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Cryoconservation of animal genetic resources at the USDA Gene Bank
Cryoconservation of animal genetic resources is a strategy wherein samples of animal genetic materials are preserved
cryogenically.
Animal genetic resources, as defined by the Food and Agriculture
Organization of the United Nations, are "those animal species that are
used, or may be used, for the production of food and agriculture, and
the populations within each of them. These populations within each
species can be classified as wild and
feral populations,
landraces and primary populations, standardised
breeds, selected lines, varieties, strains and any conserved genetic material; all of which are currently categorized as Breeds." Genetic materials that are typically cryogenically preserved include
sperm,
oocytes,
embryos and
somatic cells. Cryogenic facilities are called
gene banks and can vary greatly in size usually according to the economic resources available. They must be able to facilitate
germplasm
collection, processing, freezing, and long term storage, all in a
hygienic and organized manner. Gene banks must maintain a precise
database and make information and genetic resources accessible to
properly facilitate cryoconservation. Cryoconservation is an
ex situ conservation strategy that often coexists alongside
in situ conservation to protect and preserve livestock
genetics.
Cryoconservation of livestock genetic resources is primarily done
in order to preserve the genetics of populations of interest, such as
indigenous breeds, also known as local or minor breeds. Material may be
stored because individuals shared specific genes and phenotypes that may
be of value or have potential value for researchers or breeders.
Therefore, one of the main goals remains preserving the gene pool of
local breeds that may be threatened. Indigenous livestock genetics are commonly threatened by factors such as
globalization,
modernization, changes in production systems, inappropriate introduction of major breeds,
genetic drift,
inbreeding,
crossbreeding,
climate change,
natural disasters, disease,
cultural changes, and urbanization. Indigenous livestock are critical to
sustainable agricultural
development and
food security, due to their: adaptation to
environment
and endemic diseases, indispensable part in local production systems,
social and cultural significance, and importance to local rural
economies. The genetic resources of minor breeds have value to the local farmers, consumers of the products,
private companies
and investors interested in crossbreeding, breed associations,
governments, those conducting research and development, and
non-governmental organizations. Therefore, efforts have been made by national governments and non-governmental organizations, such as
the Livestock Conservancy, to encourage conservation of livestock genetics through cryoconservation, as well as through other
ex situ and
in situ strategies. Cryogenic specimens of livestock genetic resources can be preserved and used for extended periods of time. This advantage makes cryoconservation beneficial particularly for
threatened breeds who have low breed populations. Cryogenically
preserved specimens can be used to revive breeds that are
endangered or
extinct,
for breed improvement, crossbreeding, research and development.
However, cryoconservation can be an expensive strategy and requires long
term hygienic and economic commitment for germplasms to remain viable.
Cryoconservation can also face unique challenges based on the species,
as some species have a reduced survival rate of frozen germplasm.
Description
Cryoconservation
is the process of freezing cells and tissues using liquid nitrogen to
achieve extreme low temperatures with the intent of using the preserved
sample to prevent the loss of genetic diversity. Semen, embryos, oocytes,
somatic cells,
nuclear DNA,
and other types of biomaterial such as blood and serum can be stored
using cryopreservation, in order to preserve genetic materials. The primary benefit of cryoconservation is the ability to save
germplasms for extended periods of time, therefore maintaining the
genetic diversity of a species or breed.
There are two common techniques of cryopreservation: slow freezing and
vitrification. Slow freezing helps eliminate the risk of intracellular
ice crystals.
If ice crystals form in the cells, there can be damage or destruction
of genetic material. Vitrification is the process of freezing without
the formation of ice crystals.
Value
Cryoconservation
is an indispensable tool in the storage of genetic material of animal
origin and will continue to be useful for the conservation of livestock
into the future. Cryoconservation serves as a way to preserve
germplasms, which is particularly beneficial for threatened breeds.
Indigenous livestock may be conserved for a variety of reasons,
including the preservation of local genetics, their importance in local
traditions and their value to the culture identity and heritage of the
area.
The loss of regional livestock diversity could increase instability,
decreases future possibilities and challenge production systems.
Moreover, the maintenance of indigenous breeds can aid in the
preservation of traditional lifestyles and livelihoods, even providing
income through cultural tourism.
Indigenous breeds can contribute to local economies and production
systems by utilising land that is unsuitable for crop production to
produce food products, as well as providing hides, manure and draft
power. Therefore, the conservation and progression of these breeds are
of the utmost importance for food security and sustainability.
Another beneficial factor in cryoconservation of indigenous livestock is in terms of food security and economic development.
