6 d in the X-CELLTM

extender (P > 0. 1).

Tardif et al. (1999) reported that the overall conception rates of gilts inseminated

with optimal and suboptimal semen doses (3x109 vs 0.3x109 sperm/dose) were 87.2

±17.5% and 68.6 ± 22.8%, respectively. Whereas, fewer foetuses were obtained from

gilts inseminated with the suboptimal semen dose 5.9 ± 4.9 and with optimal dose were

8.4 ± 4.6.

Gadea (2003) recorded the fertility and litter size after insemination with semen

preserved for 4-14 and 28-38 h in BTS was 67.7, 69.8% and 11.96 and 11.73,

respectively. He found a significant difference of (p < 0.05) in litter size.

 Bracken et al. (2003) studied that the recovery of embryos and unfertilized

oocytes (UFO) from gilts inseminated with semen extended to 80 ml for each treatment

dose using a modified Modena extender and was used within 24 h of collection, for

either a single low dose of 0.5×109 spermatozoa, a single high dose of 3×109

spermatozoa or multiple high doses, two inseminations of 3×109 spermatozoa per

insemination. Embryos recovered were 4.2 ± 1.2, 8.1 ± 1.9, 9.7 ± 2.2 (p< 0.04); UFO

were 8.5 ± 1.1, 5.9 ± 1.8, 2.5 ± 2.1 (p < 0.05); Fertilization rate were 34.5 ± 8.9, 56.5 ±

14.4, 77.2 ± 16.4 % (P < 0.07) respectively.

Zhou et al. (2004) reported that both fertilization and cleavage rates did not

change significantly until day 8 and morulae were obtained with spermatozoa from day

2 and day 7 of semen storage when in vitro matured oocytes were inseminated with

spermatozoa that had been stored in ZO+PVA extender for different days. The cleavage

rates (35.0-28.5 %) obtained with semen stored up to 8 days were similar.

Anil et al. (2004) reported the association of semen ages, parity, Wean-to-

Service Interval (WSI) and number of AI with farrowing failure. As the semen age

increased, the farrowing failure increased and total born per litter decreased in sows and

gilts. The likelihood of farrowing failure among sows increased significantly

(P < 0.001) with the age of the semen. Farrowing failure was significantly (P < 0.001)

less for sows of parity 2-5 compared to sows of parity > 5, a lower total born per litter

was associated with parity > 5 (P < 0.001). The double insemination was positively and

significantly (P < 0.001) associated with the number of total born per litter.

Kadirvel et al. (2004) reported that the farrowing rate (%), average litter size

with single and double inseminations were 68 and 77 %, 7.6 ± 0.3 and 9.2 ± 0.2,

respectively with a significant difference (P ≤ 0.05) between single and double

insemination. Langendijk et al. (2005) reported that introduction of a boar during

insemination was probably an adequate way of stimulating contractility because

oxytocin release was effectively induced in all sows and because boar presence

selectively stimulates contractility in those sows with low spontaneous uterine activity.

Behan and Watson (2006) reported that, the farrowing rate and litter size could

not differ with that of natural service when LWY gilts were inseminated with 1 and 2

billions of sperm with the golden gilt catheter (IMV Technologies). They also reported

that conception rate and number of embryos in gilts after insemination twice in a single

estrus with the golden gilt catheter at reduced sperm numbers with 1, 0.5, 0.25 and 0.25

(109 spermatozoa), volume (ml) 80, 80, 80, 40 and observed conception rates were 88.2,

76.5, 12.5 and 25.0% and mean number of embryos were 11.06, 8.6, 5.50 and 7.25,

respectively.

Haugan et al. (2007) observed that the farrowing rates in multiparous sows were

84.8 % and 85.6 %, respectively by double insemination with X-cellTM and BTS.

Fitzgerald et al. (2008) recorded that the farrowing rates were 67.8 and 66.3%, while

total numbers of piglets born were 9.39 ± 0.55 and 9.74 ± 0.53 per litter for the

disposable foam-tipped intrauterine catheter and cervical catheter groups, respectively.

Hafez and Hafez (2008) reported that, fertilization rate in pigs was more than

90%. Ugwu and Igboeli (2009) reported that conception rate (%) of gilts inseminated

with semen stored in raffia palm sap extender (RPE) for 24, 48 and 72 h were 83.3, 66.6

and 16.6 %, rerspectively and average number of piglets born were 8.0, 8.0 and 6.5,

respectively with a high significant variation (p < 0.01).

Cupps (2009) reported that, the pregnancy rates in swine were 85-95% and

65- 90 %, respectively with natural mating and AI with fluid semen. Noguchi et al.

(2010) reported that the conception rates in cycling animals and pseudo pregnant

animals treated with Dinoprost were 80 and 83%, respectively after AI.
 Broekhuijse (2012) reported that, the farrowing rate (FR %) was 83.1 ± 29.9,

82.7 ± 29.2, 80.0 ± 31.8 and 84.0 ± 27.6 % and total number of piglets born (TNB)

were 12.7 ± 2.2, 12.8 ± 2.1, 13.1 ± 2.0 and 13.0 ± 2.0, respectively with 60, 70, 80 and

90% of semen motility.

Ronald et al. (2013) reported that, the conception rates were 100 and 100%

respectively with AI and NS and litter size in AI group was 8.36 ± 0.28 and natural

mating group was 10.6 ± 0.64, respectively which showed a highly significant variation.

Rogozarski et al. (2013) reported that, the farrowing rates were 94.38 and 78%

in sows inseminated applying intra-uterine (post-cervical) insemination technique with

special balloon catheters and in control group, respectively.

