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Cryopreservation of goat

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.

Individual US goat breed leaders in milk production


( Source ADGA 2008)

Breed Lactation Average Milk /day


Milk kg lactation kg
average
Alpine 2916 1083 9.6
Saanen 2987 1185 9.8
Toggenburg 3260 1024 11.9
Milk production potential of some Indian
breeds
Breed Milk Yield (kg) Reference
Jamunapari 235 Kaura, 1952
Barbari 157 Dutt, 1968
Beetal 195 Kaura, 1943
Jakhrana 122 Rout & Roy,
2006-
Goat Improvement
 We need to produce more from our goats
 How can be do that
 Breeding, Feeding , Management, and
disease control.
 Reproductive Management involves
 Use of reproductive biotechniques for
breed or goat improvement- Artificial
Insemination, Embryo transfer etc
Normal and Artificial Insemination
 Today, the artificial insemination has
become a normal method of breeding
quality cattle. A large number of cows and
buffaloes are inseminated artificially. The
technique of artificial insemination is
particularly more useful in a country like
India where the paucity of quality sires has
been the main hurdle in the way of cattle
improvement.
Goat Improvement using Normal
Reproduction
 By use of outstanding sires with good genetic
improvement ability.

 Normally fastest growing male goats gets used for meat


production and only the slow growing males are used for
breeding.Scientifically the best males which are true to
breed and having good production and reproduction
potential should be used for breeding. This is creating
negative genetic improvement.
 Use of outstanding male using normal reproduction is
useful for only a limited number of females.
Artificial Insemination using liquid
as well as frozen semen
Artificial insemination is not merely a novel method
of bringing about impregnation in females.
Instead, it is a powerful tool mostly employed for
livestock improvement. In artificial insemination
the germplasm of the bulls/bucks of superior
quality can be effectively utilized with the least
regard for their location in far away places. By
adoption of artificial insemination, there would
be considerable reduction in both genital and
non-genital diseases in the farm stock.
Advantages of Artificial Insemination
 The main advantage of artificial insemination (A.I.) is that it
increases the usefulness of superior sire/buck to an extra ordinary
degree. It makes available sires of inheritance for milk and butter fat
production to all goatmen within a limited area. Presently only a few
goat farmers get the advantage of good quality buck.
 The services of superior sires/bucks are greatly extended. By
natural services, a buck can be bred to 50 to 60 does per year. On
the other hand, by artifical insemination technique thousands of
goats can be sired in one year by one buck.
 The breeder does not need to maintain a herd buck and thus can
avoid the botherations accompanied with the management of a
buck/bull. The dairyman does not have the problem of searching
and purchasing a new herd buck every two years to avoid
inbreeding.
 The technique of artificial insemination can be made useful in cross
breeding for hybrid vigour by quickly transporting the semen by air to
different continents.
 The intensity of the spread of genital diseases is minimized if
artificial insemination is conducted under complete sanitary
conditions by the specially trained persons.
Disadvantages of A.I.