Indigenous livestock often have beneficial traits related to adaptation
to local climate and diseases that can be incorporated into major
breeds through cryoconservation practices. Cryoconservation is a favorable strategy because it allows germplasms
to be stored for extended periods of time in a small confined area. An
additional benefit of cryoconservation is the ability to preserve the
biological material of both maternal and paternal cells and maintain
viability over extended periods of time.
Cryoconservation has been successfully used as a conservation strategy
for species and breeds that have since been endangered. One drawback is
that cryoconservation can only be done if preparation has taken place in
advance.
With proper preparation of collecting and maintaining genetic material,
this method is very beneficial for the conservation of rare and
endangered livestock. Cryoconservation can serve as a contingency plan
when a breed population needs to be restored or when a breed has become
extinct, as well as for breed improvement. This process benefits
companies and researchers by making genetic materials available.
Conservation Goals
| Flexibility of country's AGR to meet changes
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Insurance against changes in production conditions
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Safeguarding against diseases, disasters, etc.
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Opportunities for genomic research
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| Genetic Factors
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Allowing continued breed evolution/genetic adaption |
Increasing knowledge of phenotypic characteristics of breed |
Minimizing exposed to genetic drafts
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| Sustainable utilization of total areas
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Opportunities for development in rural areas
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Maintenance of agro-ecosystem diversity
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Conservation of rural culture diversity
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The support of numerous stakeholders make this process possible in
the establishment and operations of cryoconservation. Before every phase
is executed, all participating stakeholders must be briefed to
understand the possible phase impending. This would include informing
the stakeholders of their responsibilities and receiving their consent
for the cryoconservation process. The possible stakeholders within the cryoconservation process could include:
- The State-the government acquires responsibility for conservation of animal genetic resources;
- Individual Livestock Keepers and Breed Associations-individual
livestock keepers are commonly the primary owners of the livestock whose
germplasm is used for processes of cryoconservation. Breed Associations
would be interested in the well-being of their respective breeds in
short and long terms. Through this interest these associations may
provide financial and organizational support for the cryoconservation
process;
- Private Companies-including, but, not limited to, commercial
breeding companies, processing companies and agricultural support
services may find value in the cryoconservation process and may striving
to become more involved;
- The National Coordinator for the Management of Animal Genetic
Resources-this particular stakeholder would possibly a member of the
National Advisory Committee on Animal Genetic Resources. This member
needs to be knowledgeable about all aspects and activities of
cryoconservation, as this stakeholder would have the responsibility of
reporting current information to the FAO.
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Methods
Collection
There are several ways to collect the genetic materials based on which type of germplasm.
Semen
Freezing semen is a commonly used technique in the modern animal agriculture industry, which is well researched with established methods Semen is often collected using an
artificial vagina,
electroejaculation,
gloved-hand technique, abdominal stroking, or epididymal sperm
collection. Preferred collection techniques vary based on species and
available tools. Patience and technique are keys to successful
collection of semen.
There are several styles and types of artificial vaginas that can be
used depending on the breed and species of the male. During this process
the penis enters a tube that is the approximate pressure and
temperature of the female's vagina. There is a disposable bag inside the
tube that collects the semen. During this process it may be beneficial
to have a teaser animal—an animal used to sexually tease but not
impregnate the animal—to increase the arousal of the male. Electroejaculation is a method of
semen collection
in the cattle industry because it yields high quality semen. However,
this process requires the animal to be trained and securely held, thus
it is not ideal when working with wild or feral animals. When performing
this process the electroejaculator is inserted into the rectum of the
male.
The electroejaculator stimulates the male causing an ejaculation, after
which the semen is collected. The glove hand collection technique is
used mainly in the swine industry. During this process, the boar mounts a
dummy, while the handler grasps the penis of the boar between the
ridges of his fingers and collects the semen.
Abdominal stroking is exclusively used in the poultry industry. During
the technique, one technician will hold the bird, while a second
technician massages the bird's cloaca. However, feces and semen both exit the male bird's body through the cloaca, so the semen quality is often low.
Embryo
Embryo
collection is more demanding and requires more training than semen
collection because the female reproductive organs are located inside of
the body cavity. Superovulation is a technique used in order to have a
female release more oocytes than normal. This can be achieved by using
hormones to manipulate the female's reproductive organs. The hormones
used are typically gonadotropin-like, meaning they stimulate the gonads.
Follicle stimulating hormone is the preferred hormone in cattle, sheep
and goats. While in pigs, equine chorionic gonadotropin is preferred.
