ASSISTED

REPRODUCTIVE
TECHNOLOGIES
Assignment#1

JULY 17, 2020

SUBMITTED TO: DR. FEHMIDA FAREED KHAN
SUBMITTED BY: BUSHRA KHAN
ROLL #: 04271713035
DEPARTMENT OF BIOTECHNOLOGY, QAU ISLAMABAD
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ASSISTED REPRODUCTIVE TECHNOLOGY

ARTs are made up of a series of surgical, hormonal, or gamete manipulations that increase the
chance of conception and implantation of an embryo. ART is basically used to alleviate infertility
problems but here we are more concerned about its role in diminishing the loss of genetic
biodiversity and elevating the number of endangered species.

TYPES OF ASSISTED REPRODUCTIVE TECHNOLOGY

Various types of ART have been described in context. Let us discuss them one by one.
Artificial Insemination
Artificial insemination is a method of treating infertility by inserting semen directly into a
womb. Medical scientists also developed the procedure to breed livestock to get better traits.

How Artificial Insemination Is Done?

Artificial insemination (AI) in cattle is a process of introducing semen, collected separately from
a bull, into the reproductive tract of the cow, allowing pregnancy to occur without physical
mating. It has been used extensively throughout the world on many other species of animals
besides cattle. Not only does it avail the opportunity of making use of genetically superior bulls
in their absence and easy introduction of new breeds into a herd, AI also reduces the risk of
spreading venereal diseases to females from males and vice versa.

FIG.1 ARTIFICIAL INSEMINATION OF COW

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Role in Reducing the Risks of Extinction

Artificial insemination could also help nurse endangered species back from the brink of
extinction.

 Hope for such efforts made a large leap forward recently with the announcement that
researchers had been able to produce Magellanic penguin chicks using artificial
insemination.
 Black-footed ferrets, giant pandas, white-naped crane and the Przewalski’s horse are
also listed in endangered species which are being gone through the process of artificial
insemination (Lincoln Park Zoo. 2015)

Gene Banking
An establishment in which both somatic and hereditary genetic material are conserved. It
stores, in a viable form, material from plants that are in danger of extinction in the wild
and cultivars that are not currently in popular use. The stored genetic information can be called
upon when required. For example, a crop may be needed that possesses a quality (e.g.
tolerance to adverse climatic conditions) which cannot be found in currently exploited cultivars
but was present in more antiquated varieties. There are various types of gene banks like seed
bank, tissue bank, cryo bank, sperm bank, ova bank, pollen bank, field gene bank etc.
How It Is Done?
The normal method of storage is to reduce the water content of seed material to around 4 per
cent and keep it at 0°C. Stored this way, the material often remains viable for 10–20 years.
Gene banks use low temperatures to stop chemical and biological activity that might break
down cells. Some banks freeze material in liquid nitrogen at –196° Celsius). This freezing
process replaces water in cells with another fluid, such as glycerol. That fluid minimizes the
development of ice crystals. Such crystals could damage cell walls. Later, during thawing,
biologists will remove the glycerol or some other fluid and return water to the cells. Dense
genotypic information for all accessions held in gene banks will facilitate access to germplasm
and guide conservation decisions. Drift during multiplication cycles may be monitored through
repeated genotyping (Rajasekharan P.E.2015).
How It Is Involved in Reduction of Extinction Risks
 In 1999 at the Audubon Center for Research of Endangered Species, a domestic
housecat gave birth to an African wildcat kitten that had been frozen as an embryo in a
DNA bank. This was the first example of interspecies birth. In 2000, the Center produced
test-tube Caracal cats from sperm that had been stored in their DNA bank.
 A variety of zoos and conservation groups have collected and stored genetic material
from endangered species. The largest DNA bank for endangered animals is housed at
the San Diego Zoo and is called the Frozen Zoo. The Frozen Zoo collected samples from
more than seven thousand threatened and endangered species, including more than
13,000 samples of semen, oocytes, and embryos (Encyclopedia., 2016).
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FIG.2 GENE BANK AS A TOOL FOR GERMPLASM CONSERVATION GUIDE.