Apic et al. (2015) reported that farrowing rates following intra cervical

insemination with 4×109 spermatozoa in volumes of 50 ml (73.3%), and with 2×109 or

4×109spermatozoa in volumes of 100 ml (76.7% or 83.3%, respectively) were not

significantly different (p > 0.05) from each other. The farrowing rates of intrauterine

insemination with doses of 50 ml were 76.7 and 83.3% for spermatozoa numbers of

2×109 and 4×109, respectively and were not significantly different (p > 0.05) from each

other. The average number of live-born piglets per litter ranged from 9.85 to 10.27

piglets in the classic intracervical insemination and 10.04 to 10.82 piglets in the

intrauterine insemination.

Patra et al. (2016) reported that the conception rate, litter size of artificial

insemination conducted at Mega Seed Project farm for Large Black 75%, 10.67;

Ghungroo 91.0%, 8.80; Hampshire 83.3%, 11.75; Hampshire-Ghungroo cross 87.5%,

11.86, respectively with an overall conception rate of 83.93% and the litter size was

10.09.
2.7 PREGNANCY DIAGNOSIS

Early identification of non-pregnant animals could facilitate rapid rebreeding or

timely removal to maximize their value as cull sows. A common pregnancy detection

technique is observation of sow for failure to return to estrus within 17-24 days after

mating. Almond and Dial (1986) reported 98 % of accuracy in predicting farrowing rate

by daily estrus detection throughout the gestation period.

Flowers et al. (1999) observed that the best time for pregnancy diagnosis is at 21

d post mating due to the accumulation of the fluid in the embryonic vesicle. De Rensis

et al. (2000) reported that the efficiency for RTU post AI was 71, 83, 75, and 91% on

days 15, 16, 17, and 18th of gestation respectively.

Miller et al. (2003) reported that by using transrectal RTU 70% of sows were

diagnosed by day 20 of gestation with > 90% accuracy and 98% of sows were

diagnosed by day 22 of gestation with 94 % accuracy. If transrectal RTU is performed

on gestation days 20th to 22nd, sows which were returning to estrus may be identified

earlier. However, the time required for performing transrectal RTU might limit its use

for pregnancy diagnosis on a routine basis. When transabdominal RTU was used 91%

of sows were diagnosed with 95% accuracy by day 24th of gestation. This procedure is

faster and easier to perform than transrectal ultrasound. They were also reported that the

fluid diameter increased to day 30, decreased to day 39 and increased thereafter. The

largest fluid vesicles occurred on day 30th with a diameter of 6.6 cm whereas on day

45th the diameter decreased to 4.5 cm.

Youngquist and Threlfall (2007) reported the accuracy of early pregnancy

diagnosis in swine by using real time ultrasonography (5 mHz sector probe) on 17-20,
21-23, 24-30, 38-44, and 52-58 days of gestation was 74.5, 97, 97.8, 98 and 98.7 %,

respectively.

Williams et al. (2008) scanned sows for pregnancy diagnosis between 17th and

24th d post-mating (PD1) and reconfirmed between 38 and 45th days of gestation (PD2).

The sensitivity, specificity and predictive value of a positive result comparing results

from PD1 with farrowing by using RTU. The efficacy between PD1-farrowing rate was

75.5%, and between PD1-PD2 was 80.6%. They were also reported that after 21 days of

gestation, RTU had over 90% sensitivity, 45% specificity and 70% of efficiency.

Cupps (2009) reported that non-return rates in sows were 90- 95 % accurate at

18-25 days after AI. Aiello and Moses (2016) reported that pregnancy was most

commonly diagnosed by noting that, the female does not return to estrus within 18-25

days, which showed 75%–85% of accuracy.

CHAPTER III

MATERIALS AND METHODS

3.1 SELECTION OF EXPERIMENTAL ANIMALS

A total of 44 (24 cycling and 20 seasonally anestrus) non-pregnant, healthy gilts

or sows of either LWY cross breed or local breed belongs to Instructional Livestock

Farm Complex, Rajendranagar and a private farm, at Mahabubnagar were selected. The

farm was located at 17º 20 longitude, 78º 24 latitude and 536 m above the mean sea

level.

Pigs were screened for reproductive tract abnormalities and pregnancy by using

ultra sound scanner (Aloka) with 5 MHz transabdominal probe. Selected pigs were

separated and properly identified, screened for brucellosis, dewormed and vaccinated as

per schedule. The animals were housed, ear tagged and reared under identical conditions

of feeding and management. All the animals were reared by intensive system in a

permanent house. Boars were always kept away from sows or gilts. The animals were

re-examined after 20 days to detect any early pregnancy, which was missed during the

first scanning.

Three mature and fertile Large White Yorkshire (LWY) cross breed boars aged

10-16 months with similar, ideal body condition score „3‟ (Fig.No. 3.1 and Table 3.1)

were subjected to breeding soundness evaluation. Physical examination and palpation of

the scrotum along with its contents were made and the scrotal size was also measured.

Boars with scrotal size (7 × 11 cm) and the ones which produced the best quality semen

were selected from the flock and raised at Instructional Livestock Farm Complex,

Rajendranagar for semen collection. Semen evaluation was done initially for
 Fig. No. 3.1 Body condition score of the pigs

Table 3.1: Body condition score index

Score Body condition

1 Emaciated sow back bone very prominent.

2 Thin backbone prominent.

3 Ideal condition during lactation and at weaning, backbone just palpable.

4 Slightly overweight. Cannot find the back bone

5 Body round, over fat.

30 days period covering 10 samples per boar to evaluate better semen producing animal.

The boars were maintained under uniform managemental conditions and feeding was

done as per NRC feed standards and constant nutritional regime and provided adlibitum

water during the period of the study. Semen was collected from each boar twice in a

week by double hand gloved method. Same boars were used for semen collection

throughout the experimental period.

