Prepared by-
Dr. S. K. Sheetal
Assistant Professor cum Jr. Scientist
Department of Veterinary Gynaecology and
Obstetrics,
Bihar Veterinary College, Bihar Animal Sciences
University, Patna-800014
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EMBRYO TRANSFER TECHNOLOGY
(ETT)
“Embryo transfer is a technique where embryos
are collected from the donor females and
transferred into uterus of recipients which
serves as a foster mother for its development
throughout the remainder period of
pregnancy.”
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Learning Objectives:
✓Define the Embryo transfer (ET)
✓Explain the steps of embryo transfer
✓List the advantages of embryo transfer
✓List the disadvantages of embryo transfer
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History of Embryo Transfer
• The first successful embryo transfer was
carried out in rabbit (1890) by Walter 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.
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Steps Involved In Embryo Transfer
1) Selection of donor
2) Selection of recipients
3) Estrus synchronization of donor and recipients
4) Superovulation (SOV) of donor (release of
multiple eggs at single estrus)
5) Artificial insemination of donor
6) Embryo collection
7) Evaluation of embryos
8) Transfer of embryos/cryopreservation of
embryos /micromanipulation
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Applications of Embryo Transfer
1) Faster genetic improvement
2) Genetic screening
3) Disease control
4) Import and export
5) Circumvention of infertility
6) Twining in cattle
7) Conservation of endangered species
8) Research/production of clones/and genetic
engineering
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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
abnormalities)
• Younger (4-8 years of age)
• Healthy and have good body weight
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• 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 interval
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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.
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3. Estrus Synchronization of Donor
• The donor cow should be synchronized to
bring into estrus OR should have palpable CL
on the ovary superovulatory process
• For this, any of the synchronization protocols
can be used.
• Ov-synch, Co-synch, select synch hybrid synch,
heat synch etc.
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4. Superovulation of Donor Cow
• 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 ova is later on fertilized and
embryo produced can be transferred to the
recipients
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• The basic principle of superovulation is to
stimulate extensive follicular development
through the use of a hormone preparation, which
is given IM or SC with FSH activity.
• In Ewe, Doe and Cow an average of 12 ovulations
can be expected. In Sow number of ovulations
could be >20.
• SOV has not yet achieved in Mares due to
ovulations occurring in at one site of ovary i.e.
ovulation fossa.
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Time of Superovulation
• For optimum response gonadotropin
treatment is initiated during mid luteal phase
of estrus cycle i.e. on days 9-14 of estrous
cycle (Day 0 is estrus)
• Donor cows can be superovulated repeatedly
at approximately 6-8 weeks interval.
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5. Insemination of Donor (A.I.)
• Donor should be inseminated artificially 2-3
times at 12 hours interval 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.
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6. Embryo Recovery
• Embryos can be collected by following
methods:
A. Surgical method
B. Non-Surgical method
C. Laparoscopic method
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A. Surgical Method
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).
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B. Non-Surgical Collection (Trans
Cervical Method)
• Commonly performed in cattle, buffalo and mare.
• It involves 2 ways or 3 ways Foley or Woerllein
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
cervix.
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Trans Cervical Embryo Collection
(Flushing of Donor)
Searching of Embryos in
Stereozoome microscope
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• Collection of bovine embryos 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 in most of the species is modified
Dubecco’s phosphate buffer saline. NS can be
used in its absence.
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• During final collection, oxytocin is
administered @ 50 i.u. intravenously.
• Give large doses of intrauterine 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 flushing.
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C. Laparoscopic Embryo Collection
• Surgical collection is choice of method in
sheep and goat due to inability to palpate
reproductive tract.
• This has lead to the use of surgical techniques
predominantly leading to adhesion formation.
• Laparoscopic is considered to results in fewer
adhesions than traditional surgery.
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7. Evaluation of Embryos
• After collection and before transfer to the recipients, the
embryos are evaluated under sterezoome microscope at
50 to 100x magnification.
• Day 7 bovine embryos (compact morulla or blastocyst)
are about150-190µm in diameter and are still within the
zona pellucida.
• Embryos are graded based on following characteristics
✓ Compactness of the cells
✓ Regularity of cells
✓ Variation in cell size
✓ Colour and texture of cytoplasm
✓ Presence of vesicles, extruded cells, cellular debris
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Morulla 16 Cell and 32 Cell Expanded Blastocyst
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Transfer of Embryo (Introduction to
Recipients)
• Recipients should be in estrus within 12 hrs. of
the donor so that is should posses good CL at the
time of transfer.
• To maximize success rate of 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 (Rt. Ovary vs. Lt. Ovary).
• Recipient is administered an epidural to relax the
muscles in the pelvic area.
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1. Surgical Method:
• It involves laparotomy incision, preferred in
sheep, goat and pig. The uterine horn ipsilateral
to ovary with CL is exposed. A small syringe fitted
with 21 gauge needle is used for transfer.
• When the embryo is placed in the uterus, the
needle is carefully inserted through the wall of
the 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.
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2. Non-Surgical Method:
• Mostly used for cattle and mare.
• Flushed embryos after inspection are loaded into ET
straw.
• If the embryo is frozen it is thawed in warm water bath
(920F) for <30s and placed in ET gun and covered with
sterile sheath.
• The ET 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 to the uterine horn.
• Pregnancy rate high when day of estrus of recipients
and donor are within 24 hours.
• The embryos are typically transferred on day 7 of the
estrous cycle.
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Storage and Cryopreservation of
Embryos
• 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 for longer than 2 hrs.
Up to 10 hrs.; a media containing 20% heat
treated serum should be used as holding media.
• If embryos are cooled at 50C (IE refrigerated
temp.) , they can be maintained for 2-4 days.
• Cryopreservation of embryo is performed for
longer period of time.
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Embryo Freezing Unit
Marking of Straws →
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Advantages of cryopreservation
• Long term storage of embryos
• Eliminates estrus synchronization in recipients
• Easy export and import
• Worldwide distribution
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• Cryoprotectants like glycerol, ethylene glycol and
DMSO are always needed for preservation of
embryos
• Thawing of Straws: Straws are thawed before
transfer of embryos to recipients. If 0.25 ml
straws→ 15 sec in air and 20 sec. in water bath at
370C
• If 0.5 ml straws→ 20 sec in air and 20 sec. in
water bath at 370C
• Exposure to air reduces damage to zona pellucida
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Advantages of ETT
• Increase the number of offspring sired from
superior females.
• Results in faster genetic progress.
• Obtain offspring from old or injured animals
incapable of breeding or calving naturally.
• Increase farm income from sale of embryos.
• Export/import of embryos is easier than with
live animals
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Disadvantages of ETT Programme
• Costly and success rate 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 diseases through recipients.
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THANK YOU
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