UNIT - II

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 Phase Solid 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

 GENE TRANSFER TECHNIQUES
• Gene transfer is a technique to stably and efficiently introduce
functional genes (that are usually cloned) into the target cells.
Genetic transfer is the mechanism by which DNA is transferred
from a donor to a recipient. Once donor DNA is inside the recipient,
crossing over can occur. The result is a recombinant cell that has a
genome different from either the donor or the recipient. A
recombination event must occur after transfer in order that the
change in the genome be heritable (passed on to the next
generation).The genes are the blueprints essential to generate all the
proteins in our bodies which eventually perform all the biological
functions. Therefore, when a gene is efficiently introduced into a
target cell of the host, the protein which is encoded by that gene is
produced .
 CALCIUM PHOSPHATE MEDIATED DNA
TRANSFER
• Calcium chloride and potassium phosphate. When the two are combined, a
fine precipitate of the positively charged calcium and the negatively charged
phosphate will form, binding the DNA to be transfected on its surface. Cells
are then incubated with precipitated DNA either in solution or in tissue
culture dish. A fraction of cells will take up the calcium phosphate DNA
precipitate by a process not entirely understood but thought to be endocytosis.
• Limitations
Frequency is very low.
Integrated genes undergo substantial modification.
Many cells do not like having the solid precipitate adhering to them and the
surface of their culture vessel.
Integration with host cell chromosome is random.
Due to above limitations transfection applied to somatic gene therapy is
limited.
 LIPOSOME MEDIATED GENE TRANSFER

• Liposomes are spheres of lipids which can be used to transport

molecules into the cells. These are artificial vesicles that can act as
delivery agents for exogenous materials including transgenes. They
are considered as spheres of lipid bilayers surrounding the molecule
to be transported and promote transport after fusing with the cell
membrane.
• Liposomes for use as gene transfer vehicles are prepared by adding
an appropriate mix of bilayer constituents to an aqueous solution of
DNA molecules. In this aqueous environment, phospholipid
hydrophilic heads associate with water while hydrophobic tails self-
associate to exclude water from within the lipid bilayer. This self-
organizing process creates discrete spheres of continuous lipid
bilayer membrane enveloping a small quantity of DNA solution.
• Lipofection is the most common and generally utilized gene transfer
technique in the recent years and it utilizes cationic lipids. The
combined DNA and cationic lipids act instantaneously to form
structures called as lipoplexes that are more complex in structure
than the simple liposomes. Cationic lipids have a positive charge
and these form cationic liposomes which interact with the negatively
charged cell membrane more readily th
• This leads to a fusion between cationic liposome and the cell surface
resulting in quick entry by endocytosis and the delivery of the DNA
directly across the plasma membrane. Cationic liposomes can be
produced from a number of cationic lipids that are commercially
available and sold as an in vitro-transfecting agent, termed
lipofectin.an uncharged liposomes.
• Advantages: Simlicity, long term stability, lo toxicity, protection of
DNA from degradation and particularly proficient at delivering
genes to dividing cells, such as cancer cells or immortalized cell
lines.
• Disadvantages: Cannot deliver genes to terminally differentiated
cells
 ELECTROPORATION
• A rapid and simple technique for introducing genes into a wide variety of
animal cells. Electroporation uses electrical pulse to produce transient
pores in the plasma membrane thereby allowing macromolecules into the
cells. These pores are known as electropores which allow the molecules,
ions and water to pass from one side of the membrane to another. The
electropores reseal spontaneously and the cell can recover. The pores can
be recovered only if a suitable electric pulse is applied.
• The formation of electropores depends upon the cells that are used and
the amplitude and duration of the electric pulse that is applied to them.
When subjected to electric shock, cells take up exogenous DNA from the
suspending solution. Once inside the cell, the DNA is integrated and the
foreign gene will express. A proportion of these cells become stably
transformed and can be selected if a suitable marker gene is carried on
the transforming DNA.
• The most critical parameters are the intensity and duration of the
electric pulse, and these must be determined empirically for different
cell types. Electric currents can lead to dramatic heating of the cells
that can results in cell death. Heating effects are minimized by using
relatively high amplitude, a short duration pulse or by using two very
short duration pulses. However, once optimal electroporation
parameters have been established, the method is simple to carry out
and highly reproducible.
• Many different factors affect the efficiency of electroporation,
including temperature, various electric-field parameters (voltage,
resistance and capacitance), topological form of the DNA, and various
host-cell factors (genetic background, growth conditions) and post-
pulse treatment.
Advantages:
• Introduction of exogeneous DNA into animal cell lines
• Genes encoding selectable marker may be used to introduce genes
using electroporation.
• To study the transient expression of molecular constructs.
• Electroporation of early embryo may result in the production of
transgenic animals.
• Hepatocytes, epidermal cells, haematopoietic stem cells, fibroblast,
mouse T and B lymphocytes can be transformed by this technique.
• Naked DNA may be used for gene therapy by applying electroporation
device on animal cells.
Disadvantages:
• Limited effective range of ~1 cm between the electrodes.
• Surgical procedure is required to place the electrodes deep into the
internal organs.
• High voltage applied to tissues can result in irreversible tissue damage
as a result of thermal heating.
• Larger numbers of cells may be required than for other methods
because, in many cases, the most efficient electroporation occurs when
there is up to 50% cell death.
 MICROINJECTION