3.2 EXPERIMENTAL SEMEN EXTENDERS PREPARATION

In the present study two different extenders were used i.e. PRIMXcell and

NBSE for semen dilution and preservation.

PRIMXcell* extender is a ready to use powdered substance available in the market. The

extender was reconstituted with double distilled water (50 gm/1lit) to prepare required

quantity of diluent solution and stirred well until the powdered form was completely

dissolved.

NBSE** extender is a ready to use powdered substance. The extender was

reconstituted with double distilled water (50 gm/1lit) to prepare the required quantity of

diluent solution and stirred well until the powdered form was completely dissolved.

The extenders prepared were kept in a water bath at 37ºC for a minimum one

hour to allow for temperature and pH equilibration (7.8). The pH was checked with the

digital pH meter, any deviation of pH if found was adjusted by using N/10 HCl solution.

*PRIMXcell IMV Technologies, France.

**NBSE (Normal Boar Semen Extender) ICAR Central Coastal Agriculture Research
Institute, Goa, India.
3.3 SEMEN COLLECTION AND EVALUATION

Semen was collected from trained LWY cross breed boars which were selected

for this study. They were maintained in the semen collection area that started one month

before beginning of the experiment. The glassware required for semen collection was

properly cleaned with non- spermicidal detergent (Labolin) and sterilized in a hot air

oven prior to usage. The preputial opening and surrounding area was cleaned by using a

single-use disposable wipe to remove any debris and the preputial fluids are evacuated

manually prior to grasping the penis for semen collection. Semen was collected

aseptically from boars using a stainless steel dummy sow (Fig.No. 3.2) by “Double

hand gloved method” into a graduated glass beaker (Fig.No. 3.3). Immediately after

semen collection it was sent to the laboratory as soon as possible for further evaluation.

In the laboratory the volume was determined by using a graduated measuring

cylinder and colour and consistency were also recorded. Semen was transferred to water

bath at 37ºC and maintained for 20-30 min. During this time the semen sample was

assessed for motility, concentration and smears were prepared for viability and

abnormality count as per the standard procedures.

3.3.1 Estimation of motility and concentration

A small drop of fresh semen was placed on a warmed (37°C/99°F) microscope

slide and overlaid with a cover slip. Sperm motility was then estimated to the nearest

5% by viewing groups of sperm in at least 4 different fields on the slide at 10X; these

readings were then averaged. Only ejaculates with at least 70% gross motility were used

for further processing. Sperm concentration was measured with Neubauer

haemocytometer by using eosin diluting fluid (Eosin Y 0.05 g, sodium chloride 1.00 g,

formaldehyde 1.00 ml, distilled water up to 100 ml).

 Fig.No3.2 Stainless steel Dummy sow

Fig.No3.3 Semen collection by Gloved Hand method

3.4 SEMEN PROCESSING AND STORAGE

After estimating the concentration, the semen was immediately extended with

the diluents to the final concentration of 3×109 spermatozoa per dose (60 ml). After

dilution, semen containing beakers were closed with sterile aluminium foil and were

shifted to the Biological Oxygen Demand incubator where temperature was maintained

at 15-18ºC.

Ejaculates having thick consistency, rapid wave motion, ˃ 70 % motility, ≥ 85%

normal sperm morphology and concentration ˃ 25 to 65×106 sperm/ml were used for

further processing.

3.5 STERILIZATION

Glassware was sterilized by using dry heat at 160ºC for one and half hour in a

hot air oven. Golden pig catheters, double distilled water were sterilized at 15 psi for 15

min, in an autoclave.

3.6 ESTRUS

The interval between weaning and onset of estrus, hormonal treatment and time

of first appearance of estrus signs were calculated.

3.6.1 Natural and Synchronized Estrus

Twenty four cycling animals were selected for natural estrus were randomly

divided into two groups having 12 pigs in each group. The animals in the first group

were kept as control and observed for natural heat and bred by natural service. The

remaining 12 animals in the second group were observed for natural estrus and

artificially inseminated with experimental diluents. Twenty selected seasonally anestrus

pigs were utilised for synchronization of estrus with 175 µg of Cloprostenol sodium

injection intramuscularly. The animals which responded to the line of treatment were

artificially inseminated.

3.6.2 Estrus detection

All the animals which were selected for the present study were kept under close

observation. Estrus detection was done twice daily regularly in pubertal gilts and in

sows after 3 days of weaning. Whereas, in case of synchronized pigs heat detection was

done regularly twice a day for 1 to 7 days from the day of treatment. The % of estrus

occurrence during the 7 days after synchronization was quantified. The visual estrus

signs like homosexual behaviour (Fig.No3.4A), Shoulder scratches (Fig.No3.4B),

vulval swelling and reddening (Fig.No3.4C), clear vaginal discharge (Fig.No3.4D),

lordosis (Fig.No3.4E), riding test (Fig.No3.4F) and boar seeking behaviour were

considered to be positive for estrus.

3.6.3 Intensity of estrus

The intensity of estrus was studied using behavioural and physiological changes

during estrus and it was scored as described by Ramakrishnan et al. (2013). The score

card used in this study was as follows:

Table 3.2: Estrus intensity grading chart

Sl.No Description Score

Off feed, keeping away from other animals, restlessness, reddening of

1
1.
vulva
Boar seeking behaviour, slight discharge from vagina, swollen vulva,
2. 2
estrus grunt
Mounting on other animals, restlessness, immobility reflex to back
3. 3
pressure
Fig.No3.4A Mounting behaviour Fig.No3.4B Shoulder scratches

Fig.No3.4C Swollen reddened vulva Fig.No 3.4D Clear and sticky mucus

Fig.No3.4E Lordosis Fig.No3.4F Riding test (standing reflex)

3.7 EXPERIMENTAL DESIGN

The sows or gilts with natural and synchronized estrus were selected to study the

conception rates using different semen diluents and were divided into five groups, one

was control group and remaining four were treatment groups corresponding to natural

and induced estrus. The semen diluents used in the study were PRIMXcell and NBSE.

