Embryo Transfer

BHATTA BR
2082
 Embryo transfer technology
• “Embryo transfer is a bio-technique where
embryos are collected from the donor
females and transferred in to the uterus
of recipients which serves as a foster
mother for its development
throughout the remainder period of
pregnancy”
Learning Objectives;

After completion of the lesson, each student

should be able to;

Define embryo transfer

 Explain the steps of embryo transfer
List the advantages of embryo transfer
List the disadvantages of embryo
transfer
 History of Embryo Transfer
The first successful embryo transfer was carried out
in rabbit (1890) by Heap.
First lamb by ETT- 1949 by Berry.
First calf by ETT- 1951 by Willet et al.
In swine – 1951 by Kvansnickii.
In Asian buffalo – 1983 by Drost et al
 Role Of ETT In Livestock Development And Breed
Improvement.

 Through ETT, one high quality cow could be made to

produce up to 32 embryos per year compared to the
conventional method of breeding where the farmer
has to wait for twelve months for a calf that could be
either male or female.

 The reproductive potential of a female newborn

calf is enormous and is estimated at 150,000
ova per cow. This reproductive potential has
largely been underutilized.
 Naturally, a cow produces about 8 to 10 calves in her
lifetime. But with embryo transfer, it is possible to
get 32 embryos per cow per year.
 Embryo transfer is a technique that can greatly
increase the number of offspring that a genetically
superior cow can produce.
 Under conventional ways, the generation interval
ranges between 6 and 7 years, but with MOET, it can
be reduced by almost half. Also useful in progeny
testing programs, due to reduction in generation
interval.
 Very Effective for the propagation of superior
genes, although factors such as lactation
status of recipient animals, time of embryo
recovery after insemination, site of embryo
placement in recipient’s uterus, embryo quality
and stage of development all influence overall
conception rate.
 Steps Involved In Embryo Transfer

1. Selection of donor
2. Selection of recipient
3. Estrus synchronization of donor and recipient
4. Superovulation of Donor with high quality
semen.(release of multiple eggs at a single estrus).
5. Artificial insemination of donor
6. Embryo collection
7. Evaluation of embryo
8. Transfer of embryo / cryopreservation of embryo /
Micromanipulation
 Applications of embryo transfer

 Faster genetic improvement.

 Genetic screening.
 Disease control.
 Import and export.
 Circumvention of infertility.
 Twinning in cattle.
 Conservation of endangered species.
 Research; production of clones and
genetic engineering.
 1. Selection Criteria Of Donor
 Superior individual performance
 Good productive performance of offspring
 Regular cyclicity
 Ovaries must be free (no adhesions)
 Intact tubular genitalia (free from any sort
of abnormalities)
 Younger (4-8 yrs. of age)
 Healthy and have good body weight
 Must have calved at least 60 days back (best 90-
100 days postpartum)
 Normal postpartum history.
 A history of no more than two breeding per
conception.
 Previous calves having been born at
approximately 365-day intervals.
 an appropriate body condition score at the time
of embryo transfer.
 2. Selection Criteria Of Recipient
 Healthy, free from infection and have good body
weight.
 Regular cyclicity.
 Intact genitalia (free from any sort of
abnormalities)
 Must have good cyclic CL of desired stage at the time
of embryo transfer.
 Exhibit calving ease, and that have good milking
and good mothering ability.
 3. Estrus Synchronization of Donor

 The donor cow should be synchronized to bring

into estrus or should have palpable corpus
Luteum in the Ovary to start the Super stimulation
procedure.
 For this, any of the synchronization
protocol can be used ( Lecture on Estrus
Synchronization)
 4. Superovulation of Donor Cow

Is the procedure for increased ovulatory

response by administration of hormones
(gonadotropins) to produce several ova instead of
one which is normally produced at each estrus.

 This large number of ova is later

on fertilized and embryo
produced can be transferred to
the other females.
The basic principle of superovulation is to
stimulate extensive follicular development
through the use of a hormone preparation,
which is given intramuscularly or
subcutaneously, with follicle stimulating
hormone (FSH) activity.

In the ewe, doe and cow, an average of 12

ovulations can be
expected. In sows, the number of ovulation could
be > 20.
 Time of Superovulation

 For optimum response

gonadotropin treatment is
initiated during mid-luteal
phase i.e. on days 9-14 (if we
consider day 0 as estrus) of a
normal estrous cycle.

