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Semen Banking for conservation of
livestock biodiversity

National GeneBank
National Bureau Of Animal Genetic Resources
Karnal – 132 001
Livestock genetic resources
India is a global paradise for fauna and flora with more than
6% of the world’s domesticated livestock biodiversity
maintained in only 2½ % of the world’s land mass
Domestication of livestock species began nearly 12,000 years
ago in India and other Asian countries for food, fibre, work,
power and other agricultural purposes
40 mammalian species for food but major production only
from 14 species
All the major species of livestock and poultry are found in India

13

8

6

37
21
42

18
Contribution of livestock in indian economy
India ranks first in world in milk production. The annual milk
production has increased to the level of 121.8 million tonnes. Livestock
contribution to National GDP and agriculture is 3.26% and 29.64%
respectively.
Livestock provides
Milk; Draft power; Organic manure; Dung as fuel ; Bones, hides &
skins ; Meat ; Employment
Total export earnings from livestock, poultry and related products
was Rs. 25,408.86 crore during 2010-11.
Production of Major Livestock Products during 2010-11
Milk
121.8 m.t.
Eggs
63,024 m.
Wool
43.0
m. kg.
Meat
4.8
m.t.
Status of livestock genetic resources in india
 Purebred populations on decline
due to indiscriminate breeding
within the native stock and with
exotic breeds

Species

Population (m)
2003
2007

Cattle

185.18

199.08

Buffalo

97.92

105.34

 No true estimates of pure bred
populations

Sheep

61.47

71.56

 Poor understanding of issues
related
to
conservation
vs
improvement

Goat

124.36

140.54

 Lack of public awareness about
value of conservation in domestic
livestock vs wild life

Pig

13.58

11.13

Horse

0.75

0.61

Camel

0.63

0.52

 Nearly 80% of indigenous animals
are non-descript
Factors affecting animal biodiversity
 Introduction of exotic germplasm
 Restricted use to a few breeds
 Degradation of ecosystems
 Disease and natural disasters
 Fluctuating market requirements
 Political unrest and instability
 Non implementation of animal breeding policies
Conservation
Conservation is the management of genetic
resources for human use so that it may yield the
greatest sustainable benefits to present generation
while maintaining its potential to meet the needs
and aspirations of future generations.
 It is always better to keep the live populations
of animals which may go on improving not only in
production potentials but also in their adaptation
to the changing environment.
Need for conservation of AnGR
To meet increased demand for animal products by growing
human population.
For providing gainful employment to people.
For preserving unique genes for future use
For economic production under prevailing low input system
For exploitation of some unique characteristics:
Disease resistance
Drought resistance
Heat tolerance
Ability to utilize coarse fodder
Other characteristics like high butterfat, therapeutic
value of milk/products
Pre-requisites / Strategies
 Degree of endangerment
 Genetic uniqueness
 Possession of unique traits
 Adaptation to specific environment
 Cultural or historical value
Taxonomic Distinctness
 Genetic characterization of breeds/populations
 Identification of genes of agricultural importance
In Today’s context it is possible to study GENERAL GENETIC
DIVERSITY and to prioritize the breeds for conservation
Categories (Population status) FAO Panel
Critical
: Breeding female <100
Endangered: Breeding female 100-1000
Vulnerable : Breeding female 1000-5000
Insecure : Breeding female 5000-10000
Normal : Breeding female >10000
Population size of a breed for its status (‘000)
Species Normal
Cattle
Buffaloes
Sheep
Goat
Camels
Horses
Pigs

25
30
50
30
20
20
10

Insecure
15-25
20-30
30-50
20-30
15-20
15-20
5-10

Vulnerable
5-15
10-20
15-30
10-20
5-15
5-15
1-5

Endangered
2-5
5-10
8-15
5-10
2-5
2-5
0.5-1.0

Critical
<2
<5
<8
<5
<2
<2
<0.5

Source: Nivsarkar A E, Gupta S C, Vij P K and Sahai R. 1994. Identification and conservation
of endangered breeds of livestock- strategies and approach. Proceedings of the national
symposium on livestock production and management held at Gujarat agricultural
university, Anand, 21 to 23 February 1994.
Sample size in preservation programmes
• Sample size in preservation programmes are influenced by
both genetic considerations and cost.
• Breeding stocks of frozen stores must be large enough to
provide a good representation of the conserved stock and to
prevent much genetic drift, or inbreeding.
• To restrict the rate of inbreeding to 0.2% per year, a
breeding herd of 10 males and 26 females would be adequate
for cattle. For frozen semen, collection from 25 sires would
be adequate for all species, when the males are used
rotationally on each other’s daughters.
Smith C.1984. Genetic aspects of conservation in farm animals.
Livestock Production Science 11:37-48.
Methods of Conservation
In Situ
Farmer’s herds/flocks
Organized herds in breed tracts