However, this is not commonly done in the swine industry because gilts
and sows (female pigs) naturally ovulate more than one oocyte at one
time. Superovulation can be difficult because not all females will
respond the same way and success will vary by species. Once the female
has released the oocytes, they are fertilized internally—in vivo—and
flushed out of her body. In vivo fertilization is more successful than
in vitro fertilization.
In cattle, usually 10 or more embryos are removed from the flushing
process. In order to flush the uterus, a technician will first seal off
the female's cervix and add fluid, which allows the ovum to be flushed
out of the uterine horns and into a cylinder for analysis. This process
typically takes 30 minutes or less.
Technicians are able to determine the sex of the embryo, which can be
especially beneficial in the dairy industry because it is more desirable
for the embryo to be a female. Vitrification is the preferred method of embryo freezing because it yields higher quality embryos.
It is crucial technicians handle the embryos with care and freeze them
within 3–4 hours in order to preserve viability of the greatest
percentage of embryos.
Oocytes
Oocytes
can be collected from most mammalian species. Conventional oocyte
collection is when ovaries are removed from a donor animal; this is done
posthumously in slaughter facilities.
The ovaries are kept warm as they are brought back to a laboratory for
oocyte collection. Keeping the ovaries warm helps increase the success
rate of fertilization. Once collected the oocytes are assessed and categorized into small, medium, and large, and then matured for 20–23 hours.
This simple, inexpensive technique can lead to about 24 oocytes
collected from a bovine. Conventional oocyte collection is especially
useful for females who unexpectedly die or who are incapable of being
bred due to injury. A second option for oocyte collection is to utilize
the transvaginal ultrasound guided oocyte collection method otherwise
known as TUGA. Collection technique varies slightly by species, but the
general methods for collection are the same; a needle is inserted into
each ovarian follicle and pulled out via vacuum. The major benefit of
using this method is the ability to expand the lifetime reproductive
productivity, or the number of productive days an animal is in her
estrous cycle. Pregnant cows and mares continue to develop new follicles until the
middle of pregnancy. Thus, TUGA can be used to substantially increase
the fitness of an individual because the female then has the potential
produce more than one offspring per gestation.
Somatic cells
Somatic
cells are an additional resource which can be retrieved for gene
banking, particularly in the cases of emergency wherein gametes cannot
be collected or stored. Tissues can be taken from living animals or
shortly after death. These tissues can be saved via cryopreservation or
dehydrated. Blood cells can also be useful for DNA analysis such as
comparing homozygosity
[36][37]
It is recommended by the FAO that two vials of blood be drawn to reduce
the chance that all samples will be lost from a particular animal. DNA
can be extracted using commercial kits, making this an affordable and
accessible strategy for collecting germplasms.
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Semen
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Semen and Oocytes
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Embryos
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| Number of samples needed to restore a breed
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2000 |
100 of each |
200
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| Backcrossing needed?
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Yes
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No
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No
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| Mitochondrial genes included?
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No
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Yes
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Yes
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| Collection Possible in livestock species
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Mostly, not always
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Yes, in some species. Operational for bovines
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Yes, in some species. Operational for bovines
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| Cost of collection
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$$
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$$
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$$$$
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| Cryopreservation possible?
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Yes
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Still in experimental stage
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Operational in bovines, horses and sheep. Promising in pigs. Impossible in poultry
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| Utilization
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Surgical or non-surgical insemination backcrossing for 4 generations
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In vitro maturation/IVF followed by surgical or non-surgical ET
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Surgical or non-surgical ET
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| Current feasibility
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High
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Medium
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High depending on available resources
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Freezing
There are two cryopreservation freezing methods: slow freezing and vitrification.
Example freezing laboratory
Slow freezing
During
slow freezing, cells are placed in a medium which is cooled below the
freezing point using liquid nitrogen. This causes an ice mass to form in
the medium. As the water in the medium freezes, the concentration of
the sugars, salts, and cryoprotectant increase. Due to osmosis, the
water from the cells enters the medium to keep the concentrations of
sugars, salts, and cryoprotectant equal. The water that leaves the cells
is eventually frozen, causing more water to diffuse out of the cell.
Eventually, the unfrozen portion—cellular—becomes too viscous for ice
crystals to form inside of the cell.
Vitrification
The
second technique for cryoconservation is vitrification or flash
freezing. Vitrification is the transformation from a liquid to solid
state without the formation of crystals. The process and mechanics of
vitrification are similar to slow freezing, the difference lying in the
concentration of the medium. The vitrification method applies a selected
medium which has a higher concentration of solute so the water will
leave the cells via osmosis. The medium is concentrated enough so all of
the intracellular water will leave without the medium needing to be
reconcentrated. The higher concentration of the medium in vitrification
allows the germplasms to be frozen more rapidly than with slow freezing.