Gamete Rescue
Post-mortem gamete rescue is a process in which gametes are collected from recently
deceased animals for cryopreservation and storage in a genome resource bank. In conjunction
with assisted reproductive technologies, it can serve to enhance the genetic diversity of ex
situ populations and also have potential to help rescue species from extinction if catastrophic
losses were to occur in the wild.
How it is Done?
For post-mortem gamete rescue in male animals, the vas deferens are ligated, then the testes,
epididymis and vas deferens are excised, cooled slowly to 5–10°C during transport to the
laboratory and processed for the recovery and cryopreservation of epididymal spermatozoa.
Offspring have been produced on a limited basis using frozen-thawed gamete rescue sperm
samples and artificial insemination in a number of domesticated and wild species.
Gamete Rescue in Endangered Specie
 Mortality rates are high among captive African black rhinoceroses, due to increased
susceptibility to disease. The ability to rescue genetic material from individuals that die
unexpectedly represents a practical approach to assist ex-situ conservation efforts
(Stoops et al., 2011).
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FIG.3 PHOTO OF A TWO-CELL AFRICAN BLACK RHINOCEROS EMBRYO WITH TWO POLAR BODIES

Cryopreservation
The process of cooling and storing cells, tissues, or organs at very low temperatures to maintain
their viability. For example, the technology of cooling and storing cells at a temperature below
the freezing point ('196' C) permits high rates of survivability of the cells upon thawing.
The primary purpose of cryopreservation is to preserve biological specimens that can be used
later on in different reproductive technologies to reduce infertility or risks of extinction.
What Procedure it Follows?
. The major steps in cryopreservation are
 The mixing of CPAs with cells or tissues before cooling.
 Cooling of the cells or tissues to a low temperature and its storage.
 Warming of the cells or tissues.
 Removal of CPAs from the cells or tissues after thawing.
 The appropriate use of CPAs is therefore important to improve the viability of the sample to be
cryopreserved (Jang et al., 2017).
Contribution Towards Decline in Extinction Rates
 Cryopreservation enables long-term conservation of critically endangered species
like Rubus humulifolius.
 Different plant organs have been used for cryopreservation of Orchidaceae, including
zygotic embryos, seeds, immature seeds, protocorms, pollen and seeds, and cell
suspensions (Cerna M. et al., 2018).
 Black-footed ferret (Mustela nigripes), 2 cheetahs (Acinonyx jubatus), 3 Eld’s deer
(Cervus eldii thamin), 4 scimitar-horned oryx (Oryx dammah), 5 tufted deer (Elaphodus
cephalophus), and 6 Przewalski horse (Equus ferus przewalskii). Ovarian tissue samples
from these species have been cryopreserved and are currently stored in the Genome
Resource Bank at the Conservation Biology Institute (Comizolli P et al., 2010)
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FIG.4 DIFFERENT METHODS OF CRYOPRESERVATION

Invitro Fertilization
IVF involves combining eggs and sperm outside the body in a laboratory. Once an embryo or
embryos form, they are then placed in the uterus. IVF is actually the process of fertilization by
extracting eggs, retrieving a sperm sample, and then manually combining an egg and sperm in a
laboratory dish.
What Procedure it Follows?
During IVF, mature eggs are collected from ovaries and fertilized by sperm in a lab. Then the
fertilized egg (embryo) or eggs are transferred to a uterus. One full cycle of IVF takes about
three weeks. Sometimes these steps are split into different parts and the process can take
longer. The procedure can be done using your own eggs and your partner's sperm. Or IVF may
involve eggs, sperm or embryos from a known or anonymous donor.