Ex situ
Organized herds outside breed tract
Cryopreserved germplasm
• Embryos
• Somatic cells
• DNA
• Spermatogonial stem cells
• Semen
Organized flocks/herds
Maintenance of small population at a place away from the
main breeding tract of the breed is the ex situ conservation of
the live animals. This may be in the form of organised herd
maintained in a research institutions, bull mother farm, state
owned livestock farm, zoo or breed park.
This population can be used in regeneration of endangered
breed, new breed development, DNA studies.
Cryopreservation of embryos
Applications
 Diploid and contain all genes, ideal for breed improvement,
conservation and revival of lost breed

Limitations
 Can not be produced in large numbers and require large
number of elite donors
 Cost of production is too high to justify for conservation of
breeds which are low producing
 Require very skilled manpower for production and transfer.
Embryos

With the present rate of one live animal production, a mean
of 8 to 13 embryos had to be frozen initially. With the
objective of obtaining 25 breedable individual of each sex for
re-creating a breed, an average of 570 to 930 embryos
respectively need to be stored. For obtaining these number of
embryos of freezable quality an average of 180 to 185 of
donor females need to be maintained.
Somatic cell banking

Applications
• These are diploid cells and contain full genetic code of an
animal.
• Can be used as genetic material for conservation of
endangered animal genetic resources.
• Can be sampled quickly even from remoter area at low cost
• Cost of maintenance is very low for large populations
• Can be used for production of producing therapeutic
proteins

Limitations
• Success rate of cloning is still very low
Cryopreservation of embryonic stem cell lines
Applications
•This can be excellent biological tool for producing live animals
•For producing genetically modified animals
•For gene and cell therapies
•For producing vital therapeutic proteins

Limitations
•Stable embryonic stem cell lines have not been successfully
generated in farm animals except in human and rodents
Storage of DNA

Cryogenic storage of DNA is another method of
preservation of genetic material. It has several advantages
over the live germplasm, avoiding the complication of
spreading of disease while its transportation, however it has
its own limitations. NBAGR has already established a DNA
bank where DNA of indigenous livestock and poultry is
cryo-preserved.
DNA storage
Applications
• Very easy to obtain and stored at low cost
• Require very less space and no chance of disease transfer etc
• Can help in conservation of gene by transgenesis or knock
out technology
• Can help in recreation of lost breeds by cross checking of
different populations or genetic material used.

Limitations
• Genome maps of different farm species are not yet available
• Life can not be created from DNA alone
Spermatogonial Stem Cells (SSCs)
Transplantation of isolated germ cells from a fertile donor male into the seminiferous
tubules of infertile recipients can result in donor-derived sperm production.
SSCs transplantation has been demonstrated in goats, dog, cow, pig, baboon and
bovine spermatogonial stem cells shown to be capable of colonizing recipient mouse
seminiferous tubules.
An in vitro system that supports the proliferation and maintenance of spermatogonial
stem cells could be used to preserve and expand spermatogonial stem cell numbers as
well as aid in genetic modification.

GS isolation
GS Culturing

GS
Transplantation
3 to 5 months

Colonization

Progeny with donor genes
Preservation of spermatozoa
Applications
For improvement of a breed
To support in situ conservation
To support in vivo populations
To study the effect of single major gene
Semen freezing infrastructure is available throughout the
country

Limitations
Spermatozoa contain haploid genome
Semen freezing/ AI is not standardized in all species
Semen harvesting and freezing

Selection of Bulls
Management of Bulls
Semen collection and freezing
Selection of Bulls
• Physical Examination
• Before procuring new bull calves/bulls for a semen station, a
thorough physical examination shall be conducted by an
accredited professional to ensure that the bulls are free from
abnormality and do not display clinical symptom(s) of any
infection or any contagious diseases.
• For every new calf procured, the measurement of scrotal
circumference and body weight should be initiated
immediately.
• Prior to introduction of new bulls for semen collection,
breeding soundness examination shall also be carried out.
• Karyotying and testing for genetically
transmitted diseases
It is necessary that all animals be karyotyped to rule out
any chromosomal defects.