 Increased labor, management and


facilities
 Accentuate poor genetics if an inferior sire
is used
Cryopreservation
 Cryo-preservation of gametes has provided has
provided a extremely useful tool for
improvement of livestock including goats at a
much faster rate. Cryopreserved semen and
embryo offer a unique tool for improving the
genetic potential of livestock including cattle,
buffalo, sheep, goat , pig, horses, camel and
other species useful to human being. The
present day breeds of livestock are a result of
continued and sustained selection of superior
animals over a period of several thousand of
years. For faster genetic improvement
cryopresed gametes provide excellent
opportunities
History of cryopreservation
 The history of cryopreservation of gametes ;No
meaningful introduction to history of
cryopreservation can be without the mention of
Anton van Leeuwenhoek who discovered sperm in
1677 using magnifying lens.Italian physiologist
L.Spallanzani in 1780 discovered that the fertilizing
power of sperm resided in the sperm carried by the
spermatic fluid. When the semn was filtered, the
liquid that passed through was sterile, but the
residue on the filter was high in fertilizing power. He
further observed that freezing stallion semen in
snow or winter cold did not necessarily kill the “
spermatic vermiculi” but held them in a motionless
state until exposed to heat, after which they
continued to move for seven and a half hours (1803)
Early 20th century
 Philipsand Lardy (1940) brought forth egg
yolk as a semen dilutor to protect the
sperm cells against damage during
cooling. Almquist et al (1949) introduced
the addition of the antibiotics penicillin and
streptomycin to semen to control
pathogenic microorganisms.
 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).
Goat Improvement
 We need to produce more from our goats
 How can be do that
 Breeding, Feeding , Management, and
disease control.
 Reproductive Management involves
 Use of reproductive biotechniques for
breed or goat improvement- Artificial
Insemination, Embryo transfer etc
Normal and Artificial Insemination
 Today, the artificial insemination has
become a normal method of breeding
quality cattle. A large number of cows and
buffaloes are inseminated artificially. The
technique of artificial insemination is
particularly more useful in a country like
India where the paucity of quality sires has
been the main hurdle in the way of cattle
improvement.
Goat Improvement using Normal
Reproduction
 By use of outstanding sires with good genetic
improvement ability.

 Normally fastest growing male goats gets used for meat


production and only the slow growing males are used for
breeding.Scientifically the best males which are true to
breed and having good production and reproduction
potential should be used for breeding. This is creating
negative genetic improvement.
 Use of outstanding male using normal reproduction is
useful for only a limited number of females.
ARTIFICIAL INSEMINATION

A.I. is the deliberate


introduction of semen into
a female repro-ductive
tract for the purpose of
achieving a pregnancy
through fertilization by
means other than mating/
natural service.
Cryopreservation of semen
This is the technique by which the semen is
stored/preserved for long time at subzero
temperature (-196˚C)
Artificial Insemination using liquid
as well as frozen semen
Artificial insemination is not merely a novel method
of bringing about impregnation in females.
Instead, it is a powerful tool mostly employed for
livestock improvement. In artificial insemination
the germplasm of the bulls/bucks of superior
quality can be effectively utilized with the least
regard for their location in far away places. By
adoption of artificial insemination, there would
be considerable reduction in both genital and
non-genital diseases in the farm stock.
Cryopreservation
 Cryo-preservation of gametes has provided has
provided a extremely useful tool for
improvement of livestock including goats at a
much faster rate. Cryopreserved semen and
embryo offer a unique tool for improving the
genetic potential of livestock including cattle,
buffalo, sheep, goat , pig, horses, camel and
other species useful to human being. The
present day breeds of livestock are a result of
continued and sustained selection of superior
animals over a period of several thousand of
years. For faster genetic improvement
cryopresed gametes provide excellent
opportunities
History of cryopreservation
 The history of cryopreservation of gametes ;No
meaningful introduction to history of
cryopreservation can be without the mention of
Anton van Leeuwenhoek who discovered sperm in
1677 using magnifying lens.Italian physiologist
L.Spallanzani in 1780 discovered that the fertilizing
power of sperm resided in the sperm carried by the
spermatic fluid. When the semn was filtered, the
liquid that passed through was sterile, but the
residue on the filter was high in fertilizing power. He
further observed that freezing stallion semen in
snow or winter cold did not necessarily kill the “
spermatic vermiculi” but held them in a motionless
state until exposed to heat, after which they
continued to move for seven and a half hours (1803)
History
Year Events Researcher
1677 Discovered spermatozoa. Leeuwenhoek

1780 Inseminated a bitch Spallanzani


1930 Development of AV for bull, stallion and rams Milovanov

1936 Birth of lamb after AI Arthur Walton

1940 Developed phosphate-buffered egg yolk Phillips

1941 Developed citrate-buffered egg yolk Salisbury

1949 Glycerol as a cryo-protectant Polge et al

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)

Sperm membrane becomes more fusogenic

Acrosomal Reaction
and Upreti et al., (1999)

Chromatin decondensation

Sawyer & Brown, (1995)


