UNIT - II

Part 1
 ARTIFICIAL INSEMINATION (AI)
• Artificial insemination (AI) is the process of collecting sperm cells from a
male animal and manually depositing them into the reproductive tract of a
female.
• Artificial insemination is not merely a novel method of bringing about
impregnation in females, instead, it is a powerful tool mostly employed for
livestock improvement.
• By adoption of artificial insemination, there would be considerable
reduction in both genital and non-genital diseases in the farm stock.
• All bull semen used for AI is cryopreserved allowing long storage times and
easy distribution, and inseminations are generally done by trained
inseminators.
• AI has been most widely used for breeding dairy cattle; 253 million
frozen AI doses and 11.7 million liquid doses are produced worldwide
every year
 STEPS INVOLVED IN ARTIFICIAL
INSEMINATION
• Bull fertility assessment
A careful selection of bulls ensures high quality specimens both from the
reproductive and physical point of view. This includes requirements
concerning physical fitness, condition of genital organs, semen and sexual
behavior.
• Semen Collection
Semen is usually collected from bulls by using an artificial vagina (AV),
electro-ejaculation and transrectal massage. Semen is collected into a
prewarmed insulated or jacketed tube through a funnel or Cone. All
surfaces coming into contact with semen should be clean, warm, dry and
free of Spermatoxic agents. The collection of semen is performed in a
specially prepared place called the "manege"
• Semen Evaluation
Semen is suspension of spermatozoa in seminal fluid. Semen evaluation
has great diagnostic value in determining the cause, severity, and degree
of testicular and accessory gland pathology or infertility as well as being of
value in estimating the fertility of the male.

• Macroscopic\physical test
Visual evaluation for volume, colour, consistency density, odour and
observation for presence of foreign material (blood, pus cells, dung, hair
etc.) is made and recorded. Semen evaluation is described as very good
(80-100% motile sperm cells), good (60- 80% motile sperm cells) fair (40-
60% motile sperm cells), poor (20- 40% motile sperm cells), very poor (10-
20% motile sperm cells). Semen should have a minimum of 30 percent
vigorous, motile sperm when diluted and viewed through the microscope.
Temperature, shock and other factors can greatly interfere with motility
scores.
• Semen processing
It is important to work as rapidly and effectively possible to extend the life
of the sperm. Diluters must be isotonic with semen and must protect the
sperm from cold shock injury. Milk and egg yolk, are basic ingredients of
most extending media.
• Preservation of Spermatozoa
The life span of spermatozoa of most species can be prolonged more
conveniently by cooling to a temperature well below ambient, freezing
(cryopreservation), and suspending the metabolic activity of sperm while
maintaining it at ambient temperature.
• Timing of insemination
When a heifer becomes sexually mature the ovaries begin to function in a
cycle of activity. This cycle involves a sequence of events in preparation for
mating, conception and pregnancy. The cycle repeats in preparation for a
new mating cycle if pregnancy does not occur. The cycle has an average
length of 21 days. Success in insemination timing is dependent upon a
good heat detection program. Proper oestrus detection is critical to the
success of AI.
• Preparation of insemination gun
Check the identity of the animal obtain the cows previous history
including inseminations, calving, fertility and disease. Once the semen
dose has left the container it should be thawed in a 30°C to 37°C water
bath for 10-60 seconds and dry the straw and maintain the temperature at
37°C. Put a straw in the insemination gun sealed end first. Cover the gun
with a plastic sock to prevent contamination of the instrument. Push the
plunger of the insemination gun slowly until the semen is visible at the
open end of straw.
• Insemination of semen
The technique of inseminating a cow is a skill requiring adequate
knowledge, experience and patience. Early method of AI involved
deposition of the semen in the vagina, Fertility is low and greater numbers
of sperm are required as would occur in natural mating. Another method
which gained popularity was the "speculum" method. This method is
easily learned, but proper cleaning and sterilizing of the equipment is
necessary, making it more impractical to inseminate than with the
rectovaginal.
• Recto vaginal method
The safe and best method of insemination is “Recto vaginal method of
insemination”. In the recto-vaginal technique a sterile, disposable catheter
containing the thawed semen is inserted into the vagina and then guided
into the cervix by means of a gloved hand in the rectum. The inseminating
catheter is passed through the spiral folds of the cow's cervix into the
uterus. Part of the semen is deposited just inside the uterus and the
remainder in the cervix as the catheter is withdrawn. Expulsion of the
semen should be accomplished slowly and deliberately to avoid excessive
sperm losses in the catheter. The body of the uterus is short; therefore,
care should be taken not to penetrate too deeply which might cause
physical injury. In animals previously inseminated, the catheter should not
be forced through the cervix since pregnancy is a possibility.
 SUPEROVULATION
• Superovulation is a reproductive technology used in the dairy industry to
increase the reproductive rate of superior females. It is also called
superstimulation. Superovulation is the primary requirement for
physiologically low ovulation rates (cattle, sheep, goats, and horses) in
animals for the successful application of embryo transfer.