• Quarantine
A quarantine period of minimum 60 days* is compulsory
before bringing new bulls into a semen station. Each new
animal in quarantine station will be tested against major
contagious diseases before its entry to resident herd e.g.
TB, JD, Brucellosis, Campylobacteriosis and
Trichomoniasis.
• During quarantine period, the bulls shall be vaccinated
against FMD, HS, BQ, Theileriosis and Anthrax.
However, vaccinations against bacterial diseases shall
be done only if there is an outbreak or prevalence of a
particular disease.
• Once the quarantine period is over, all bulls shall be
introduced to the young bull rearing station.
Management of Bulls
• The objective of daily care of bulls is to ensure a satisfactory
state of cleanliness.
 The bulls shall be kept under hygienic conditions at all
times.
 The coat of the bulls shall be kept clean and generally short.
 The hooves shall be regularly trimmed.
 The length of the tuft of hairs at the preputial orifice, which
is invariably soiled, shall be cut to about 2 cm.
 Bulls shall be brushed and groomed regularly, and where
necessary, special attention shall be given to the underside of
the abdomen, a day prior to semen collection.
 Cleaning of the prepuce with sterile normal saline
solution may be done every ten days if the microbial
load is within the prescribed limits.
 Cleaning prior to the day of collection can be practiced
if the microbial load in frozen semen is beyond the
prescribed limit.
 In the event of obvious soiling, careful cleaning of the
preputial orifice and the adjoining areas with soap or a
detergent is recommended; followed by thorough
rinsing and drying.
 Scientific feeding schedule shall be followed for the
bulls.
Semen Collection
• Ideally, the floor of the collection yard shall be
made of concrete layer at a depth of one foot
from the ground level.
Handling, processing & freezing of semen
•
•
•
•
•
•
•
•
•

Premises
Equipment
Personnel Hygiene
Diluents
Evaluation & Processing
Colour Specifications
Printing of Straws
Post thaw motility
Quality Checks for frozen semen
Semen freezing
The biophysical principles that apply to cryopreservation of living cells and
tissues also apply to cryopreservation of sperm.
The sperm may be damaged during cryopreservation and/ or thawing either
by the formation of large intracellular ice crystals or by the increased
intracellular concentration of solutes and accompanying changes that result
from the dehydration of cells during cryopreservation (solution effects).
The damage to sperms during cryopreservation by crystal formation and/
or solution effect can be minimized by freezing sperms at an optimal freezing
rate.
Adding cryoprotectants such as glycerol or dimethyl sulfoxide to the
freezing medium results in freezing at lower temperatures. This probably
retards dehydration of cells and the resultant harmful solution effects; thus
sperms may be cooled slowly enough to prevent the formation of large ice
crystals.
Quality control in GeneBank
Status of the frozen semen quality to be deposited
in GeneBank for long term cryopreservation
Semen Parameter
Post thaw motility(%)
Acrosomal integrity(%)
Abnormal sperm(%)
Hypo osmotic swelling test(%)
(responsive cells)
Microbial load

Minimum acceptable level
40
50
< 20%
40
<5000CFU/ml
Quantity of semen doses to be preserved
for breed conservation
At NBAGR, total number of frozen semen doses to be kept in National Animal
Gene Bank for different animal species is as given below.
 Cattle and Buffalo: A total of 30,000 semen doses (2000 doses each from 15
unrelated bulls) for a breed are to be preserved. Fifty percent of these semen
doses are proposed to be kept at National Gene Bank and rest 50% at Regional
Centres in respective state.
 Sheep and Goat: A total of 25,000 semen doses (1000 doses each from 25
unrelated breeding males) for a breed are to be preserved. Fifty percent of these
semen doses are to be kept at National Gene Bank and rest 50% at Regional
Centres in respective state.
Quality control in GeneBank
 Quality of frozen semen stored in GeneBank is an important issue
and is taken care seriously.
 The males used for semen collection must be free from infectious
diseases like FMD, Contagious Bovine Pleuropneumonia,
Tuberculosis, Brucellosis, Vibriosis, Trichomoniasis and Blue
Tongue.
 In addition to it the frozen semen in GeneBank is monitored
regularly for semen quality parameters so as to access the fertility
status of frozen semen.
Information regarding germplasm
The available details of bull pedigree, physical
characteristics and bull’s semen quality is procured from
cooperating centers for documenting the details of frozen
semen stored in GeneBank.