Milk extender
Plasma membrane triglyceride
+
triglyceride of milk

Hydrolysis BUSgp 60

Oleic acid production

Toxic to sperm

Bellicer-Rubio et al., (1997);


Combarneus (1998)
Factors affecting AI
 Breed
 Age of buck and doe
 Condition of buck and doe
 Nutrition
 Season of insemination
 Use of fresh, cooled, chilled, frozen semen
 Technical\labour involved
 Time of insemination
 Extender used
 Dose of inseminated semen
 Method (vaginal, cervical, cervico-uterinal or
laparoscopic) used
Cryopreservation

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

Temperature reduction Membrane lipid phase changes and


depolymerization of cytoskeleton
Increased solute Osmotic shrinkage
concentration
Increased ionic concentration Direct effects on membranes, including
solubilization of membrane proteins
Dehydration Destabilization of the lipid bilayers
Gas bubble formation Mechanical damage to membranes and the
cytoskeleton

Increased solution viscosity Possible limitation of diffusion processes

Changes in pH Denaturation of proteins

Direct contact between cells Membrane damage


Process of apoptosis
High ROS and Low antioxidant enzyme

Externalization of phospatidylserine

This marked the cell for phagocytosis

Activation of Caspases
i) Initiator Caspase- 2,8,9,10
ii) Effector Caspase- 3,6, 7
FAS receptor activate Effector Caspase

Apoptosis/ cell death Said et al., (2010)


Source of reactive oxygen species

Major source of ROS


production

Activation of aromatic amino


acid oxidase & death of bull
NADH/NADPH OXIDASE sperm or from leucocytes.

Aitken et al., (1997) Upreti et al., (1998)


Factors affecting cryopreservation
 Generation of Reactive oxygen species

 Formation of intracellular ice crystals

 Formation of extracellular ice crystals

 Osmotic stress

 Cooling rate

 Extender composition

 Storage temperature

 Cryoprotectant concentration

 Seminal plasma composition

 Hygienic control
Parks and Graham, (1992)
Types of damages

1. Membrane damage

2. Mitochondrial damage

3. DNA damage
Methods of assessing damage

Simple optical microscopy

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

Staining of cells with membrane


impermeable dyes.

Hypo-osmotic swelling test.

Estimation of enzyme leakage.

Fluorescence microscopy.

Annexin –V

Electron microscopy.

Pawar et al, (2011)


Acrosomal staining

 Live acrosome intact


 Dead acrosome intact
 Dead acrosome
denuded
Cryocapacitation

Membrane Increase Cryocapaciat Abnormal cell


damage Membrane membrane ion like
AffectsCa++ function &
during permeability leakiness & changes (not
regulation Inclusively
cryopreservati increases Specific affect the cell death
on channels motility)
2. Mitochondrial damage
Induction of Apoptosis

Mitochondrial pores open

Decrease mitochondrial potential

Cause release of proapoptotic factor in cytoplasm

Modulation of redox status

Decrease in energy production

Disturbed in Ca ++Homeostasis
Spermatozoa that were analysed
utilizing JC-1 stain

Non-functional mitochondria Functional Mitochondria

Schulte et al., (2010)


3. DNA damage
Aberrant chromatin packaging during
spermatogenesis

Defective apoptosis

Excessive reactive oxygen species


production

Decreased seminal antioxidants

Certain drugs, fever/high testicular


temperature/varicocele

Advance age
DNA and Membrane damage
Assessment of DNA damage
TUNEL assay

ISNT (Insitu Nick translation)

SCD (sperm chromatin dispersion test)

Single cell gel electrophoresis/COMET assay

SCSA (Sperm chromatin structure assay)

Acridine orange staining

Aniline blue staining

CMA3 (Chromomycin A3) staining

Toluidine blue
DNA breakage using single-cell
microgel electrophoresis
Fig 1-
Negatively charged DNA
strand breaks away from
the nucleus toward the
anode.

The tail represents the


extent of migration from
the sperm nucleus.

Fig 2- Healthy sperm


sperm nucleus is seen as a
bright fluorescent nuclear
core.