• Superovulation is achieved by using follicle-stimulating gonadotropins, FSH

and LH hormones to promote the development of subordinate follicles.
Superovulation with gonadotropins is an essential assisted reproductive
technology that increases the number of oocytes to achieve high
pregnancy rates. Oral or injectable administration of gonadotropins to
females is widely used for treatment and increasing the number of
offspring from animals.
 METHODS OF SUPEROVULATION
• Superovulation technology have evolved significantly over the last 40–50
years. The production of commercial pituitary extracts and prostaglandins
(PG) in the 1970s, and partially purified pituitary extracts and progesterone
in the 1980s and 1990s resulted in development of most modern
techniques. Furthermore, improved knowledge about follicular wave
dynamics through use of real-time ultrasonography.
• Two main types of gonadotropin preparations have been used to induce
superovulation in the cow: gonadotropins from extracts of domestic animal
pituitaries (mainly porcine) and equine chorionic gonadotropin (eCG,
previously known as pregnant mare serum gonadotrophin, PMSG).
• Pituitary extracts contain follicle-stimulating hormone (FSH) with
variable concentrations of luteinizing hormone (LH). The biological half-
life of FSH in the cow has been estimated to be 5 hours or less, so it must
be injected twice daily over multiple days to successfully induce
superovulation. The usual treatment regimen is twice-daily intramuscular
injections of FSH for 4 or 5 days, with a total dose of 260–400 mg, and
PGF2α administration towards the end of FSH treatment to induce
luteolysis. Estrus occurs in 36–48 hours, with ovulations beginning 24–36
hours later.
• Equine chorionic gonadotropin is a complex glycoprotein with both FSH
and LH activity. It has been shown to have a half-life of 40 hours in the
cow and persists for up to 10 days in the bovine circulation; thus, it is
normally injected intramuscularly once followed by a PGF2α injection 48
hours later. Recommended doses of eCG range from 1500 to 3000 IU.
While a distinct advantage in terms of its practical use, the long half-life of
eCG is associated with continued ovarian stimulation, unovulated follicles,
abnormal endocrine profiles, and reduced embryo quality.