 Donor cows can be

superovulated repeatedly at
approximately 6-8 weeks
intervals .
 Superovulation Protocol For Cattle And Buffalo

[Link] FSH
Days 10-13th of estrous cycle FSH is administered either as a
equal dose of 5 mg in morning and 5 mg evening (total
dose 40 mg) or as a reducing dose.
[Link]
H

[Link]
eCG
 Superovulation Protocol For Sheep
[Link]
eCG

Using eCG with

Progesterone
 Superovulation Protocol For Goat
1. Using eCG and
Progesterone

[Link] FSH and

Progesterone
 Superovulation Protocol For Swine
[Link] Progesterone and
eCG

Superovulation Protocol In Mare

 Crude equine pituitary gonadotropins are
used in combination with hCG. In this
species eCG is not recommended because it
has low binding capacity to FSH and LH
receptors and therefore not sufficient for super
ovulatory treatment.
 5. Insemination Of Donor (A.I)
 Donor should be inseminated artificially 2-3
times at 12 hours, 24 hours and 36 hours
interval, beginning at 8-10 after the onset of
estrus. This is required because ovulation can
occur over an extended time period.
Fresh semen is preferred.
If frozen semen- then use double insemination dose
at
each insemination.

6. Embryo Recovery
Embryo can be collected by following methods;
1. Surgical method
2. Non-surgical method
3. Laparoscopy
 Surgical method is most often used in sheep, goat and
swine through mid-ventral incision under general
anesthesia. The method can be performed on day 3-4 after
estrus in sheep and goat (8 -cell embryo or less) and on 2-3
days after estrus in swine (4-cell stage).

Normal embryos will have between 2 and 64

cells.
 [Link] collection in cattle & buffalo
 The CL is located by rectal palpation and
the flank ipsilateral to the CL is clipped,
washed with soap and water, and sterilized
with iodine and alcohol.
 About 60 ml of 2 percent procaine is given along
the line of the planned incision. (flank incision is
far more practical than mid-ventral under GA).
 laparotomy incision at flank, reproductive tract is
exposed
A clamp or thumb and forefinger can be used to
block the
distal one-third of the uterine horn.
PBS, 20 ml injected in to that segment can
be forced through the oviduct with gentle milking
Procedure can be carried out prior to 5th day of
estrous
cycle.

2. Non-surgical collection (Trans cervical

method)

 Commonly used in cattle, buffalo and mare.

 involves two ways or three ways Foley’s catheter
which allows flushing fluids to pass into the
uterus and at the same time allows fluids
to be returned from the uterus to a collecting
receptacle.
 A small balloon near the end of catheter can be
inflated just inside the uterine horn to prevent
the flushing fluid from escaping through the
Foley’s Catheter for Embryo Recovery
from Donor
Collection of bovine embryo should be made at
6-8 days post-breeding at compact morula or
blastocyst stage.
6-7 days post-ovulation at blastocyst stage in
mare.
 The best flushing medium for embryo collection for
most of the species is modified
Dulbecco’s phosphate buffer saline. NS can
be used in its absence.
 During final collection, oxytocin is administered @
50 i.u.
I/V.
 Large doses of antibiotics to prevent infection.

 Injection of PGF2α is also recommended to speed

recoveries of ovaries and to prevent pregnancy, if
viable embryos are not dislodged by the flush.
 [Link] embryo collection

Surgical collection is choice of method in sheep and

goat due
to inability to palpate RTs.
This has lead to the use of surgical
techniques predominately leading to
adhesion formation.
Laparoscopy is considered to results in fewer
adhesions than traditional surgery.
 7. Evaluation of embryo
After collection and before transfer to the
recipients, the embryos are evaluated under
stereomicroscope at 50-100 x magnification.
Day 7 bovine embryos (compact morula or
blastocyst) are about 150-190µm in diameter and
are still within the zona- pellucida

Embryos are graded based on following

Characteristics.
 Compactness of the cells
 Regularity of shape
 Variation in cell size
 Color and texture of cytoplasm
 Presence of vesicles, extruded cells, cellular
debris
 Using these criteria, the
embryosare graded
Grades Types as: Characteristics
I Excellent Symmetrical, compact, distinct outline, no blastomere
extrusion, even granulation, neither very light nor very
dark

II Good Somewhat asymmetric, even granulated with distinct

outline,
some blastomere extrusion
III Fair Hazy outline, extruded cell, asymmetric

IV Poor Uneven granulation, hazy outline, abnormal shaped

V Degenerated Developmental stage difficult to determine

 Transfer of embryo (Introduction to recipient)

 Recipient should be in estrus within 12 hours of

the donor so that it should posses good CL at
the time of transfer.