The animals were assigned into the following groups.

3.7.1 Group 1 (n=12)

This group was assigned as control group and consists of 12 natural estrus pigs

and were bred by natural service using a fertile LWY 75% cross breed boar.

3.7.2 Group 2 (n=12)

A total of 12 cycling pigs that were observed for natural estrus and bred by CAI

after 12 and 24 h and 24 and 36 h after onset of estrus in gilts and sows, respectively

using diluted semen with 2 diluents. This group was further subdivided into two

subgroups based on the diluents used.

3.7.2.1 Subgroup 2A (n=6)

A total of 6 pigs were bred by semen diluted with PRIMXcell diluent which was

utilised within 24 h of dilution of the semen.

3.7.2.2 Subgroup 2B (n=6)

A total of 6 pigs were bred by semen diluted with NBSE diluents which was

utilised within 24 h of dilution of the semen.

3.7.3 Group 3 (n=20)

This group comprised of 20 seasonally anestrus pigs (sows that failed to exhibit

estrus two weeks after weaning and gilts which obtained optimum body weight and

failed to exhibit estrus) were treated with 175 µg of Cloprostenol sodium*** and AI

was done in pigs which exhibited estrus after hormonal treatment by CAI 12 & 24 h

and 24 & 36 h after onset of estrus in gilts and sows, respectively using diluted semen

with 2 diluents. This group was further subdivided in to two subgroups based on the

semen diluents used.

3.7.3.1 Subgroup 3A (n=10)

A total of 10 pigs that exhibited estrus after synchronization were bred by semen

diluted with PRIMXcell diluent which was utilised within 24 h of dilution of the semen.

3.7.3.2 Subgroup 3B (n=10)

A total of 10 Pigs that exhibited estrus after synchronization were bred by semen

diluted with NBSE diluent which was utilised within 24 h of dilution of the semen.

***VETMATE: Akums Drugs and Pharamaceuticals Limited, Haridwar.

3.8 METHOD OF ARTIFICIAL INSEMINATION

Cervical artificial insemination was done in pigs with a golden pig catheter and

20 ml glass syringe. KY-jelly was used as lubricant (Fig.No3.5). Pigs which were

allotted to group 2 and 3 were bred by using 60 ml of semen containing

3×109spermatozoa at 12 & 24 h and 24 and 36 h after onset of estrus in gilts and sows.

The female which was to be inseminated was brought to an area where she could smell,

see or hear a boar and backpressure was applied to make sure that she was in the

standing heat. The vulval region of the animal was cleaned with single use disposable

wipe so that no dirt or manure was introduced into the reproductive tract when the

insemination rod was inserted. The golden pig catheter was inserted at an angle of 30º to

the backbone after proper lubrication until resistance was felt (Fig.No3.6). The

inseminating catheter was gently twisted counter clockwise until the tip was properly

locked into the cervix. The glass syringe was connected to the catheter and the liquid

semen was deposited slowly with the help of the syringe by simultaneously rubbing the

flank and underline region to stimulate the female to suck semen into the uterus

(Fig.No3.7). After complete deposition of the semen catheter was gently withdrawn by

simultaneously twisting it in a clockwise direction. The second insemination was done

12 h after the first AI with semen collected from the same boar and diluted in the same

extender which was used in the first AI.

 Fig. No. 3.5 Equipment used for estrus synchronization and AI in swine

(e)

(d)

(a)
(c)

(b)

(a).Vetmate (b).Golden pig catheters (c).Glass syringes (d).K-Y jelly (e).Semen

extenders.

Fig. No. 3.6 Inserting golden pig catheter at 30º angle

3.9 PREGNANCY DIAGNOSIS

Early pregnancy diagnosis was done on the basis of non-return rates and

ultrasound scanning after 30 days of AI.

3.9.1 Non-return rates

To detect pigs returning to estrus, all the pigs inseminated were detected for

estrus at the next expected estrus cycle (18-25 days) based on behavioural and visual

signs of estrus twice daily. Pigs which were not returning to estrus were considered as

pregnant.

3.9.2 Ultrasound scanning

Pregnancy diagnosis was performed by using ultrasound scanner* with 5.0 MHz

transabdominal probe 30 days after AI. The results were confirmed by rescanning at

45th and 60th days after AI. (Fig.No3.8)

3.10 STASTISTICAL ANALYSIS

A Chi- square test was used to analyze the conception rates among the groups at

5% level of significance using SPSS software. A paired sample t-Test was used to

analyze and correlate estrus parameters.

*Aloka pro sound, Hitachi Aloka Medical Ltd, Japan.

Fig. No. 3.7 Cervical Artificial Insemination method

Fig. No. 3.8 Transabdominal ultrasonography

 CHAPTER IV

RESULTS

The present work was undertaken to select the best semen diluent for AI and to

study the conception rates after CAI in natural and synchronized estrus in sows or gilts

of either LWY cross breed or local breed, by utilising three LWY cross breed boars and

forty four sows or gilts. Experiments were designed to study the conception rate with

two semen extenders i.e, NBSE and PRIMXcell in both natural and induced estrus. At

the same time estrus (natural and synchronized) parameters were evaluated to know the

efficacy of the Cloprostenol (VETMATE) for inducing estrus in pigs.

4.1 ESTRUS

Estrus parameters like weaning to estrus interval, farrowing to estrus interval,

duration of estrus were recorded. At the same time to know the efficiency of estrus

synchronization with 175 µg Cloprostenol administered by intramuscular route.