• In microinjection DNA can be introduced into cells or protoplast

with the help of very fine needles or glass micropipettes having the
diameter of 0.5 to 10 micrometer. The DNA may be directly
delivered into the nucleus or in the cytoplasm. Some of the DNA
injected may be taken up by the nucleus. The desired gene in the
form of plasmid or alone is injected directly into the animal cells.
• Injection into the nucleus is more efficient than that into the
cytoplasm. Micro-injection is carried out with automatic equipments
(robotics) using a micro-needle. Computerized control of holding
pipette, needle, microscope stage and video technology has
improved the efficiency of this technique.
• Microinjection is more commonly used in animal cells. It is ideally
useful for producing transgenic animals.
Advantages:
• Frequency of stable integration of DNA is far better as compare to
other methods.
• Method is effective in transforming primary cells as well as cells in
established cultures.
• The DNA injected in this process is subjected
• to less extensive modifications. Mere precise integration of
recombinant gene in limited copy number can be obtained.
Limitations:
• Costly and skilled personal required.
• Embryonic cells preferred for manipulation.
• Process causes random integration.
• Rearrangement or deletion of host DNA adjacent to site of integration
are common.
• The technique is laborious, technically difficult, and limited to the
number of cells actually injected.
 VIRAL VECTORS FOR GENE TRANSFER

• The use of viruses as vectors for transduction, i.e. the introduction of

genes into animal cells by exploiting the natural ability of the virus
particle, within which the transgene is packaged, to adsorb to the
surface of the cell and gain entry is being employed . Due to the
efficiency with which viruses can deliver their nucleic acid into cells
with high levels of replication and gene expression.
• viruses have been used as vectors not only for gene expression in
cultured cells but also for gene transfer to living animals. Four
classes of viral vector have been developed for use in human gene
therapy and have reached phase 1 clinical trials. These are the
retrovirus, adenovirus, herpesvirus and adenoassociated virus
(AAV) vectors.
• Transgenes may be incorporated into viral vectors either by addition
to the whole genome or by replacing one or more viral genes. This is
generally achieved either by ligation (many viruses have been
modified to incorporate unique restriction sites) or homologous
recombination.
• For many applications, it is favourable to use vectors from which all
viral coding sequences have been deleted. These amplicons (also
described as ‘gutless vectors’) contain just the cis-acting elements
required for packaging and genome replication. The advantage of
such vectors is their high capacity for foreign DNA and the fact that,
since no viral gene products are made, the vector has no intrinsic
cytotoxic effects.
• The choice of vector depends on the particular properties of the
virus and the intended host, whether transient or stable expression is
required and how much DNA needs to be packaged.
Advantages:
• Viral vectors that especially efficient at transducing the intended cell
type but not other cell types.
• Retroviruses are particularly proficient at delivering genes to
dividing cells, such as cancer cells or immortalized cell lines.
Limitations:
• Cannot deliver genes to terminally differentiated cells.
• Viral vectors may be highly immunogenic
 IMPORTANCE & METHODS OF PRODUCING
TRANSGENIC ANIMALS