 To maximize success rate of the transfer, the

recipient’s
estrus should be in sync with that of the donor.
Process Of Transferring Embryos

 The recipient is palpated to determine the

presence and location of the CL (right vs. left).
Recipient is administered an epidural
(lidocane) to relax the muscles in the pelvic
Transfer of embryo (Introduction to recipient)

Surgical Non
Surgical
[Link]
method:
 Involves laparotomy incision, preferred in sheep,
goat and pig. The uterine horn ipsilateral to the
ovary with CL is exposed. A small syringe fitted
with 21 gauge needle is used to make the
transfer.
 When the embryo is placed in the uterus, the
needle is carefully inserted through the wall of
uterine horn whereas, when embryo is placed in
oviduct then the needle is inserted through the
infundibulum into the ampulla where the
embryo is deposited.
 [Link]-surgical method
 Mostly used for cattle and mare.
 Flushed embryos that pass inspection are loaded
into an
AI straw.
 If the embryo is frozen it is thawed in a warm
water bath (92°F) for < 30 sec and placed in a
specially designed transfer gun and covered
with a sterile sheath.
 The transfer gun is passed through the vagina,
cervix, and into the uterine horn on the side as
the CL. The embryo is deposited 1/3 the way up
the uterine horn.
 Pregnancy rates are greatest when the day of
the estrous cycles of donor and recipient are
within 24 hours. Recipients should be in heat
24 hours before to 12 hours after the donor
Storage And Cryopreservation Of Embryo
 Embryos can be maintained at near body
temperature in the media used for flushing
during the period between recovery and
transfer.
 If embryos are to be held longer than 2 hrs. up to
10 hrs., a media containing 20% heat treated
serum should be used as a holding medium.
 If embryos are cooled at 5ºC (I.E. refrigerated
temperature), they can be maintained for 2-4
days.
 Cryopreservation of embryo is performed for
longer period
of time.
Advantages of Cryopreservation.
 long term storage
 eliminates estrus synchronization in recipients
 Cryoprotactants like glycerol, ethylene glycol and
DMSO (Dimethyl sulpho-oxide) are always
needed for preservation of embryos.

Thawing Of Straws.
 Straws are thawed before transfer of
embryo to the recipients.
 If 0.25ml straw – 15 sec. in air and 20 sec in
water bath at 37 ºC
 If 0.5 ml straw- 20 sec. in air and 20 sec in water
bath at 37 ºC
 Exposure to air reduces damage to the zona
pellucida.
 Advantages ETT Program

Increase the number of offspring sired from

superior females.
Results in faster genetic progress.
Increase the frequency of desired mating,
capitalizing on
excellence of a mating.
Obtain offspring from old or injured animals
incapable of
breeding or calving naturally.
Increased farm income through embryo
sales.
Exportation and/or importation of embryos is
easier than with live animals.
Disadvantages of EET Program
Can be cost prohibitive and success rates are less
than AI.
Cost and maintenance of recipient females.
Requires a technician with the skills to flush
embryos from the reproductive tract.
Possible spread of disease through recipients.
Micromanipulation

 Bisection of embryo
 Sexing of embryos
 Nuclear transfer/gene
transfer.
Bioinformatics for animal breeding
Bioinformatics is the application of computer science,
mathematics, and statistics to the analysis and interpretation
of biological data, such as DNA sequences, gene expression,
or protein structures. Bioinformatics can help animal breeders
to identify and characterize the genetic variants that affect
the traits of interest, such as growth, health, or behavior. For
example, bioinformatics can be used to perform genome-wide
association studies (GWAS), which can reveal the associations
between genetic markers and phenotypes across a large
number of individuals. Bioinformatics can also help to design
and implement genomic selection (GS), which is a method of
selecting animals based on their genomic estimated breeding
values (GEBVs), rather than their phenotypic performance or
pedigree information.
Systems biology for animal breeding

Systems biology is the study of the interactions and

dynamics of biological components, such as genes,
proteins, cells, or organs, within a system, such as an
organism, a population, or an ecosystem. Systems
biology can help animal breeders to understand and
predict the complex and nonlinear effects of genetic
and environmental factors on the traits of interest. For
example, systems biology can be used to build and test
mathematical models that simulate the biological
processes and pathways that underlie the phenotypic
variation and response to selection. Systems biology
can also help to integrate and interpret the data from
Benefits of bioinformatics and systems biology for
animal breeding

Bioinformatics and systems biology can offer several

benefits for animal breeding programs, such as
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Additionally, they can improve the animal welfare and
environmental sustainability of animal production, by
optimizing the adaptation and performance of animals
Challenges of bioinformatics and systems
biology for animal breeding

Bioinformatics and systems biology present several

challenges for animal breeding programs, such as the
need for high-quality and high-throughput data
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inaccessible for some breeders and researchers.
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How to start using bioinformatics and systems biology for
animal breeding

If you are interested in using bioinformatics and systems biology

for your animal breeding program, there are a few steps you
should take. First, define your objectives and criteria, and identify
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