Vitrification is considered to be the more effective technique of
freezing germplasms.
Facility design and equipment
Facility design
Example of animal holding and collecting facility
When designing a facility, there are several things that should be
kept in mind including biosecurity, worker safety and efficiency, and
animal welfare. Diverse infrastructure is required in order to
successfully collect and store genetic material. The buildings needed
depend on the size of facilities as well as the extent of the
operations.
Biosecurity
Biosecurity,
a management measure used to prevent the transmission of diseases and
disease agents on the facility, is important to keep in mind when
designing a facility.
In order to achieve a high level of biosecurity, collection facilities
should be placed as far as possible from one another, as well as from
farms. According to the FAO's recommendations, facilities should be "at
least 3 km from farms or other biological risks and 1 km from main roads
and railways".
Separation between collection facilities and surrounding farms can
improve biosecurity as pests, such as flies and mice, have the potential
to travel from farm to facility and vice versa. Other disease agents
may be able to travel through the air via wind, furthering the
importance of separation of farms and proper air sanitation and
ventilation. Additionally, a
perimeter fence
is used to prevent potential threats that could cause contamination to
germplasms, such as unauthorized personnel or unwanted animals, from
entering the facilities. Animals may be housed in
pens
located inside or outside of a barn as long as they are contained
within the perimeter fence. When interaction with outside objects, such
as feed trucks or veterinary personnel, is necessary, complete
sanitation is required to decrease the risk of contamination. There is
always the possibility of disease spreading among the animals whose
biological data is being collected or from animal to human. An example
of a disease that can easily spread through germplasm is Porcine
Reproductive and Respiratory Syndrome, otherwise known as PRRS. A highly
contagious disease between swine, PRRS causes millions of dollars to be
lost annually by producers. The disease can be spread through boar
semen.
Therefore, biosecurity is particularly important when genetic material
will be inserted into another animal to prevent the spread of such
diseases.
Human considerations
Worker
safety is always a priority when handling livestock. Escape routes and
alternative access throughout the facility are crucial for both the
handlers and livestock.
Germplasm storage and collection sites must include locker rooms for
staff, which provide lockers, showers, and storage of clothing and
footwear, in order to meet sanitation requirements.
Animal considerations
Animal
housing practical when collecting germplasms because they keep donor
animals in an easily accessible area, making the process of collecting
germplasms easier and more efficient. The species and breeds of animals
housed should be considered while planning the facility; facilities
should be big enough to meet animal welfare standards, yet small enough
to reduce human contact and increase ease of handling while reducing
stress of the animal. As the process of collecting germplasm may take
several days, the animal may become stressed causing a lower quality of
genetic material to be obtained. Thus, training the animal to become
familiar with the process is key.
Holding facilities for animals may also serve as a quarantine.
Quarantine facilities are necessary in order to prevent the transmission
of disease from animal to animal, animal to germplasm, germplasm to
germplasm, and germplasm to animal. Introducing quarantine to separate
the diseased animal(s) from the healthy should be done immediately.
However, a quarantine does not always prevent the spread of disease.
Temperature control and ventilation
Temperature control and
ventilation
should be included in the design of the holding and collection
facilities to keep the animals comfortable and healthy, while limiting
stress during the germplasm collection process. Ventilation serves as an
effective way to keep clean airflow throughout the facilities and
eliminate odors Temperature control helps regulate the air quality and
humidity level inside the barn.
Equipment
A
freezing and processing laboratory for genetic materials can be on the
same site as the holding and collecting facility. However, the
laboratory must have higher sanitation standards. According to the FAO, a
proper germplasm laboratory should include the following:
- Washable work surfaces, floors (non-slip) and walls;
- Sufficient lighting and ventilation;
- Hot and cold, purified water;
- Electrical sockets;
- Adequate storage for consumable materials.
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Cryopreservation requires equipment to collect biological material
and test tubes for storage. Price is highly variable based on the
quality of the collection and storage materials. The life expectancy of tools should be considered when determining costs. In addition to traditional laboratory equipment, the FAO also suggests the following:
- Disposable coveralls;
- Portable incubator;
- Haemocytometer;
- Semen straws and filling/sealing equipment;
- Liquid nitrogen storage tank;
- Liquid nitrogen;
- Liquid nitrogen dry-shipper;
- Equipment for determining sperm concentration (one or more of the following three);
- Spectrophotometer (fixed or portable);
- Makler counter chamber (or disposable counting chamber);
- Haemocytometer;
- Straw filling and sealing equipment;
- Freezing equipment (manual or programmable);
- Carbon dioxide incubator (for embryos);
- Laminar flow benches (for embryos);
- Dry liquid nitrogen shipping tanks;
- Long-term liquid nitrogen storage tanks.