• During the past decades various methods have been developed to

generate the transgenic animals. With the advent of gene sequencing
many sequences have been determined bringing the knowledge of
promoters and gene of interest for various species. The advent of
genomics, proteomics and the new generation of reproductive
biotechnologies hold the promise of successful application as
transgenesis to domestic animals.
• The techniques and methodologies to be implemented in the
generation of the transgenic animal depend upon the targeted use of
the animals
TRANSGENIC ANIMALS HAVE BEEN PRODUCED
BY A NUMBER OF TECHNIQUE
• ˆPronuclear microinjection ˆ
• Nuclear transfer ˆ
• Retroviral mediated gene transfer ˆ
• Lentiviral mediated gene transfer ˆ
• Sperm mediated gene transfer ˆ
• Transposon mediated gene transfer ˆ
• Stem cell mediated gene transfer
 APPLICATION OF TRANSGENIC ANIMALS
• Livestock production
Enhanced prolificacy and reproductive performance increased feed
utilization and growth rate, improved carcass consumption,
improved milk production or composition and increased disease
resistance are practical application of transgenesis.
• Modification of milk
Advances in recombinant DNA technology have provided the
opportunity to change the composition of milk, increase in milk
volume and to produce entirely novel proteins in milk. These
changes may add value, as well as increase the partial uses of milk.
Clearly altering the charactertics of one of the components to
enhance a particular processing feature
• Modification of growth and carcass composition
Introduction of porcine growth hormone (PGH) genes into swine
genome increases the growth rate, without increased arthritis and
abnormal skeletal growth. An alternative approach was performed
hypertrophy of numerous muscles while reducing body fat by
introduction of the chicken ski mutant oncogene, which was
previously shown to cause. This strategy, however, has resulted in
limited success although muscle hypertrophy has been observed in
some transgenic pig and transgenic cattle .The myostatin (growth
differentiation factor-8, GDF-8) gene normally present in mouse
skeletal muscle. Myostatin serves as autocrine prenatal inhibitor of
myoblast differentiation and growth, when it knocks out result in an
increase in lean muscle mass. Such genes also present in livestock
hence same approaches can be used made in livestock to improve
their lean muscle production
Modification of disease resistance
• Identification of single gene in the major histocompatibility complex
(MCH), which influences the immune responses, was instrumental
in the reorganization up genetic basis of disease resistance or
susceptibility. It has only been realized recently that there are many
aspects of disease resistance or susceptibility in livestock that are
genetically determined. manipulation of the MCH in farm animal
through ES cell or NT transgenesis could have a major beneficial
effect on disease resistance for livestock producers.
• Mice and mouse fibroblast cell line that contain the Mx protein were
shown to be resistant to infection with influenza virus. The Mx
cDNA has been introduced into porcine fertilized ova, producing
pigs that are resistant to influenza infection.
• The knocking out technique is performed in sheep and cattle to
improve the disease resistance to scrapie and bovine spongiform
encephalitis.
Modification of wool production
Alteration of the protein composition of wool fiber via transgenesis
with sheep wool keratin and keratin associated protein (KAP) gene
may lead to the production of fiber types with improved processing
and wearing qualities. These authors obtained wool fibers with
higher luster and reduced crimp as a result to alteration in their
micro and macro structure due to a high level of cortical specific
expression of a wool type II intermediate filament (f) keratin gene.
Modification of digestion
Phytase is an enzyme that cleaves inorganic phosphorous form to
organic form which increases the phosphorous availability to the
animals. This enzyme normally presents in ruminates but absent in
monogastric animal. By additional supply of this enzyme along with
feed make better utilization of feed. produced the transgenic pig
which expresses the phytase enzyme in their saliva, which enhance
the digestion and reduce the feed cost in pig production.
 TRANSGENIC MICE
• The mice that have been artificially modified at a genetic level to
include a foreign sequence, or transgene. This often involves the
insertion of a human gene into the mouse’s genome to create a humanized