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)

Australia 1000 Egg yolk Tris 30 (ut) 40 60

France 580000 Skim milk 100 30 65

Italy 5000 Modified Skim milk 100 … …

Norway 940 Modified Skim milk 100 >50 64

Poland 51 Modified milk 200 50 75

Switzerland 1635 Egg yolk Tris 180 50 55-65


Status of goat AI
80

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

2007-2012 • Comparison of 6% glycerol S.K.Jindal et. al


cryoprotectants -- best
• Comparison of different 10% -- optimum
egg yolk level
• Used of Dextran with 3%with glycerol–
glycerol best
Work done in CIRG cont..
Year Work done Finding Researcher

2012-13 Use of specially Conception rate S.D. kharche et


design A.I crate Without crate - al
44.44%
With crate-
64.28%
Precautions during storage
 LN2 level above 5''.

 Never lift a canister above the frost line.

 Removed semen from the tank should be


placed immediately in the thaw box.

 Never expose semen to direct ultraviolet


light.

 Never refreeze semen.

 Check for proper identification.

 Always wear gloves and goggles.


Remedies for improvement in post
thaw quality

 Antioxidants
Antioxidants
 Chelating agents
 Chelating agents
 Metabolic stimulants

 Metabolic stimulants
 Detergents


 Membrane stablizers
Detergents

 Membrane stablizers
1-Antioxidants
Antioxidants Level Researcher
Vitamin E 0.3mg/ml Shukla & Mishra (2005)

Ascorbic Acid 2.5mM Li et al., (2009)

Cysteine 0.125% Tiwari et al, (1991)

Catalase 200unit/ml Ahmet Atessahin et al., (2008)

Selenium 0.3mg/ml Kolev (1997)

Glutathion peroxidase 9mM Ahmet Atessahin et al., (2008)

Hyaluronan 500ul/ml Bucaka et al., (2009)

Glutamine 2.5mM Bucaka et al., (2009)


Antioxidants
Antioxidants Level Researcher
Taurine 25 mM A. Atessahin et al., (2008)

carnitine 7.5mM Bucak et al, (2010)

Inositol 7.5mM Bucak et al, (2010)

Methionine 2.50mM Patra et al, (2001)

Sodium pyruvate 1.25mM Fabbrocini et al., (1991)

Lycopene 0.1mg/ml Rosato et al., (2012)

n propyl gallate 15µM Shukla & Mishra (2005)


2-Chelating agents
 0.1% EDTA. Dhami and Sahni (1993)

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.

 Difficulty in insemination due to tortuous cervix.

 Timing of AI is critical.

 Lack of interest on the part of concerned agencies to encourage


the concept of mixed AI centers.

 Illiteracy among farmers & religious taboos.

 Higher cost.

 Lack of trained technical and extension workers in AI of goats at


the national and provincial level.
FUTURE PROSPECTS
 To evolve a simple, rapid & economic method for processing and freezing of
buck semen with acceptable fertility.

 Technique of deep cervical and intra-uterine insemination needs perfection for


higher fertility.

 Critical studies to establish the minimum number of sperm per inseminating


dose for acceptable fertility (>50%).

 Strict quality control of the frozen semen at various stages of production,


processing, storage and final use.
FUTURE PROSPECTS
 Periodic andrological examination of breeding bucks and observance
of utmost hygienic precaution during collection & handling of semen.

 Attention needs to be given for preputial washing with sterile normal


saline solution prior to semen collection which will reduce bacterial
contamination and increase the freezabilty.

 Suitably organized breeding programmes, proper training and


education of extension workers and awareness on the part of goat
farmers on the economic advantage of using elite bucks should be
strengthened.
Conclusion
 The sperm cells damages as a result of freezing and thawing indicates that
despite considerable progress in recent years, cryopreservation still
imposes great stress on spermatozoa.

 Further studies being required to improve semen cryopreservation


protocols in various species.