Date of birth
Sire index (if tested)
Age at first collection
Any abnormality detected in karyotyping
Physical characteristics alongwith photograph of bull
Health status alongwith Any abnormality in genital organs
Semen quality
Livestock conservation through National/
Regional semen banks
National Gene Bank at NBAGR Karnal
20% of total
Initially 1000 doses of semen each from 15 unrelated bulls (Cattle and
Semen doses
Buffalo); 500 doses each of 25 unrelated breeding males(Sheep and
For use in field
Goats). Subsequent years 20% replacement semen doses

State Livestock Development Boards, Conservation cell

20% of total
Semen doses
For use in field

Initially 1000 doses of semen each from 15 unrelated bulls (Cattle and
Buffalo); 500 doses each of 25 unrelated breeding males(Sheep and
Goats). Subsequent years 20% replacement semen doses

Regional Frozen Semen Production units/ Banks
Semen supply for genetic improvement and breeding of females in
field
Artificial Insemination Centres
Semen available in GeneBank
No. of species

Cattle, Buffalo , Goat, Sheep, Camel, Yak & Equine

7

No. of Breeds

21 breeds (Cattle)
9 breeds ( Buffalo )
3 breeds ( Goat)
2 breeds (Equine)
1 breed each from Sheep, Camel & Yak

38

No. of
Bulls/Rams/Bucks/Stallions

Cattle (115)
Buffalo (79)
Goat (38)
Sheep (20)
Camel (15)
Yak (3)
Equine(6)

282

No. of doses

Cattle - 62,854
Buffalo- 38,153
Goat - 11,593
Sheep - 8375
Camel- 928
Yak - 360
Equine- 1220

1,23,283
Semen Banking for conservation of  livestock biodiversity

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Semen Banking for conservation of livestock biodiversity