mouse. Methods for creating transgenic mice are versatile, making it
possible to create transgenic mice for many different kinds of research. In
translational cancer research, transgenic mice are a powerful tool in
assessing the potential validity of targeted therapy because the targets can
be precisely inactivated in the setting of a developing or developed tumor.
• In order to understand transgenic mice, it is first necessary to learn about
transgenes. Transgenes are genes that have been taken from one
organism and transferred to the genetic makeup of another. For
example, a human gene can be copied and transferred to the genetic
makeup of a mouse in order to study human disease in a model biological
system.
• The most common methods of introducing a transgene into an
animal model involve genetic manipulation, such as by pronuclear
injection of embryos or by homologous recombination in cells. As a
result, the use of genetically enhanced transgenic mice has become a
crucial part of the science behind finding new treatments for human
diseases.
• Two basic approaches exist for creating transgenic mice. The first
involves delivery of the transgene into a single cell of the mouse
embryo via pronuclear injection. Through this method, researchers
can overexpress new genes, effectively creating a transgenic mouse.
The second method is through the modification of embryonic stem
cells via homologous recombination, and the injection of the
targeted ES cells into mouse blastocysts.
• In recent days to obtain more accuracy result transgenic mice are
produced through Knockin Permissive Locus Model and
CRISPR/Cas9 System.
 TRANSGENIC FISH
• Attempts to produce transgenic fish started in 1985 and some
encouraging results have been obtained.
• The genes that have been introduced by microinjection in fish
includes.
– Human or rat gene for growth hormone.
– Chicken gene for delta crystalline protein.
– E.Coli gene for beta galactosidase
– E.Coli gene for neomycin resistance
– Winter flounder gene for antifreeze protein
– Rainbow trout gene for growth hormone
• The technique of microinjection has been successfully used to
generate transgenic fish in many species such as common carp,
catfish, goldfish and salmon in other animals usually direct
microinjection of cloned DNA into male pronuclei of fertilized eggs
has proved very successful but in most fish species studied so far,
pronuclei can not be easily visualized so that the DNA needs to be
collected and placed into a separate dry container.
• Fertilization is intiated by adding water and sperm to eggs, with
gentle stirring to facilitate the fertilization process.
• Egg shells are hardened in water. About 106 to 108 molecules of
linearized DNA in a volume of 20 ml or less are microinjected into
each egg within the first few hours after fertilization.
• Following microinjection, eggs are incubated in appropriate
hatching trays and dead embryos are removed daily.
• Since in fish, fertilization is external, in vitro culturing of embryos
and their subsequent transfer into foster mothers is not required.
Further, the injection into the cytoplasm is not as harmful as that
into the nucleus, so that the survival rate in fish is much higher. ·
• Human growth hormone gene transferred to transgenic fish allowed
growth that was twice the size of their corresponding non transgenic
fish.
• Similarly antifreeze protein gene was transferred in several cases
and its expression was studied in transgenic salmon.
• It was shown that the level of AFP gene expression is still too low to
provide protection against freeze. There is also a report of the
production of transgenic zebra fish from an Indian laboratory.
• In this attempt a plasmid containing rat growth hormone gene was
microinjected into fertilized zebra fish eggs and its presence
confirmed in adult fish.
 ANIMAL AS BIOREACTOR
• The production of therapeutic proteins represents the first
application of recombinant DNA technology, by the 2003; the
European Union had approved 88 products. However none of this
approved product was obtained in transgenic system. Despite this,
domestic animals represent on efficient production system for large
and complex (and biologically active) recombinant protein, which
would be used to treat or prevent human disease.
• The production of these pharmaceutical proteins in the mammary
gland of livestock originated the term biopharming or gene
pharming. Various investigators have produced transgenic rabbit,
sheep, goat, pig and cattle express heterologous protein successfully.