• Challenges in conventional Superovulation protocol

– The requirement to have trained personnel for detection of estrus, both
before and after initiating treatments.
– The necessity to have all donors in estrus at the same time in order to
begin the super stimulatory treatments at the most appropriate time in
groups of cows.
• Methods to facilitate superstimulation
– Administration of estradiol and progesterone, which has recently been
incorporated into protocols that permit fixed-time AI of donors. It involves
the administration of 2.5–5 mg estradiol-17β or 2–2.5 mg estradiol
benzoate plus 100 or 50 mg progesterone by intramuscular injection at the
time of insertion of an intravaginal progestin device. However, estradiol
cannot be used in many countries because of concerns about the effects of
estrogenic substances in the food chain.
– Follicle ablation using ultrasound-guidance to eliminate the suppressive
effect of the dominant follicle. Super stimulatory treatments are then
initiated 1–2 days later, at the time of emergence of a new follicular wave.
Although follicle ablation has been shown to be highly effective,
ultrasound equipment and trained personnel are required.
– Administration of GnRH can be used, which will also induce emergence of
a new follicular wave. However, only around 60% of animals will respond
and ovulate. This protocol consists of the administration of PGF2α at the
time of insertion of a progestin device. Seven days later (with the progestin
device still in place), GnRH is administered to induce ovulation of the
persistent follicle and synchronization of follicle wave emergence. FSH
treatments are initiated 36 hours after the administration of GnRH.
 INVITRO FERTILIZATION
• The process of retrieval of eggs and sperms from the male and
female animals and placing them together in a laboratory dish to
facilitate fertilization under controlled environment.
• Animal research in IVF improves the techniques for use in other
animals.
• IVF in valuable livestock allows production of more offsprings from
genetically superior animals.
• To produce offsprings from cows that are infertile due to old,
disease of the reproductive tract and cystic ovaries.
 STEPS INVOLVED IN IVF
• Oocyte (egg) collection
• In-vitro Maturation (IVM) of eggs
• In-vitro Fertilization (IVF) of eggs
• In-vitro culture (IVC) of embryos
• Embryo transfer
 Oocyte (egg) collection

• From Ovaries
• Surgically removed from a cow
• Removed from cows post mortem (after death)
• By transvaginal ultrasound guided aspiration
– Donors might be stimulated with FSH
– Allows repeated collection because the cow’s reproductive
tract remain intact
– Oocytes with cumulus cells just after aspiration
• Invitro Maturation of eggs
• The collected oocytes are placed in culture medium filled dishes and
allowed a period of 24h to reach maturity.
• The glass dishes are incubated at 38.5C, which is normal body
temperature of cow.

• Invitro fertilization
• After 24h incubation of oocytes in IVM medium.
• The oocytes are washed and moved into new dishes with IVF
culture medim.
• The sperm cells are added at a concentration of optimal for sperm
capacitation and fertilization
• Followed the dishes are incubated for 18h to allow fertilization to
occur.
• In-vitro culture of embryos
• After 18h incubation in IVF medium.
• The culture dishes are checked for the sperm hyperactivation.
• The zygotes are washed and transferred to IVC medium.
• The culture dishes are incubated for 7 days and monitored
periodically to ensure contamination free and embryo development
(cleavage)
• After seven days, the blastocyst stage embryo is ready to transfer for
any recipients.
 EMBRYO TRANSFER IN CATTLE
• Embryo Transfer involves the removal of an embryo from a female of
superior genetics and the placement of the embryo into the reproductive
tract of a female of average genetics.

• The goal of ET is to obtain the maximum number of genetically superior

embryos in a minimum amount of time.

• Embryo Transfer (ET) is a expensive procedure, costing around $300 for

each flush and approximately $270 for each calf born.

• ET is a complicated procedure with a fairly high difficulty level.

• ET should only be performed by trained professionals.

 BENEFIT OF EMBRYO TRANSFER
• Traditionally, cows produce only one calf per year.
• ET allows the production of many offspring within a year from a
single cow.
• ET can increase the genetic potential of a herd in a relatively short
period of time.
• ET can increase milk production in dairy herds.
• ET can increase weaning weights in beef and dairy herds.
• ET allows other producers to take advantage of superior genetics
because frozen embryos can be shipped almost anywhere.
• ET preserves superior genetics for future generations due to
embryo freezing.
 SELECTION OF DONOR AND
RECIPIENT COWS
• Donor cows : The donor cows will contribute the embryos to be
transferred. A donor cow should have superior trait including high
milking ability, high growth rate and outstanding reproductive
capacity.

• Recipient cows: Recipient cows serve as surrogate (foster) mothers

to the calves, but contribute no genetic information. However, the
recipient cow must be able to maintain her pregnancy to term and
produce an adequate milk supply for her calf.
SYNCHRONIZING THE ESTROUS CYCLE
• It is important to synchronize estrous cycles because the
reproductive environments of the donor and recipients must be
identical in order for the embryo to survive the transfer.

• The estrous cycle is controlled by the production and secretion of

hormones at the proper time during the cycle. • Prostaglandin
(PGF2α) is the hormone used to synchronize the estrous cycles of
the donor and recipient cows.