  • 1. Semen Banking for conservation of livestock biodiversity National GeneBank National Bureau Of Animal Genetic Resources Karnal – 132 001
  • 2. Livestock genetic resources India is a global paradise for fauna and flora with more than 6% of the world’s domesticated livestock biodiversity maintained in only 2½ % of the world’s land mass Domestication of livestock species began nearly 12,000 years ago in India and other Asian countries for food, fibre, work, power and other agricultural purposes 40 mammalian species for food but major production only from 14 species
  • 3. All the major species of livestock and poultry are found in India 13 8 6 37 21 42 18
  • 4. Contribution of livestock in indian economy India ranks first in world in milk production. The annual milk production has increased to the level of 121.8 million tonnes. Livestock contribution to National GDP and agriculture is 3.26% and 29.64% respectively. Livestock provides Milk; Draft power; Organic manure; Dung as fuel ; Bones, hides & skins ; Meat ; Employment Total export earnings from livestock, poultry and related products was Rs. 25,408.86 crore during 2010-11. Production of Major Livestock Products during 2010-11 Milk 121.8 m.t. Eggs 63,024 m. Wool 43.0 m. kg. Meat 4.8 m.t.
  • 5. Status of livestock genetic resources in india  Purebred populations on decline due to indiscriminate breeding within the native stock and with exotic breeds Species Population (m) 2003 2007 Cattle 185.18 199.08 Buffalo 97.92 105.34  No true estimates of pure bred populations Sheep 61.47 71.56  Poor understanding of issues related to conservation vs improvement Goat 124.36 140.54  Lack of public awareness about value of conservation in domestic livestock vs wild life Pig 13.58 11.13 Horse 0.75 0.61 Camel 0.63 0.52  Nearly 80% of indigenous animals are non-descript
  • 6. Factors affecting animal biodiversity  Introduction of exotic germplasm  Restricted use to a few breeds  Degradation of ecosystems  Disease and natural disasters  Fluctuating market requirements  Political unrest and instability  Non implementation of animal breeding policies
  • 7. Conservation Conservation is the management of genetic resources for human use so that it may yield the greatest sustainable benefits to present generation while maintaining its potential to meet the needs and aspirations of future generations.  It is always better to keep the live populations of animals which may go on improving not only in production potentials but also in their adaptation to the changing environment.
  • 8. Need for conservation of AnGR To meet increased demand for animal products by growing human population. For providing gainful employment to people. For preserving unique genes for future use For economic production under prevailing low input system For exploitation of some unique characteristics: Disease resistance Drought resistance Heat tolerance Ability to utilize coarse fodder Other characteristics like high butterfat, therapeutic value of milk/products
  • 9. Pre-requisites / Strategies  Degree of endangerment  Genetic uniqueness  Possession of unique traits  Adaptation to specific environment  Cultural or historical value Taxonomic Distinctness  Genetic characterization of breeds/populations  Identification of genes of agricultural importance In Today’s context it is possible to study GENERAL GENETIC DIVERSITY and to prioritize the breeds for conservation
  • 10. Categories (Population status) FAO Panel Critical : Breeding female <100 Endangered: Breeding female 100-1000 Vulnerable : Breeding female 1000-5000 Insecure : Breeding female 5000-10000 Normal : Breeding female >10000
  • 11. Population size of a breed for its status (‘000) Species Normal Cattle Buffaloes Sheep Goat Camels Horses Pigs 25 30 50 30 20 20 10 Insecure 15-25 20-30 30-50 20-30 15-20 15-20 5-10 Vulnerable 5-15 10-20 15-30 10-20 5-15 5-15 1-5 Endangered 2-5 5-10 8-15 5-10 2-5 2-5 0.5-1.0 Critical <2 <5 <8 <5 <2 <2 <0.5 Source: Nivsarkar A E, Gupta S C, Vij P K and Sahai R. 1994. Identification and conservation of endangered breeds of livestock- strategies and approach. Proceedings of the national symposium on livestock production and management held at Gujarat agricultural university, Anand, 21 to 23 February 1994.
  • 12. Sample size in preservation programmes • Sample size in preservation programmes are influenced by both genetic considerations and cost. • Breeding stocks of frozen stores must be large enough to provide a good representation of the conserved stock and to prevent much genetic drift, or inbreeding. • To restrict the rate of inbreeding to 0.2% per year, a breeding herd of 10 males and 26 females would be adequate for cattle. For frozen semen, collection from 25 sires would be adequate for all species, when the males are used rotationally on each other’s daughters. Smith C.1984. Genetic aspects of conservation in farm animals. Livestock Production Science 11:37-48.
  • 13. Methods of Conservation In Situ Farmer’s herds/flocks Organized herds in breed tracts Ex situ Organized herds outside breed tract Cryopreserved germplasm • Embryos • Somatic cells • DNA • Spermatogonial stem cells • Semen