Parameters like estrus response rate, interval between hormone treatment and onset of

estrus, duration of induced estrus and estrus intensity were recorded.

4.1.1 Weaning to Estrus Interval (WEI)

The interval between weaning to onset of estrus was measured. The average

WEI was 5.66 ± 1.20 days (Mean ± SE) and ranged from 3 to 14 days. A negative

correlation was observed between WEI and duration of estrus (r = -0.414), indicating

that longer weaning to estrus intervals had shorter duration of estrus (Table 4.1, 4.2 and

4.3).
4.1.2 Interval between hormonal (PGF2α) treatment and onset of

estrus

The onset of synchronised estrus was calculated from the time of administration

of hormone to the time of first appearance of estrus symptoms. The mean interval

between Cloprostenol treatment and the onset of estrus was 3.75 ± 0.44 days ranging

from 3-7 days (Table 4.3).

4.1.3 Interval between farrowing to estrus

The average period between farrowing to onset of estrus was 52.16 ± 2.94 days

(Mean ± SE) and ranged from 42 to 70 days after farrowing. A positive correlation was

observed between weaning to estrus interval and farrowing to estrus interval (r = 0.398),

indicating that increase in weaning to estrus interval, increased the farrowing to estrus

interval. A strong negative correlation was noticed between farrowing to estrus interval

and duration of estrus (r= -0.616; p < 0.05) which is statistically significant, indicating

an increased FEI decreased the DE (Table 4.1 and 4.2).

4.1.4 Duration of Estrus

The interval between first acceptance of mounting and last acceptance of

mounting by a boar. The mean duration of estrus observed in natural estrus was 57.83 ±

2.22 h (Mean ± SE) and 42.00 ± 1.80 h in induced estrus (Table 4.3) which was

significantly different (p < 0.05).

4.1.5 Estrus response rate

Estrus response rate was calculated by number of pigs in estrus divided by

number of pigs treated (weaning and hormonal treatment) and multiplied by hundered.
 All the animals (sows or gilts) in the control group and in group 2 selected for natural

estrus exhibited estrus (100%). The animals (sows or gilts) which responded to

synchronization of estrus were 12 out of 20 i.e., 60% of animals expressed estrus within

3-7 days after Cloprostenol injection (Table 4.3).

4.1.6 Estrus intensity

Estrus intensity was graded from 1 to 3 (grade 3 was better). All the pigs (100%)

in control as well as group 2 selected for natural estrus expressed 2nd and 3rd grade estrus

intensity.

Out of 20 pigs synchronized for estrus 25% (5/20) of the pigs expressed of 2nd

and 3rd grade estrus intensity and 35% animals (7/20) expressed 1st grade estrus intensity.

The remaining 40% (8/20) animals could not respond to the hormonal treatment (Table

4.1 and Fig.No 4.3).

Table 4.1: Weaning to Estrus Interval and Farrowing to Estrus Interval in
natural estrus

Natural estrus Mean ± SE Range

WEI (days) 5.66±1.20 3-14

FEI (days) 52.16 ± 2.94 42-70

Table 4.2: Correlation between Weaning to Estrus Interval, Farrowing to Estrus

Interval and Duration of Estrus with natural estrus

Weaning to Farrowing to Estrus Duration of estrus

Estrus Interval Interval
WEI --- 0.398# -0.414*

FEI --- --- -0.616**

Duration of estrus --- --- ---

#A moderate positive correlation exists between WEI and FEI.

*A moderate negative correlation exists between WEI and duration of estrus.

**A large negative correlation is observed between FEI and duration of estrus.
Table 4.3: Comparison of estrus parameters (mean ± SE) between natural and synchronized estrus in LWY cross

bred or local pigs.

Estrus parameters Natural estrus Induced estrus

Estrus response rate (%) 100a 60b

Onset of estrus (days) 5.66±1.20a 3.75±0.44b

Duration of estrus (h) 57.83±2.22a 42.00±1.80b

Estrus intensity (%) 100a 25b

The values with different superscripts in rows differ significantly (p ≤ 0.05)

 Fig. No. 4.1 Estrus intensity grading in natural and synchronised estrus

Estrus intensity for natural and synchronised estrus

14
12
12

10
No.of
animals 8
7
6 Natural estrus
5
4 Synchronised estrus

2
0
0
1st grade 2nd and 3rd grade

Estrus intensity
4.2 CONCEPTION RATE

The conception rate was calculated as % ratio of number of pigs positive for

pregnancy to number of pigs inseminated. In group 2A, the conception rate obtained

using diluted semen utilised within 24 h of dilution with PRIMXcell was 100%. While

in group 3A, with synchronized estrus was 33.33% with an overall conception rate of

66.66% for PRIMXcell diluents (Table 4.4, Fig.No. 4.2 and 4.3).

In group 2B, the conception rate obtained using diluted semen utilised within 24

h of dilution with NBSE was 100% while in group 3B, with synchronized estrus was

50% with an overall conception rate of 75.00% for NBSE diluted semen (Table 4.4,

Fig.No. 4.2 and 4.3).

The overall conception rate in group 2 by using both the diluents was 100%. In

group 3 conception rate with PRIMXcell was 33.33% and with NBSE was 50%

(Fig.No. 4.2) with an overall mean of 41.65% which was statistically significant (p <

0.05). This indicated that the conception rate with natural estrus was higher than

synchronized estrus (Fig.No. 4.4), but the diluents did not differ significantly (P ˃ 0.05)

in the conception rate for synchronized estrus (Table 4.4). However, NBSE semen

diluent group had showed better conception rate over the PRIMXcell semen diluent

group.

In group 1 the overall conception rate with natural estrus and natural service by a

boar was 100 %. When group 1 is compared with group 2A, 3A and with group 2B, 3B

the chi square value was 0.862 with (P ˃ 0.05) no significant difference in overall

conception rates (Table 4.4). Hence, this indicates that AI at natural esrus with both the

diluents used in the experiment yielded similar results of conception rates with that of

natural service by a boar.

