Professional Documents
Culture Documents
semen:
Present status and future
prospects
S.K. Jindal
PR&SM Division,
CIRG, Makhdoom
IMPORTANCE OF GOATS IN RURAL
ECONOMY
Goats are reared by more than 70 % landless,
marginal and small farmers of the rural India.
They significantly contribute to the agrarian
economy and play a very vital role in the
livelihood security of the small and marginal
farmers and landless labourers especially in
arid, semi-arid and mountainous regions of the
country.
The socio-economic value of goat rearing as
compared to other livestock for poor farmers is
immense.
The low input, high fecundity, easy
marketing and unprejudiced social
acceptance of its products are few of
many advantages of this enterprise that
provides assured higher income. Goat
also is one of the main meat animals in
India, and its meat is most preferred.
Goats have distinct social, economical,
managerial and biological advantages
over other livestock species.
Milk production per cow during the past 25 years in
USA ( National Average (Kg)
1950 2415
1955 2,655
1960 3195
1965 3772
1970 4431
1975 4706
1979 5227
(Foote, 1981)
Our goats are low producers in terms of milk and meat as
compared to European breeds of goats.
1952 First calf born after frozen thawed bull Chris Polge and
semen Tim Rowson
1963 Developed Tris-buffered egg yolk-glycerol Davis et al.
Artificial insemination technique based on the
principle of cryopreservation originated before
the second world war in several European
countries.By 1945, the use of artificial
insemination was not successful as a tool for the
genetic improvement but it did get cows in calf.
Therefore, for progeny testing of dairy bulls
artificial insemination became an important
scientific tool. However, it was soon realized that
it took so long to accomplish ‘proof’ that most of
the bulls tested had long since disappeared from
the scene. A major development which took
place at that time revolutionized the dairy
industry around the world in coming years.
2nd half of 20th Century
C.Polge (1949) discovered a practical method
for the long term preservation of the semen of
certain species by deep freezing to
temperatures of -790C by means of dry ice. In
fact, this was a demonstration that semen can
be preserved in frozen state. Later
developments in the field of semen freezing lead
to the worldwide acceptance and use of this
technique in artificial breeding programmes
resulting in considerable improvement in milk
yield of livestock species.
. Frozen semen of goats has not been used as
successfully as in cattle. Most of the procedures
developed for cryopreservation and insemination
of cattle semen has been extrapolated for use
with goat semen. However, the efficacy of
several of them for goat spermatozoa has not
been proved beyond doubt. Therefore, the use
of A.I. in goats is limited because of a variety of
management problems, prejudice, economics
and less well developed technology.
Characteristics of a good extendor
It should provide nutrients as a source of energy.
It should protect the spermatozoa against the harmful
effects of rapid cooling.
It should provide good buffering capacity to prevent
harmful shifts in pH as lactic acid is formed.
It should maintain the proper Osmotic pressure and
Electrolyte balance.
It should be able to inhibit bacterial growth
It should increase the volume of the semen so that it can
be used for multiple inseminations.
The extendor should be easy to prepare, low in cost and
give a clear picture of spermatozoa or embryos under
microscope and render no problem in the cleaning of
glassware and other containers.
It should also ensure a long shelf life of spermatozoa
/embryos on storage.
Principles of cryopreservation
It is well known that low temperature storage of any
material ensures longer keeping quality of any material
including foodstuffs. Refrigeration storage of food
material is a common knowledge. The storage at 40C or
lower temperatures like deep freeze delays the harmful
bacterial growth which spoils the food or other material.
This applies to preservation of gametes as well.
Therefore, earlier attempts to extend diluted semen at
refrigeration temperature extended the life span of
spermatozoa by few days. This period was sufficient for
transport of semen upto a distance of few hundred
kilometers for insemination of cattle at a distant place.
However, the quality soon deteriorated
Freezing at below refrigeration temperature resulted in
death of spermatozoa or embryos because of formation
of ice crystals. As you might be aware that ice occupies
more space than the water from which it is formed
resulting in bursting of the vessel in which it is contained.
Therefore, the availability of cryoprotectants which
prevent the formation of ice crystals during freezing and
developments of such procedures which cause
minimum damage to the cells during freezing became
key to such long term preservation of gametes.
The availability of cryoprotectants is very useful to
prevent the damage from cold shock. Glycerol is the
most commonly used cryoprotectant although ethylene
glycol and dimethy sulfoxide are also considered good.
The procedure for cryopreservation
includes initial exposure to and equilibration
with the cryoprotectants, cooling to sub zero
temperatures, storage, thawing . Cells must
maintain structural integrity throughout the
cryopreservation procedure. Storage is
usually at -1960C , the temperature of liquid
nitrogen. Above a temperature of -800C,
cells gradually lose viability , but below -
1300C, there is insufficient energy for most
reactions.
Step wise dilution procedure is sometimes
preferred as compared to single step
method for removal of cryoprotectant.