  • 14. Organized flocks/herds Maintenance of small population at a place away from the main breeding tract of the breed is the ex situ conservation of the live animals. This may be in the form of organised herd maintained in a research institutions, bull mother farm, state owned livestock farm, zoo or breed park. This population can be used in regeneration of endangered breed, new breed development, DNA studies.
  • 15. Cryopreservation of embryos Applications  Diploid and contain all genes, ideal for breed improvement, conservation and revival of lost breed Limitations  Can not be produced in large numbers and require large number of elite donors  Cost of production is too high to justify for conservation of breeds which are low producing  Require very skilled manpower for production and transfer.
  • 16. Embryos With the present rate of one live animal production, a mean of 8 to 13 embryos had to be frozen initially. With the objective of obtaining 25 breedable individual of each sex for re-creating a breed, an average of 570 to 930 embryos respectively need to be stored. For obtaining these number of embryos of freezable quality an average of 180 to 185 of donor females need to be maintained.
  • 17. Somatic cell banking Applications • These are diploid cells and contain full genetic code of an animal. • Can be used as genetic material for conservation of endangered animal genetic resources. • Can be sampled quickly even from remoter area at low cost • Cost of maintenance is very low for large populations • Can be used for production of producing therapeutic proteins Limitations • Success rate of cloning is still very low
  • 18. Cryopreservation of embryonic stem cell lines Applications •This can be excellent biological tool for producing live animals •For producing genetically modified animals •For gene and cell therapies •For producing vital therapeutic proteins Limitations •Stable embryonic stem cell lines have not been successfully generated in farm animals except in human and rodents
  • 19. Storage of DNA Cryogenic storage of DNA is another method of preservation of genetic material. It has several advantages over the live germplasm, avoiding the complication of spreading of disease while its transportation, however it has its own limitations. NBAGR has already established a DNA bank where DNA of indigenous livestock and poultry is cryo-preserved.
  • 20. DNA storage Applications • Very easy to obtain and stored at low cost • Require very less space and no chance of disease transfer etc • Can help in conservation of gene by transgenesis or knock out technology • Can help in recreation of lost breeds by cross checking of different populations or genetic material used. Limitations • Genome maps of different farm species are not yet available • Life can not be created from DNA alone
  • 21. Spermatogonial Stem Cells (SSCs) Transplantation of isolated germ cells from a fertile donor male into the seminiferous tubules of infertile recipients can result in donor-derived sperm production. SSCs transplantation has been demonstrated in goats, dog, cow, pig, baboon and bovine spermatogonial stem cells shown to be capable of colonizing recipient mouse seminiferous tubules. An in vitro system that supports the proliferation and maintenance of spermatogonial stem cells could be used to preserve and expand spermatogonial stem cell numbers as well as aid in genetic modification. GS isolation GS Culturing GS Transplantation 3 to 5 months Colonization Progeny with donor genes
  • 22. Preservation of spermatozoa Applications For improvement of a breed To support in situ conservation To support in vivo populations To study the effect of single major gene Semen freezing infrastructure is available throughout the country Limitations Spermatozoa contain haploid genome Semen freezing/ AI is not standardized in all species
  • 23. Semen harvesting and freezing Selection of Bulls Management of Bulls Semen collection and freezing
  • 24. Selection of Bulls • Physical Examination • Before procuring new bull calves/bulls for a semen station, a thorough physical examination shall be conducted by an accredited professional to ensure that the bulls are free from abnormality and do not display clinical symptom(s) of any infection or any contagious diseases. • For every new calf procured, the measurement of scrotal circumference and body weight should be initiated immediately. • Prior to introduction of new bulls for semen collection, breeding soundness examination shall also be carried out.
  • 25. • Karyotying and testing for genetically transmitted diseases It is necessary that all animals be karyotyped to rule out any chromosomal defects. • Quarantine A quarantine period of minimum 60 days* is compulsory before bringing new bulls into a semen station. Each new animal in quarantine station will be tested against major contagious diseases before its entry to resident herd e.g. TB, JD, Brucellosis, Campylobacteriosis and Trichomoniasis.
  • 26. • During quarantine period, the bulls shall be vaccinated against FMD, HS, BQ, Theileriosis and Anthrax. However, vaccinations against bacterial diseases shall be done only if there is an outbreak or prevalence of a particular disease. • Once the quarantine period is over, all bulls shall be introduced to the young bull rearing station.