• Prostaglandin is produced naturally by the cow. However, a

synthetic version called Lutalyse is given in one or two injections
intra muscular in the neck or hip to synchronize estrous cycles.
 PREPARATION OF DONOR COW
• Before the donor cow is flushed, she is superovulated with a series
of injections of Follicle Stimulating Hormone (FSH).

• When donor shows sign of estrus (riding other cows, clear vaginal
mucus, and pacing the fence) she is ready to be bred.

• After breeding, the donor is not disturbed for 5-6 days, which
facilitates growth of multiple embryos in the donor.

• During this time the embryos also travels down the reproductive
tract from the oviduct (the site of fertilization) to the uterus where
they can be flushed out.
 FLUSHING EMBRYOS FROM DONOR
• On the seventh day after breeding, the embryos are ready to be
removed. This process is called flushing.
• Embryo professionals use a non-surgical method to remove the
embryos. The process requires experience and a patient, steady
hand.
• An injection of lidocaine is given prior to the flush to reduce
pressure and stress on the donor cow and to make the flush easier
• To begin the flush, a catheter is passed through the cervix into one
uterine horn.
• The catheter contains a balloon that is inflated with a saline solution
in order to seal the entrance to the uterus so fluid and embryos are
not lost.
• The uterine horn is filled with flush media and massaged to allow
the embryos to flow out of the tract. This process is repeated several
times in each uterine horn.
 COLLECTION OF EMBRYOS
• Embryos are carried out of the reproductive tract through plastic
tubes and collected in a filter with the flush media.
• The pores in the filter are smaller than the embryos so excess fluid
drains out of the filter without losing the embryos.
• After the embryos have been flushed out, uterus injected with
penicillin to kill any missed embryos or infections.
• An average of 7-10 embryos is collected from each flush. •
However, the number of embryos obtained from a single flush may
range anywhere from 0-60.
 SCREENING EMBRYOS QUALITY
• In the lab, embryos are separated from the
flush media and examined under a microscope
to determine their quality and stage of
development.
• Embryos are microscopic in size (about 0.2
mm). Only undamaged embryos at proper
maturity should be transferred.
 EMBRYO TRANSFER TO RECIPIENT
• The embryo to be transferred is put into a small, plastic straw and
then loaded into an embryo transfer gun.
• The embryo is then inserted into either the left or right uterine horn
depending on which ovary has a corpus lutuem (CL)
• The corpus lutuem is a structure on the ovary that secretes the
hormone progesterone which is needed to maintain the pregnancy.
• Embryos should be transferred as soon as possible after the flush
(within 8 hours at least).
• Embryos can also be frozen for later implantation and stored in
liquid nitrogen tanks.
 PREGNANCY DIAGNOSIS AFTER IMPLANTATION
In both beef and dairy cattle, pregnancy diagnosis is an important tool to measure the success
of a reproductive management, to allow for early detection of problems and to achieve
resynchronization of nonpregnant cows.

• Estrus Detection- Cows are commonly said to show estrus approximately every 21 days (20
days for a heifer). If a cow is pregnant after insemination, the corpus luteum will not regress,
progesterone concentrations will remain high, and the cow will not return to estrus.
• The use of estrus detection following insemination is useful to detect nonpregnant cows and
allow for re-synchronization of such a group of cows.

• Use of Milk or Plasma Progesterone - The corpus luteum regresses in cows that do not
become pregnant and the cow returns to estrus within approximately 20-23 days after
insemination.

• Therefore, it is possible to diagnose cows for pregnancy based on milk or plasma

progesterone concentrations at about 21 days after insemination.