Procedure of goat semen
cryopreservation
Collect the semen using artificial vagina
Evaluation of semen for motility
Dilution of semen in extender using 10 % egg
yolk and 6 % glycerol as cryoprotectant.
Cool the diluted semen to 5 degree centigrade.
Fill the semen in straws (0.25ml ) french straws.
Expose the straws to liquid nitrogen vapors for
10 minutes
Dip the straws in liquid nitrogen ( -196 degree
centigrade).
Methods of A.I.
1- Intravaginal
2- Intracervical
3- Intrauterine
4- Intratuboperitoneal
5- Intratubal
6- Intraperitoneal
Egg yolk extender
EYCE
Yolk lecithin Fatty acid & Lysolecithin
Hydrolysis
Iritani & Nishikawa,(1961, 1993)
Acrosomal Reaction
and Upreti et al., (1999)
Chromatin decondensation
Hydrolysis BUSgp 60
Toxic to sperm
DNA
damage
Sperm Damage to
motility MitochondrIa
Leakage Protein
of damage
enzymes
Premature
capacitation and
acrosomal
reaction
(Farber et al., 1990; Aitken et al., 1999; Bailey et al.,2000;
Dutta et al., 2000)
Sources of injury from freeze-thawing cells
Stress encountered Potential cellular response
Externalization of phospatidylserine
Activation of Caspases
i) Initiator Caspase- 2,8,9,10
ii) Effector Caspase- 3,6, 7
FAS receptor activate Effector Caspase
Osmotic stress
Cooling rate
Extender composition
Storage temperature
Cryoprotectant concentration
Hygienic control
Parks and Graham, (1992)
Types of damages
1. Membrane damage
2. Mitochondrial damage
3. DNA damage
Methods of assessing damage
Fluorescent microscopy
Electronmicroscopy
Flowcytometry
Westernblotting
Immuno cytochemistry
Computer assisted semen
analysis
Spermchromatin structure
assay
1. Memberane damage
Cascade of the
chemical reaction
ROS attacks
polyunsaturated Lipid
fatty acids (PUFA) peroxidation
in cell membrane
Assessment of membrane integrity
Fluorescence microscopy.
Annexin –V
Electron microscopy.
Disturbed in Ca ++Homeostasis
Spermatozoa that were analysed
utilizing JC-1 stain
Defective apoptosis
Advance age
DNA and Membrane damage
Assessment of DNA damage
TUNEL assay
Toluidine blue
DNA breakage using single-cell
microgel electrophoresis
Fig 1-
Negatively charged DNA
strand breaks away from
the nucleus toward the
anode.
Fluorescence
photomicrograph
Young et al., (2003)
TUNEL assay & SCD test
Blue sperm are TUNEL negative Non-fragmented DNA form large halos
No halo indicating DNA
Green sperm are TUNEL positive fragmentation.
indicating DNA fragmentation
Schulte et al., (2010)
Status of goat AI-World
Country Number Semen extenders Number of Motility Fertility(%
of sperm (%) )
insemin inseminated
ations (million)
70
60
50
40
Motility (%)
30
Fertility(%)
20
10
0
Work done in India & Abroad
Additives
Freezing
rate
Glycerol
Egg yolk
Diluter
Work done in CIRG
Year Work done Finding researcher
2001-2003 • Standardization of • freezing rate 20-25 N.K Sinha et. al
freezing rate after C/min is better
equilibration •Conception rate 30-
35%
2003-200 • Effect of different • Post thaw motility N.K Sinha et. al
concentration of higher in 50-100
spermatozoa million sperm/dose
Metabolic stimulants
Detergents
Membrane stablizers
Detergents
Membrane stablizers
1-Antioxidants
Antioxidants Level Researcher
Vitamin E 0.3mg/ml Shukla & Mishra (2005)
3- Metabolic stimulant
Caffeine 7mM Bhosrekar et al, (1990)
Bradykinin (2ng/ml) Shukla and Mishra (2007)
4-Detergents
• Triethanolamine lauryl sulphate
Bhosrekar et al. (1990)
5-memberane stablizers
Chloroquine diphosphate (0.54mM) Kumar et al (2003)
Chlorpromazine hydrochloride (0.1mM)
Paudel et al. (2008)
6. Cholesterol loaded cyclo dextrin
Limitation of AI in Goat
Conception rate with AI is lower.
Timing of AI is critical.
Higher cost.
The true rational approaches defining the optimal conditions for semen
freezing, thawing and accurate evaluation recently developed need to be
continued and applied.
World’s current population of goats is
around 807.6 million (FAO, 2005). India
possesses about 124.4 million goats
making about 15 % of the world population
and stands second to China.
IMPORTANCE OF GOATS IN RURAL ECONOMY
1950 2415
1955 2,655
1960 3195
1965 3772
1970 4431
1975 4706
1979 5227
(Foote, 1981)
World’s current population of goats is
around 807.6 million (FAO, 2005). India
possesses about 124.4 million goats
making about 15 % of the world population
and stands second to China.
IMPORTANCE OF GOATS IN RURAL ECONOMY