 When compared in between two diluents in terms of conception rate, there is no

significant difference (P ˃ 0.05) between diluents. Hence, it was concluded that LWY

cross bred boar semen can be diluted with PRIMXcell or NBSE without much variation

in the fertility when utilised within 24 h of dilution for artificial insemination.

Table 4.4: Conception rate in LWY or local pigs with artificial insemination using diluted semen with two

different diluents and with natural and synchronized estrus.

Diluents Control Natural estrus Synchronized estrus

Overall conception rates
Group 1 Group 2 Group 3
(subgroup 2A) (subgroup 3A)
PRIMXcell 66.66%(8/12)NS
a b
100%(6/6) 33.33%(2/6)
100 %(12/12)
(subgroup 2B) (subgroup 3B)
NBSE 75.00%(9/12)NS
a b
100%(6/6) 50.00%(3/6)

Overall 100%(12/12) 100%(12/12)a 41.65%(5/12)b 70.82%(17/24)NS

NS – Non significant

The values in the table, with different superscripts vary significantly (p < 0.05) and with similar superscripts do not vary
significantly (p ˃ 0.05).
Fig.No. 4.2 Conception rate in LWY or Local pigs artificially inseminated
to natural and synchronized estrus.

100%(6/6) 100%(6/6)
100
90
80
70
60 50%(3/6) NBSE
50 PRIMXcell
40 33.3%(2/6
30 )
20
10
0
Natural estrus Synchronized estrus
estreseestrus

Fig.No. 4.3 Overall conception rate with AI using NBSE and PRIMXcell
extenders

76

74

72

Conception
70
rate (%)
Observed
68 conception rate
(%)
66

64

62
NBSE PRIMXcell
Fig.No. 4.4 Comparison of conception rate in pigs artificially inseminated to natural and synchronized heat with natural service.

100 %(12/12) 100 %(12/12)

100

90

80

70

60

50 41.6 %(5/12)

40

30

20

10

0
Natural service Natural heat & AI Synchronized
heat & AI
4.3 PREGNANCY DIAGNOSIS

4.3.1 Non Return Rate

The animals were observed 18-25 days post AI for non-return to estrus and was

calculated based on return to estrus with 80 % accuracy.

4.3.2 Ultrasound Scanning

Early pregnancy diagnosis was done on 21st day post AI with ultrasound scanner

using 5 MHz transabdominal probe. Rescanning was done on 30th, 45th and 60th days.

Accuracy of pregnancy diagnosis was 75, 90, 97 and 100%, respectively at 21, 30, 45

and 60th day of gestation. (Fig.No. 4.5,Table 4.5)

4.3.2.1 Fetal Parameters

Several fetal parameters like amniotic vesicle diameter (AVD), fetal crown rump

length (CRL), biparietal diameter (BPD) were measured. The mean AVD was 2.31 ±

0.17, 4.88 ± 0.61 and 4.80 ± 0.37 cm at 21st day (Fig.No. 4.6), 30th day (Fig.No. 4.7)

and 45th day (Fig.No. 4.8) of gestation, respectively. BPD and CRL were 3.50 ± 0.01

cm and 3.88 ± 1.25 cm, respectively at 60th day of gestation (Table 4.5; Fig.No. 4.9 and

4.10). Ribs and spine were visible at 78th day of gestation (Fig.No. 4.11).
Fig.No. 4.5 Accuracy of pregnancy diagnosis with transabdominal ultrsonography by using (5MHz) linear

probe in relation to gestation perod in days.

Pregnancy diagnosis by ultrasonography

100
97

90

75

60
45
30
21

1 2 3 4

Days of gestation Accuracy (%)

 Table 4.5: Fetal parameters during transabdominal ultrasonography at different days of gestation

Amniotic vesicle diameter(AVD) Bi parietal diameter(BPD) Crown rump length(CRL)

Days of gestation
(cm) (cm) (cm)

21 2.31 ± 0.17 -- --

30 4.88 ± 0.61 -- --

45 4.80 ± 0.37 -- --

60 -- 3.50 ± 0.01 3.88 ± 1.25

 Fig. No. 4.6 Anechogenic smaller amniotic vesicles at 21 days of gestation

Fig. No. 4.7 Anechogenic amniotic vesicle with maximum size at 30th day of
gestation
 Fig. No. 4.8 Echogenic embryo within the anechogenic amniotic vesicle (45 days)

Fig. No. 4.9 An ultrasonographic measurment of CRL of fetus at 60 days of

gestation
Fig.No. 4.10 An ultrasonographic measurement of BPD of a fetus at 60 days of
gestation

Fig. No. 4.11 An ultrasonograhy of fetus at 78 days of gestation with ribs and spine
 CHAPTER V

DISCUSSION

In the present experiment, conception rate in pigs after artificial insemination

with diluted semen was studied using two different semen extenders i.e, NBSE and

PRIMXcell for both natural estrus and synchronized estrus with single injection of 175

µg Cloprostenol (Vetmate). At the same time estrus (natural and synchronized)

parameters were evaluated to know the efficacy of the Cloprostenol (VETMATE) for

inducing estrus in pigs.

5.1 Weaning to Estrus Interval

The average WEI in the sows was 5.66 ± 1.20 days. The WEI in the present

study was in accordance with the studies of Youngquist and Threlfall (2007) and Hafez

and Hafez (2008). A negative correlation was noticed between increased weaning to

estrus interval and duration of estrus. The similar findings were also reported in the

studies of Weitze et al. (1994), Steverink et al. (1999) and Kemp and Soede (1996).