• Usually, samples are collected at 21 and 24 days after insemination. If either one of those
samples is considered to have low progesterone concentrations, cows are diagnosed as not
pregnant. Thus, the use of progesterone tests is a good diagnosis tool to detect non-pregnant
cows. If both samples have high progesterone concentrations, however, there is still a certain
probability that the cow is not actually pregnant.
• Ultrasonography -In the 1980s, real time utrasonography was developed for use in
domestic animals. An ultrasound machine resembles a radar device. A probe is inserted
through the rectum and positioned above the uterus. This probe generates pulses of
ultrasound that are transmitted to adjacent tissues. These pulses are then reflected back to
the probe from different tissue surfaces.
• Structures that contain fluid (such as the fluid-filled placenta) absorb most of the ultrasound
pulses and the result is a black image on the video screen. On the other hand, more dense
structures (such as an embryo) are more ecogenic (i.e., have greater reflectivity) and result
in a light gray or white image on the screen.
-The main advantages of the use of ultrasound for pregnancy diagnosis are 1) the
high reliability of the results that are generated and 2) the fact that pregnancy diagnosis may
be conducted relatively early after insemination (i.e., as early as 25 days after
insemination).

• Rectal Palpation -Palpation of the uterine contents rectally is probably the most commonly
used method for pregnancy diagnosis.
• Pregnancy diagnosis after insemination can be conducted as early as 30 days in heifers and
35 days in cows, although much practice is necessary to be able to determine pregnancy at
that stage.
-Several palpable structures are indicative of pregnancy. Due to accumulation of fluids
within the pregnant uterine horn, one of the initial signs of pregnancy is a difference in size
of uterine horns (i.e., uterine asymmetry). Also, it is possible to feel the slipping of the
chorioallantoic membrane (fetal membrane) along the greater curvature within the uterus
(i.e., membrane slip).
-As pregnancy progresses, it becomes possible to feel the presence of the fetus within the
pregnant horn. After about day 150, the fetus is too far forward in the body cavity to palpate
the entire fetus although fetal structures can be palpated.
 EMBRYO SEXING
• Before the implantation of embryo its sex is detected from the biopsy
sample.
• The presence of Y chromosome makes the offsprings male and that of X
makes the female
• PCR technique is used in sex detection. PCR amplifies DNA sequence of
Y chromosomes and reaction products can be seen directly.
• Handyside et al.(1989) isolated single blastomere from early embryo from
a womb, amplified DNA sequences of Y chromosomes and carried out
embryo sexing before implantation into uterus.
• Now the sexed embryos are commercially but these are very costly.
 EMBRYO SPLITTING

• The embryos of blastocyst stage is split into equal halves

• The demi-embryo are transferred into the oviduct of synchronised recipient

for normal embryo transfer to produce identical twins. At this stage it is
necessary to know the situation for successful embryo implantation that the
wall of synchronized recipient is waiting for embryo. Thus , embryo
splitting technology has increased the rate of pregnancy.

• Embryo biopsy –is the removal of small number of cells for genetic
analysis should be combined with splitting so that the twins which will be
produced should be identical and of known genotype.

• It is necessary in breeding so that sex and genetic diseases could be

detected. In case the embryo has any genetic disease, it can be prevented
from implantation in recipient females.
 EMBRYO
CRYOPRESERVATION
Cryopreservation enables to store the embryos and sperms by arresting or slowing metabolic
activities until the subsequent thawing procedure.
Embryo cryopreservation is a means of long-term storage of valuable strains. This technique
can be used to safeguard strains of animals from genetic drift, prevent loss due to disease or
catastrophe and reduce animal housing costs.
HISTORY
• In 1891, Walter Heape (1855-1929), a professor and physician at the University of
Cambridge, England, reported the first known case of embryo transplantation : Working with
two species of rabbits, he flushed embryos from the oviducts (rabbit fallopian tubes) of one
breed (Angora) and placed them into the uterus of a recently mated Belgian hare. In the
resulting litter, there were 4 Belgians and 2 Angoras. He proved that it was possible to take
preimplantation embryos and transfer them to a gestational carrier without affecting their
development.
• Gregory Pincus and colleagues were the first to show how eggs of various animals would
undergo maturation if released from their follicle and cultured in a laboratory. In 1939, he
reported that human eggs would mature in the laboratory within 12 hours.
• Cryopreservation era arised with the accidentally understanding of the value of
cryoprotectants in 1949 by Christopher Polge.
• By the early 1970s, Wilmut and Whittingham developed independent methods for freezing
mouse embryos in DMSO.
• In 1985, new approach, termed vitrification, in which highly concentrated cryoprotective
agents were used, was successfully applied on mouse embryos.
 Cryopreservation Of Embryo

