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

1842

21

813

6

37

Contribution of livestock in indian economyIndia 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

Nearly 80% of indigenous animals are non-descript

No true estimates of pure bred populations

Poor understanding of issues related to conservation vs improvement

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

SpeciesSpecies Population (m) Population (m)

2003 20072003 2007

Cattle 185.18 199.08

Buffalo 97.92 105.34

Sheep 61.47 71.56

Goat 124.36 140.54

Pig 13.58 11.13

Horse 0.75 0.61

Camel 0.63 0.52

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

Factors affecting animal biodiversity

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.

Conservation

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 <100Endangered: Breeding female 100-1000Vulnerable : Breeding female 1000-5000Insecure : Breeding female 5000-10000Normal : Breeding female >10000

Population size of a breed for its status (‘000)

Species Normal Insecure Vulnerable EndangeredCritical

Cattle 25 15-25 5-15 2-5 <2Buffaloes 30 20-30 10-20 5-10 <5Sheep 50 30-50 15-30 8-15 <8Goat 30 20-30 10-20 5-10 <5Camels 20 15-20 5-15 2-5 <2Horses 20 15-20 5-15 2-5 <2Pigs 10 5-10 1-5 0.5-1.0

<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 SituFarmer’s herds/flocksOrganized herds in breed tracts

Ex situOrganized herds outside breed tractCryopreserved germplasm

• Embryos

• Somatic cells

• DNA

• Spermatogonial stem cells

• Semen

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.

Organized flocks/herds

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 isolationGS Culturing

GS Transplantation

Progeny with donor genes

Colonization

3 to 5 months

Preservation of spermatozoa

Applications For improvement of a breedTo support in situ conservationTo support in vivo populationsTo study the effect of single major geneSemen freezing infrastructure is available throughout the country

LimitationsSpermatozoa contain haploid genome Semen freezing/ AI is not standardized in all species

Semen harvesting and freezing

Selection of BullsManagement 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 diseasesIt 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 Minimum acceptable level

Post thaw motility(%) 40Acrosomal integrity(%) 50Abnormal sperm(%) < 20%Hypo osmotic swelling test(%) 40(responsive cells)Microbial load <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 Karnal20% 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 andFor use in field Goats). Subsequent years 20% replacement semen doses

State Livestock Development Boards, Conservation cell

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 andFor use in field 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