Limitations
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Cryoconservation
is limited by the cells and tissues that can be frozen and successfully
thawed. Cells and tissues that can be successfully frozen are limited
by their surface area. To keep cells and tissues viable, they must be
frozen quickly to prevent ice crystal formation. Thus, a large surface
area is beneficial.
Another limitation is the species being preserved. There have been
difficulties using particular methods of cryoconservation with certain
species. For example, artificial insemination is more difficult in sheep
than cattle, goats, pigs, or horses due to posterior folds in the
cervix of ovines. Cryopreservation of embryos is dependent on the species and the stage
of development of the embryo. Pig embryos are the most difficult to
freeze, thaw, and utilize produce live offspring due to their
sensitivity to chilling and high lipid content.
Legal issues
The
collection and utilization of genetic materials requires clear
agreements between stakeholders with regards to their rights and
responsibilities.
The FAO and others, such as Mendelsohn, suggests that governments
establish policies with regards to livestock genetic resources and their
collection, storage, distribution, and utilization are governments.
The FAO also recommends that national or regional livestock industries
establish an advisory committee to advise and provide recommendations on
policy. Livestock are traditionally a private good; in order to obtain
ownership of genetic materials, gene banks have several strategies that
they can deploy. Gene banks may either:
- buy the livestock in order to obtain and preserve genetic information;
- have the germplasm donated by livestock owner;
- pay a fee to the livestock owner for germplasm rights;
- develop a contract with the livestock owner in order to obtain
germplasm ownership only after set period of time, in order to prevent
immediate acquisition of germplasm by competitors.
One of the key elements of cryoconservation of livestock is open
access to genetic materials, to make the resources of these conserved
genetic materials accessible for utilization. Utilization should be
based on sustainable use, development, and conservation, as well as
improvement for the livestock industry.
Government and non-governmental organizations recommend that genetic
information should have open access for the following purposes:
- national public need;
- non-research breeding by non-governmental organizations or private entities;
- research for breed improvement, conservation of endangered breeds, and potential recovery of extinct breeds.
Examples
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Hungarian Grey cattle
An example of the use of cryoconservation to prevent the extinction of a livestock breed is the case of the
Hungarian Grey cattle,
or Magya Szurke. Hungarian Grey cattle were once a dominant breed in
southeastern Europe with a population of 4.9 million head in 1884. They
were mainly used for draft power and meat. However, the population had
decreased to 280,000 head by the end of
World War II and eventually reached the low population of 187 females and 6 males from 1965 to 1970. The breed's decreased use was due primarily to the
mechanization of agriculture and the adoption of major breeds, which yield higher milk production.
The Hungarian government launched a project to preserve the breed, as
it possesses valuable traits, such as stamina, calving ease, disease
resistance, and easy adaptation to a variety of climates. The government program included various conservation strategies, including the cryopreservation of semen and embryos.
The Hungarian government's conservation effort brought the population
up to 10,310 in 2012, which shows significant improvement using
cryoconservation.
The Gaur
Gaur, also known as the Indian bison, is the heaviest and most powerful of all wild cattle native to South and Southeast Asia. It is indicated in field data that the population of mature animals is about 5,200–18,000. Male and female Gaur both have distinctive humps between the head and shoulders, a dorsal ridge, prominent horns, and a
dewlap
which extends to the front legs.The Gaur grows 60% faster than domestic
cattle, meaning farmers meat can be harvested at a faster rate, making
beef production two to three times more profitable. Gaur meat is
preferred over other breeds' meat among local people. Another benefit of
the bovine is that it has the ability to sweat and tolerates heat well.
The Gaur population experienced a drastic decline of about 90%
between the 1960s and 1990s due to poaching, commercial hunting,
shrinking habitat, and the spreading of disease. According to the International Union for Conservation of Nature's Red List, the Gaur is a
vulnerable species due to its declining population in Southeast Asia.
Although the global Gaur population has declined by 30% over the past
30 years, the Gaur has a relatively stable population in India, due to
protective efforts such as cryoconservation. The American
Association of Zoos and Aquariums,
Integrated Conservation Research (ICR), and Advanced Cell Technology
have made efforts to use cryopreserved specimens of the Gaur through
artificial insemination, embryo transfer, and cloning, respectively.
Hybridization with domestic cattle has been successfully achieved by
ICR, in order to create higher yielding, heat resistant cattle.