Optimum lactation length, balanced feeding practice during lactation might have caused

early follicular development and this might be the reason for shorter WEI. But,

Tantasuparuk et al. (2000) and Cavalcante-Neto et al. (2008) observed an increase in

litter size in the following estrus, because in longer WEI sows had a longer period to

recover from a catabolic stage around weaning. Hence, this increased BCS and may be

the reason for increased litter size. These above parameters might have been affected

due to lactation length (Marby et al., 1996), management, season etc.

5.2 Farrowing to Estrus Interval

The average FEI observed in the present study was 52.16 ± 2.22 days. A

significant negative correlation between FEI and duration of estrus was noticed. In

contrary, Correa et al. (2014) reported an increase of litter size than the present study as

FEI increased. This variation might have been due to loss of BCS during lactation,

nutrition, management.

5.3 Synchronized Estrus

The efficacy of Cloprostenol (VETMATE) for induction or synchronization of

estrus in seasonally anestrus pigs was discussed in terms of estrus response rate, interval

between hormonal treatment and onset of estrus, duration of estrus and intensity of

estrus.

5.3.1 Estrus Response Rate

The percentage of pigs that responded to hormonal (PGF2α) treatment and

exhibited estrus was 60.00%. With PGF2α analogue, similar results were also noticed

by Guthrie and Polge (1976) and Chakurkar (2009). Higher estrus response rate than the

present study was recorded by Guthrie and Polge (1978), Jochie et al. (1983) and

Noguchi et al. (2010). In contrary to this Pena et al. (2001) and Stancic et al. (2014)

observed lower estrus response rate.

The variation in estrus response observed by the researchers might be due to

variable dosage of PGF2α, different analogues of PGF2α, day of estrous cycle during

which PGF2α was injected (Connor et al., 1976, Estill et al., 1993), boar exposure

(Breen et al., 2005) etc. It is known that porcine CL would not respond to

prostaglandins prior to day 12th of estrous cycle. However, in this study Vetmate was
administered randomly without knowing the stage of estrus cycle. This might be the

reason for low degree of estrus synchronization after PGF2α treatment of pigs in the

unknown stage of spontaneous estrous cycle.

5.3.2 Interval between hormone treatment and estrus onset

The period between Cloprostenol treatment and the onset of estrus in the present

study was 3.75 ± 0.44 days which was similar to the findings of Moeljono et al. (1976)

and Chakurkar (2009). Higher interval from hormone treatment to estrus onset than the

present study was noticed by Kraeling et al. (1975), Guthrie and Polge (1978), Noguchi

et al. (2010) and Konch et al. (2012). Variable response in the interval between

hormonal treatment and estrus onset might be attributed to differences in breed,

nutrition, season, boar exposure (Breen et al., 2005) after hormone treatment and dosage

of hormone.

5.3.3 Duration of estrus

The mean duration of estrus in the present study in natural estrus was 57.83 ±

2.2 h and in synchronized estrus was 42.00 ± 1.8 h. With respect to the duration of

estrus similar results were found by Weitz et al. (1994), Soede and Kemp (1997)

Roberts (2004), Hafez and Hafez (2008) for natural estrus and by Noguchi et al. (2010)

for synchronized estrus.

A longer duration of estrus than the present study was reported by Moeljono et

al. (1976) and Konch et al. (2012) for synchronized esrtus. In contrary to this, shorter

duration of estrus than the present study was recorded by Steverink et al. (1997) and

Konch et al. (2012) for natural estrus.

 Variations in the duration of estrus might be attributed to parity, season,

weaning-to-estrus interval and farm management, age (Roberts, 2004), dosage and drug

(Stancic et al., 2014) might have been the reason for variation in synchronized estrus.

5.3.4 Estrus intensity

In the present study, estrus intensity was graded from 1 to 3 (lowest to highest)

based on intensity of estrus symptoms exhibited by pigs.

Similar estrus intensity grading system was also reported by Ramakrishnan et al.

(2013). The behavioural signs which were used to grade the estrus intensity were

similar to the findings of Youngquist and Threlfall (2007) and Roberts (2004). Pigs

showing high estrus intensity were easily identified at heat detection in the present

study.

5.4 CONCEPTION RATE

Conception rate in the present study with both the diluents (PRIMXcell and

NBSE) was 100% in natural estrus (group 2A, group 2B) which was similar to the

findings of Ronald et al. (2013) with BTS diluent, Hafez and Hafez (2008), Cupps

(2009). In contrary, lower conception rate than the present study was recorded by

Johnson (1998), Tardif et al. (1999), Bracken et al. (2003), Gadea (2003), Behan and

Watson (2006), Ugwu and Igboeli (2009) and Patra et al. (2016).

Conception rate in the present study in synchronized estrus pigs inseminated

with PRIMXcell (group 3A) was 33.33% and with NBSE diluted semen was 50%

(group 3B). Whereas, higher conception rate than the present study with synchronized

estrus followed by artificial insemination was reported by Guthrie and Polge (1976b),

Guthrie and Polge (1978) and Noguchi et al. (2010).

 However, overall conception rate with PRIMXcell was 66.66% and with NBSE

diluents was 75%. The overall conception rate for an artificial insemination with semen

diluted to 3x109 spermatozoa in 60 ml volume dose inseminated within 24 h of semen

collection in the present study was 70.82%, which was similar to the observations of

Johnson et al. (1988) and Johnson (1998).

Higher conception rate than that of the present study was found by Ronald et al.

(2013), Tardif et al. (1999), Bracken et al. (2003), Behan and Watson (2006), Ugwu

and Igboeli (2009) and Patra et al. (2016). In contrary to this lower conception rate was

observed by Gadea (2003).