 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

 The goats around the world contributed


12438.4 TMT of milk, 4562.1 TMT of meat
and 985.9 TMT of fresh skins per annum.
India produced 2700 TMT of milk, 475 TMT of
meat, 130 TMT of skins, 8.5 MT of Pashmina
and 400 TMT of manure from goats. The goat
sector in India generates about 5 % rural
employment and about 20 million smallholder
families are engaged in goat keeping. In
addition a large number of commercial goat
farms have been established in different parts
of the country.
 The contribution of goats and its products at aggregate
level in monetary terms has been estimated to be Rs.74,
777 million annually to the national economy accounting
for 7.6% of GDP from livestock sector, which contributes
around 23% of GDP from agriculture sector. Among
different products meat has the largest share
contributing more than 50 % of total value of goat
products, followed by milk and manure which contribute
26.64 and 10.43 % of total value of goat products,
respectively. Goatskin is indeed another important
output contributing about Rs.4510.80 million annually to
the National Economy.
Early 20th century
 Philipsand Lardy (1940) brought forth egg
yolk as a semen dilutor to protect the
sperm cells against damage during
cooling. Almquist et al (1949) introduced
the addition of the antibiotics penicillin and
streptomycin to semen to control
pathogenic microorganisms.
Advantages of Artificial Insemination
 The main advantage of artificial insemination (A.I.) is that it
increases the usefulness of superior sire/buck to an extra ordinary
degree. It makes available sires of inheritance for milk and butter fat
production to all goatmen within a limited area. Presently only a few
goat farmers get the advantage of good quality buck.
 The services of superior sires/bucks are greatly extended. By
natural services, a buck can be bred to 50 to 60 does per year. On
the other hand, by artifical insemination technique thousands of
goats can be sired in one year by one buck.
 The breeder does not need to maintain a herd buck and thus can
avoid the botherations accompanied with the management of a
buck/bull. The dairyman does not have the problem of searching
and purchasing a new herd buck every two years to avoid
inbreeding.
 The technique of artificial insemination can be made useful in cross
breeding for hybrid vigour by quickly transporting the semen by air to
different continents.
 The intensity of the spread of genital diseases is minimized if
artificial insemination is conducted under complete sanitary
conditions by the specially trained persons.
Advantages of AI
 Spread elite genetic material throughout a population.

 Facilitate progeny testing.

 Eliminates the necessity of keeping buck.

 Kids of desired sex can be produced through sperm sexing.

 Greatly reduce transmission of diseases or parasites.

 Has paved the way for other reproductive biotechnologies.


Disadvantages of A.I.

 Increased labor, management and


facilities
 Accentuate poor genetics if an inferior sire
is used
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 potential of some Indian
breeds
Breed Milk Yield (kg) Reference
Jamunapari 235 Kaura, 1952
Barbari 157 Dutt, 1968
Beetal 195 Kaura, 1943
Jakhrana 122 Rout & Roy,
2006-
Our goats are low producers in terms of milk and meat as
compared to European breeds of goats.

Individual US goat breed leaders in milk production


( Source ADGA 2008)

Breed Lactation Average Milk /day


Milk kg lactation kg
average
Alpine 2916 1083 9.6
Saanen 2987 1185 9.8
Toggenburg 3260 1024 11.9
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)
 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

 The goats around the world contributed


12438.4 TMT of milk, 4562.1 TMT of meat
and 985.9 TMT of fresh skins per annum.
India produced 2700 TMT of milk, 475 TMT of
meat, 130 TMT of skins, 8.5 MT of Pashmina
and 400 TMT of manure from goats. The goat
sector in India generates about 5 % rural
employment and about 20 million smallholder
families are engaged in goat keeping. In
addition a large number of commercial goat
farms have been established in different parts
of the country.
 The contribution of goats and its products at aggregate
level in monetary terms has been estimated to be Rs.74,
777 million annually to the national economy accounting
for 7.6% of GDP from livestock sector, which contributes
around 23% of GDP from agriculture sector. Among
different products meat has the largest share
contributing more than 50 % of total value of goat
products, followed by milk and manure which contribute
26.64 and 10.43 % of total value of goat products,
respectively. Goatskin is indeed another important
output contributing about Rs.4510.80 million annually to
the National Economy.