FIG.5 PROCESS DEPICTING IVF

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Invitro Fertilization as a Tool to Reduce Extinction Rate

 Dr. Jennifer Barfield an assistant professor of reproductive physiology at CSU has
successfully used IVF to reintroduce pure Bison with Yellowstone genetics to the
northern Colorado prairie at the Laramie Foothills Bison Conservation Herd.
 Today, millions of human babies are conceived with IVF science, but this procedure
wasn’t always so common. The first human baby who was conceived by IVF, Louise Joy
Brown, turns 40 this July (History.com)
 IVF is commonly used in the cattle industry to breed robust herds by creating embryos
using eggs from the best dairy-producing cows. In fact, bovine IVF is now used at a scale
comparable to human IVF.
 Scientists have saved the genetic material of Sudan (the last male white northern rhino)
with the prospect of using in vitro fertilization to create a viable embryo with eggs from
the two remaining Northern white rhinos, his daughter and granddaughter (Tunstall et
al., 2018). It was possible to combine Sudan’s sperm with an egg from a living female
Northern white rhino, creating an embryo that would save the subspecies by creating a
new generation (Hildebrandt, T. B et al., 2018).

Embryo Transfer
Embryo transfer is one step in the process of removing one or more embryos from the
reproductive tract of a donor female and transferring them to one or more recipient females
(Garzo V Gabriel et al., 2006).Embryo transfer techniques have been applied to nearly every
species of domestic animal and to many species of wildlife and exotic animals, including
humans and non-human primates.
How Embryo Transfer Takes Place?
Virtually all commercial embryo transfers use nonsurgical recovery of the embryos rather than
the surgical techniques.
The process involves several steps and considerable time as well as variable expense. The steps
are as follows (Bó G.A (2018).
i. Selection of the Donor Cow
ii. Superovulation of the Donor Cow
iii. Insemination of the Cow
iv. Flushing the Embryos
v. Evaluation of the Embryos
vi. Selection and Preparation of Recipient Females
vii. Transfer of the Embryos
viii. Expected Embryo Transplant Results
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FIG.6 FLOW CHART DEPICTING THE PROCESS OF EMBRYO TRANSFERE

Embryo Transfer to Alleviate Extinction Risks

 Scientists at the Smithsonian Conservation Biology Institute (SCBI) and the Columbus
Zoo have successfully transferred cheetah embryos produced by in vitro fertilization
(IVF) to a surrogate cheetah mom for the first time. Two cubs were born Feb. 19 to 3-
year-old mom Izzy, though the cubs’ biological mom is 6 1/2-year-old Kibibi.
 TIME LINE: NOV 14,2019 TO FEB 19,2020 (washingtonpost.com)
 Interspecies embryo transfer was most successful between the cow (Bos taurus) and
zebu (B. indicus). Zebu is a domesticated species adapted to the hot climate. These two
species are very close in genetics and reproductive biology and it is not surprising that
interspecies transfer was successful.
 In addition to successful embryo transfer between different cattle species, in the
Cincinnatti Zoo, the embryos were transferred between two large antelopes: bongo
(Tragelaphus euryceros) and eland (T. oryx). Note that the bongo is an endangered
species (Amstislavsky et al., 2006).

Nuclear Transfer
The development of a single celled fertilized zygote to an animal capable of reproduction
involves not only cell division but the differentiation or specialization to numerous cell types
forming each tissue and organ of the adult animal. The technique of nuclear transfer allows the
reconstruction of an embryo by the transfer of genetic material from a single donor cell, to an
unfertilized egg from which the genetic material has been removed (Campbell K. H. (2002).
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What Process Does It Involves?

The following basic steps are involved in nuclear transfer (Tian et al., 2003)
i. Cells are collected from donor
ii. Cultured in vitro.
iii. A matured oocyte is then enucleated.
iv. A donor cell is transferred into the enucleated oocyte.
v. The somatic cell and the oocyte are then fused
vi. The embryo is allowed to develop to a blastocyst in vitro.
vii. The blastocyst can then be transferred to a recipient
viii. Cloned animals are born after completion of gestation.