• The production of biopharmaceuticals present the most varied
purposes: for treating such disease as multiple sclerosis, hepatitis,
cystic fibrosis, blood disorders, some type of cancers, haemophilia,
thrombosis, growth disorders, pompe’s disease, osteoporosis,
Paget’s disease and anemia and for improving infant’s formula.
• Initially the use of transgenic animals as bioreactors focused on the
use of mammary glands as target but today blood, bladder, eggs and
male accessory glands have all have been considered as bioreactors
for pharmaceutical proteins.
 Importance of Transgenic Mammary
gland
• Advantages of pharmaceutical proteins expressed in milk include
• The mammary gland is a prodigious production system that is cable of
generating between 23 gm (dairy cattle) and 205 gm (rat) of protein / kg
of body weight during peak lactation.
• Milk is clearly the least complicated body fluid to collect especially from
ruminants.
• Another advantage of producing biologically active products using the
mammary gland is the isolation of the mammary gland from circulatory
system. It is argued that bioreactor animals would be protected from the
potentially untoward effect of biologically active compound because those
compounds would be sequestered in the mammary gland and there fore
would be unavoidable to the circulatory system.
• Proteins which are expressed through mammary gland are able to post
translation modification and also biologically active.
 Expression of therapeutic proteins in
Animals
• Human Lysozyme
Milk lysozyme has three times more lytic activity than that of egg white
lysozyme because it possesses a greater positive charge than latter.
Lysozyme, which helps to increase the level of beneficial microorganism
in the infants and strength of their disease resistance. Successfully
expressed the human lysozyme in the milk of transgenic mice and also it
is biologically active. It can be express in cows milk that with helpful to
create disease resistance in many orphan child.
• Human Growth Hormone
The transgenic rabbit for production of human growth hormone using
whey acid protein gene promotor. The growth hormone enhances the
growth retarded children. Recombinant pig growth hormone causes a
massive in muscle mass. Hence it also used for increase the body weight
in pig.
• Lactoferrin
Lactoferrin present in human milk is an iron binding glycoprotein, which has
bacteriostatic and bactericidal effect on the gram positive and gram negative
bacteria. This helps to prevent bacterial infections that cause digestive
problem that harm or kill millions of newborns around the world. Now
transgenic bull was developed for human lactoferrin gene and it was named
as Herman. Herman has now fathered for many calves. Herman is produced
by gene pharming Europe, B.V. Company (Netherland).
• α 1- protease inhibitor
Air contains many living organism where it enters the lungs. The lungs
contain large numbers of neutrophils. These neutrophils secrete elastase and
clear the organism. The walls of the alveoli in the lungs contain elastin,
which maintains the elasticity of the lungs and this can be also be broken
down by elastase and cause emphysema of the lung. To prevent this from
happening there is secreted into the blood serum an enzyme called α 1-
protease inhibitor (previously called α1- antitrypsin) or α PI. Now this 1-
antitrypsin successfully expressed in transgenic sheep and it named as Tracy
grazes by PPL therapeutic company (UK). That can be purified and used for
treatment of emphysema of lung.
• Biosteel
Biosteel is an extra ordinary new product that may soon be used in bullet
proof vests and in suture silk for closing up of wounds. Fundamentally
biosteel is spider web, which is among the strongest fiber on the earth.
The gene of spider silk has been successfully transferred into goat by
nexia biotechnologies (Canada) and those goat’s milk contains the protein
that make up spider silk.
• Human blood clotting factor IX
Polly is the transgenic sheep that express the human blood clotting factor
IX in their milk. It is produced by nuclear transfer technique.
• Human antithrombin – III
Antithrombin-III is normally presents in human blood and it prevents
clotting in the veins. Some people with an inherited deficiency (lack) of
this protein are more prone to blood clots, which are dangerous when they
break free lodge in lung or brain. To prevent this that patient need to take
repeated injection at AT- III. Now Genzyme Corporation company and
also produced AT – III producing goat farm successfully express it in goat
milk.