The variation in conception rate with present study might be due to variations in

the dosage of semen (Alexopolus et al., 1996; Tardif et al., 1999 and Bracken et al.,

2003), semen extenders (Machaty et al., 1992; Kuster and Althouse 1999), time of

insemination (Roozeboom et al., 1997; Nissen et al.,1997; Gracia et al., 2007 and Roca

et al., 2011) relative to ovulation, variable duration of estrus, presence of boar

(Lagendejik et al., 2005) at the time of insemination, parity (Anil et al., 2004), age,

number of inseminations (Steverink et al.,1999 and Kadirvel et al., 2004), storage

period (Gadea, 2003; Ugwu and Igboeli 2009) and temperature of storage (Zohu et al.,

2004).

5.5 Pregnancy Diagnosis

The accuracy of non-return rate at 18-25 days was 80% which was similar to the

findings of Aiello and Moses (2016), but lesser than the present study was reported by

Cupps (2009). This variation might be due to habituation of standing reflex by the pigs

for back pressure test and also continuous exposure to the boar.
 Pregnancy diagnosis by ultrasonography was 75% accurate as early as 21 days

of gestation, which was similar to the findings of Youngquist and Threlfall (2007) and

Williams et al. (2008).

On 30th day the accuracy of pregnancy diagnosis was 90%, contrary to this

higher accuracy than the present study was reported by Miller et al. (2003), Youngquist

and Threlfall (2007) and Williams et al. (2008). This variation might be due to expertise

with the technique. Accuracy for pregnancy diagnosis on 45th day was 97% and on 60th

day was 100% which was similar to the findings of Youngquist and Threlfall (2007).

AVD increased from 2.31 ± 0.17 cm (21 days) to 4.88 ± 0.61 cm by 30th day of

gestation and then decreased slightly to 4.80 ± 0.37 cm at 45th day of gestation which

was similar to the observations of Miller et al. (2003). Maximum AVD at 30th day in the

present study was 4.88 ± 0.61 cm which was in contrary to the observations (6.6 ± 0.5)

of Miller et al. (2003). These variations in pregnancy diagnosis might be due to

expertise with ultrasonography and the efficiency of the equipment used.

5.6 Conclusion

The results of the present study indicated that both the diluents can be used for

storage of liquid boar semen for about 24 h without much difference in fertility with

both natural and synchronized estrus. Hence, it can be concluded that acceptable

conception rates may be obtained with artificial insemination using liquid boar semen

stored at 15-18ºC. However, NBSE semen diluent is recommended for Indian

conditions as it had showed higher conception rate than the PRIMXcell diluent. Added

advantage to the NBSE diluents is, it is cost economical when compared to PRIMXcell

diluent.
 CHAPTER VI

SUMMARY

The present study discusses about the efficacy of semen diluents (PRIMXcell a

newly launched short term extender prepared by IMV and NBSE a short term extender

prepared by ICAR central costal research institute, Goa, India) on the fertility of the

LWY crossbred boar diluted liquid semen at 15-18ºC with natural and synchronized

heat. Forty four non-pregnant pigs (gilts or sows) were selected after screening the

animals with ultrasound for pregnancy; the boars of 1-3 years of age were selected and

trained with a dummy sow for semen collection.

Twelve animals kept as control (group 1) observed for natural estrus and bred by

natural service with a fertile boar, rest of the animals (n=12) were observed for natural

heat and were inseminated (group 2) with NBSE diluted semen (n=6) and also using the

PRIMXcell diluted semen (n=6). The other (n=20) animals (group 3) which could not

exhibit estrus within 14 days after weaning or gilts after attaining optimum body weight

were subjected to estrus synchronization with 175 µg of Cloprostenol (VETMATE)

intramuscularly.

Semen was collected from the boars during early hours of the day and was

subjected for routine laboratory examinations like mass motility, individual motility,

viability and concentration. The fresh undiluted semen with ≥ 75% motility, 80%

normal morphology and 70% viability was processed for dilution and utilised for AI

within 24 h after the dilution.

The WEI, FEI and duration of estrus were studied for natural estrus. The average

WEI was 5.66 ± 1.20 days, average FEI was 52.16 ± 2.94 days and average duration of

estrus was 57.83 ± 2.22 h.

 The estrus response rate was 100 % in animals selected for natural estrus with

2nd and 3rd grade estrus intensity. However, estrus response rate with Cloprostenol was

60% (12/20) and average time taken to exhibit estrus after hormone treatment was 3.75

± 0.44 days with an average estrus length of 42.00 ± 1.80 h. However, only 25%

animals exhibited estrus behaviour with 2nd and 3rd grade and 35% animals showed 1st

grade estrus intensity, remaining 40% animals could not exhibit estrus.

Pregnancy diagnosis was done based on non-return rates and ultrasonography.

Accuracy of non return rate was 80% and that of ultrasonography was 75% on 21st, 90%

on 30th, 97% on 45th and 100% at 60th day of gestation.

Conception rate with both the diluents was 100% in natural estrus and with

synchronized estrus for NBSE and PRIMXcell was 50% and 33.33%. The overall

conception rate with NBSE was 75% and with PRIMXcell was 66.66%. The results

indicated that the overall conception rate for both the diluents were not significantly

different. Conception rate with natural service and AI with natural estrus was similar

but, AI with synchronized estrus was significantly lower.

The study concluded that both the diluents could be used for dilution of boar

semen at 15-18ºC. However, NBSE is recommended over the other diluent. Conception

rate with natural estrus was far higher than the synchronized estrus, indicating that there

is a need for standardization of PGF2α (Cloprostenol) synchronization protocol in pigs.

Diluents for long term storage of boar semen at lower temperatures (0-5ºC) for

cryopreservation of boar semen, the use of cryopreserved boar semen for artificial

insemination and synchronization protocols for pigs need to be further investigated in

the future studies.

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