  • 27. Management of Bulls • The objective of daily care of bulls is to ensure a satisfactory state of cleanliness.  The bulls shall be kept under hygienic conditions at all times.  The coat of the bulls shall be kept clean and generally short.  The hooves shall be regularly trimmed.  The length of the tuft of hairs at the preputial orifice, which is invariably soiled, shall be cut to about 2 cm.  Bulls shall be brushed and groomed regularly, and where necessary, special attention shall be given to the underside of the abdomen, a day prior to semen collection.
  • 28.  Cleaning of the prepuce with sterile normal saline solution may be done every ten days if the microbial load is within the prescribed limits.  Cleaning prior to the day of collection can be practiced if the microbial load in frozen semen is beyond the prescribed limit.  In the event of obvious soiling, careful cleaning of the preputial orifice and the adjoining areas with soap or a detergent is recommended; followed by thorough rinsing and drying.  Scientific feeding schedule shall be followed for the bulls.
  • 29. Semen Collection • Ideally, the floor of the collection yard shall be made of concrete layer at a depth of one foot from the ground level.
  • 30. Handling, processing & freezing of semen • • • • • • • • • Premises Equipment Personnel Hygiene Diluents Evaluation & Processing Colour Specifications Printing of Straws Post thaw motility Quality Checks for frozen semen
  • 31. Semen freezing The biophysical principles that apply to cryopreservation of living cells and tissues also apply to cryopreservation of sperm. The sperm may be damaged during cryopreservation and/ or thawing either by the formation of large intracellular ice crystals or by the increased intracellular concentration of solutes and accompanying changes that result from the dehydration of cells during cryopreservation (solution effects). The damage to sperms during cryopreservation by crystal formation and/ or solution effect can be minimized by freezing sperms at an optimal freezing rate. Adding cryoprotectants such as glycerol or dimethyl sulfoxide to the freezing medium results in freezing at lower temperatures. This probably retards dehydration of cells and the resultant harmful solution effects; thus sperms may be cooled slowly enough to prevent the formation of large ice crystals.
  • 32. Quality control in GeneBank Status of the frozen semen quality to be deposited in GeneBank for long term cryopreservation Semen Parameter Post thaw motility(%) Acrosomal integrity(%) Abnormal sperm(%) Hypo osmotic swelling test(%) (responsive cells) Microbial load Minimum acceptable level 40 50 < 20% 40 <5000CFU/ml
  • 33. Quantity of semen doses to be preserved for breed conservation At NBAGR, total number of frozen semen doses to be kept in National Animal Gene Bank for different animal species is as given below.  Cattle and Buffalo: A total of 30,000 semen doses (2000 doses each from 15 unrelated bulls) for a breed are to be preserved. Fifty percent of these semen doses are proposed to be kept at National Gene Bank and rest 50% at Regional Centres in respective state.  Sheep and Goat: A total of 25,000 semen doses (1000 doses each from 25 unrelated breeding males) for a breed are to be preserved. Fifty percent of these semen doses are to be kept at National Gene Bank and rest 50% at Regional Centres in respective state.
  • 34. Quality control in GeneBank  Quality of frozen semen stored in GeneBank is an important issue and is taken care seriously.  The males used for semen collection must be free from infectious diseases like FMD, Contagious Bovine Pleuropneumonia, Tuberculosis, Brucellosis, Vibriosis, Trichomoniasis and Blue Tongue.  In addition to it the frozen semen in GeneBank is monitored regularly for semen quality parameters so as to access the fertility status of frozen semen.
  • 35. Information regarding germplasm The available details of bull pedigree, physical characteristics and bull’s semen quality is procured from cooperating centers for documenting the details of frozen semen stored in GeneBank.        Date of birth Sire index (if tested) Age at first collection Any abnormality detected in karyotyping Physical characteristics alongwith photograph of bull Health status alongwith Any abnormality in genital organs Semen quality
  • 36. Livestock conservation through National/ Regional semen banks National Gene Bank at NBAGR Karnal 20% of total Initially 1000 doses of semen each from 15 unrelated bulls (Cattle and Semen doses Buffalo); 500 doses each of 25 unrelated breeding males(Sheep and For use in field Goats). Subsequent years 20% replacement semen doses State Livestock Development Boards, Conservation cell 20% of total Semen doses For use in field Initially 1000 doses of semen each from 15 unrelated bulls (Cattle and Buffalo); 500 doses each of 25 unrelated breeding males(Sheep and Goats). Subsequent years 20% replacement semen doses Regional Frozen Semen Production units/ Banks Semen supply for genetic improvement and breeding of females in field Artificial Insemination Centres
  • 37. Semen available in GeneBank No. of species Cattle, Buffalo , Goat, Sheep, Camel, Yak & Equine 7 No. of Breeds 21 breeds (Cattle) 9 breeds ( Buffalo ) 3 breeds ( Goat) 2 breeds (Equine) 1 breed each from Sheep, Camel & Yak 38 No. of Bulls/Rams/Bucks/Stallions Cattle (115) Buffalo (79) Goat (38) Sheep (20) Camel (15) Yak (3) Equine(6) 282 No. of doses Cattle - 62,854 Buffalo- 38,153 Goat - 11,593 Sheep - 8375 Camel- 928 Yak - 360 Equine- 1220 1,23,283