• Embryo cryopreservation is a means of long-term storage of valuable

strains.
• This technique can be used to safeguard strains of mice or rats from
genetic drift, prevent loss due to disease or catastrophe and reduce animal
housing costs.
• Embryos are preserved in a cryoprotectant then slowly frozen. Embryos
are then transferred to liquid nitrogen tanks for long-term storage.
• Cryopreserved embryos can be recovered at any time the investigator
desires. The Transgenic Animal Facility can cryopreserve embryos from
either mice or rats, provide for their long-term storage in liquid nitrogen
and recover strains on demand.

Embryo cryopreservation has the following steps:

• Assessment of animal colony to assure multiple young or proven males are
available for matings and young females are available for superovulation.
• Superovulation of females to obtain fertilized embryos.
• Cryopreservation of embryos using a controlled-rate freezer.
• Storage of embryos in liquid nitrogen tanks.
• Recovery of live animals from a strain by thawing and surgically
transferring the frozen embryos into pseudopregnant recipients.
•Embryos were assessed based on their morphology. Good quality embryos are
preserved in glycerol by slow-cooling methods. Embryos are stored in plastic straws
within liquid nitrogen tanks.

• It is recommend that freezing 100-125 embryos if homozygous or heterozygous

embryos are preserved.

• The embryo is tested for viability of each line by thawing one straw of embryos and
transferring them into a pseudopregnant recipient. Embryo transfers will be done if
necessary.

•The main techniques used for embryo cryopreservation are vitrification (fast
frrezing) and slow programmable freezing (SPF).
 Cryopreservation of the pre-implantation
embryo

• Controlled rate freezing or slow

programmable freezing

• Vitrification
SLOW FREEZING

⮚ Expensive equipment
⮚ Higher incidence of intracellular ice
formation
⮚ Low concentration of cryoprotectant, less
toxic
⮚ Not cost-effective ??
 VITRIFICATION

Vitrification is the solidification of a solution at low temperature

without ice crystal formation

Liquid Solid phase

Phase

Amorphous
state/ Vitrified
state /Glassy
state
No ice crystal

Possible osmotic stress

Absence of mechanical
injuriy

CRYOPROTECTANT TOXICITY??
Cryopreservation Of Mouse Embryo
 Cloning for conservation of endangered species
•Reproductive cloning, or the production of offspring by nuclear transfer, is
often regarded as having potential for conserving endangered species of
wildlife.
•Cloning can reverse the extinction of animals.
•Cloning animals has been possible for many years by splitting the embryo.
•Lately cloning has become possible using adult cells, by copying the DNA
and inserting it into an egg.
•Using this technology, we can create exact replicas of living animals.
•This may allow us to clone endangered or animals.
•Gaurs are endangered animals that live in the woodland of Asia.

This is Noah. He is a gaur that was cloned in

2001.
•Approximately 100 species become extinct a day. Despite increasing interest in using cloning to
rescue endangered species, successful interspecies nuclear transfer has not been previously
described, and only a few reports of in vitro embryo formation exist.

•Somatic cell cloning methods used to restore endangered, or even extinct species and
populations.

•Interspecies nuclear transfer can be used to clone an endangered species with normal karyotypic
and phenotypic development through implantation and the late stages of fetal growth.

•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 transferred to surrogate mothers. Three of the fetuses
were selectively 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.
 Figure 1 This example highlights the importance of considering the fate of mitochondria in
relation to trans-species cloning.

William V Holt et al. Reproduction 2004;127:317-324

© 2004 Society for Reproduction and Fertility

 Figure 2 This example shows how the problem of mitochondrial inheritance, shown in Fig. 1,
could be avoided, while still using oocytes derived from a common species such as a cow.

William V Holt et al. Reproduction 2004;127:317-324

© 2004 Society for Reproduction and Fertility