FIG.7 NUCLEAR TRANSFER PROCESS

Contribution Towards Extinction Control

 This technique was implemented on sturgeons (one fish family bearing some of the
most critically endangered species). Single cells were transplanted enzymatically
isolated from a dissociated fin-fragment of the Russian sturgeon (Acipenser
gueldenstaedtii) into non-enucleated eggs of the sterlet (Acipenser ruthenus), two
species bearing different ploidy (4n and 2n, respectively). Up to 12% of the transplanted
eggs underwent early development, and one feeding larva (0.5%) was successfully
produced (Fatira et al., 2018).
 Somatic cells from a gaur bull (Bos gaurus), a large wild ox on the verge of extinction,
(Species Survival Plan < 100 animals) were electrofused with enucleated oocytes from
domestic cows. Twelve percent of the reconstructed oocytes developed to the
blastocyst stage, and 18% of these embryos developed to the fetal stage when
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transferred to surrogate mothers. Three of the fetuses were electively removed at days
46 to 54 of gestation, and two continued gestation longer than 180 (ongoing) and 200
days, respectively. Microsatellite marker and cytogenetic analyses confirmed that the
nuclear genome of the cloned animals was gaurus in origin (Lanza et al., 2000).

FIG.8 NUCLEAR TRANSFER USING CELL LINE OF ENDANGERED SPECIE (HOLT ET

AL.,2004).

FUTURE PROSPECTS
The list of assisted reproductive technologies has been expanding each year and will continue
to do so in the future. The transgenic cow, pig, and goat have become a prominent driving force
within the biotechnology industry, particularly with respect to biomedical research. As new
technologies such as IVF, sperm sorting, embryo sexing, genetic testing, and NT become more
available to the livestock industry, livestock producers will use basic nonsurgical ET procedures
pioneered in the mid-1970s with greater frequency. There is still much to be studied and
learned in the use of assisted reproductive technologies to maximize reproductive potential in
genetically valuable animals. Now that repeatable oocyte retrieval methods have been fine-
tuned, the next obstacle to overcome will be development of a chemically defined embryo
culture system that supports high in vivo development rates following transfer of fresh and
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frozen-thawed (or vitrified) IVP embryos. It will be exciting to follow new developments and
new applications of assisted reproductive technology in the decades ahead (Godke et al., 2014).

BIBLIOGRAPHY

 Lincoln Park Zoo. (2015, August 13). Critically endangered species successfully
reproduced using frozen sperm from ferret dead for 20 years: Genetic diversity of the
species significantly increased providing fresh hope for the future survival of this near-
extinct species. ScienceDaily.
 Rajasekharan P.E. (2015) Gene Banking for Ex Situ Conservation of Plant Genetic
Resources. In: Bahadur B., Venkat Rajam M., Sahijram L., Krishnamurthy K. (eds)
Plant Biology and Biotechnology. Springer, New Delhi
 "DNA Banks for Endangered Animals." World of Forensic Science. Retrieved July 16,
2020 from Encyclopedia.com: 
https://www.encyclopedia.com/science/encyclopedias-almanacs-transcripts-and-
maps/dna-banks-endangered-animals
 Stoops, M. A., O'Brien, J. K., & Roth, T. L. (2011). Gamete rescue in the African black
rhinoceros (Diceros bicornis). Theriogenology, 76(7), 1258–1265.
https://doi.org/10.1016/j.theriogenology.2011.05.032
 Jang, T. H., Park, S. C., Yang, J. H., Kim, J. Y., Seok, J. H., Park, U. S., Choi, C. W., Lee, S. R.,
& Han, J. (2017). Cryopreservation and its clinical applications. Integrative medicine
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https://doi.org/10.1016/j.imr.2016.12.001
 Cerna, M., Valdivieso, P., Cella, R., Mátyás, B., & Aucapiña, C. (2018). Cryopreservation
of orchid seeds through rapid and step freezing methods. F1000Research, 7, 209.
https://doi.org/10.12688/f1000research.13622.1
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 https://www.washingtonpost.com/dc-md-va/2020/02/24/first-cheetah-cubs-are-born-
ivf-national-zoo-facility-virginia/
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