Embryo Transfer Technology
History
1890 -Transfer embryos from donor to recipients first demonstrated in rabbit
1951- First successful bovine embryo transfer (ET) by surgical collection of embryos
1964- First non-surgical collection
1983 -Successful In-vitro fertilization of a bovine ovum
Definition of Terms
Embryo Transfer (ET)-The process of collecting embryos (flushing) from one female (the
donor) and transferring to other females (the recipient) to complete the gestation period.
Embryo- A fertilized ovum that hatch, 9 to 10 days after fertilization, from the protective
“shell-like” coating (the zona pellucida) i.e. the hatched blastocyct.
Superovulation – Induction of development and maturation of multiple follicles in ovaries by
treating the donor with gonadotropic hormone (FSH or GnRH).
Synchronization- Matching the stage of estrous cycle of a donor and recipients by the
injection of prostaglandin (PGF2α) to both the donor and recipients.
Advantages and disadvantages of an Embryo Transfer (ET) program in cattle
Advantages
a) Increase the number of offspring from superior parentage.
b) Results in faster genetic progress within a population compared to natural breeding
or AI. NB: the superior qualities of the parents are passed to offspring; therefore by
carefully selecting high quality donor and the sire (e.g. bull whose daughters are high
milk yielders and a donor with good body conformation), superior animals are
produced over a short period.
c) Increase the frequency of desired mating (superior cows and bulls)- since donor is
used only to produce embryos without passing through gestation, she produces more
fertile ova in her lifespan.
d) Obtain offspring from old or injured animals incapable of breeding or calving
naturally.
e) Increased income through embryo sales.
f) It is easier to export and/or import high quality embryos than live animals.
Disadvantages
a) ET is costly and success rates for fertility are lower than AI.
b) High cost of maintenance of recipient females.
c) Requires specially trained personnel
d) Possible spread of diseases and genetic disorders to offspring from recipients.
Purpose of Embryo Transfer (ET)
1
1. It is easier and cheaper to import/export embryos than superior quality animals
2. ET enables quicker testing for inherited defects in bulls at AI stations- by mating the
bull to six or eight of his daughters and observing for recessive traits in the second
generation.
3. Treatment of infertility. Reproductive traits have a low heritability- dams rarely pass
infertility to their daughters in ET i.e. embryos from donors with incidences of
infertility rarely develop the reproductive abnormality.
4. The recipient animals’ genetic or phenotypic traits do not affect the implanted embryo
during gestation e.g. can transfer a frieshian embryo to a zebu recipient… calf remains
frieshian!
Selection of Donor and receipients
Donor: animal that donates embryos
1) Selection criteria are used to select a donor e.g. genetic merit (milk yield, body
conformation, resistance to diseases etc), reproductive performance, progeny
performance etc.
a. NB: Embryo from a donor cow does not guarantee a superior calf as offspring
and dam differ genetically (half genes in offspring are from the sire!).
b. Each embryo has a different genotype and more than likely a different
phenotypic expression due to environment (gestation in different animals).
2) Reproductive soundness –Selected animals must be exhibiting regular estrous cycles at
least 60 to 90 days post calving.
Recipient: animal that receives embryo from donor.
1) Recipients (also known as surrogate) are the greatest single cost of an ET program
and
a. Are Synchronized to be at the same stage of the estrous cycle as the donor when
she donates the embryos.
b. should be healthy, in good body condition and vaccinated for common diseases
c. Should have been through at least two normal estrous cycles before use.
d. Between 10 to 14 surrogates are selected for each donor
2) Pregnancy rates are greatest when
a. Day of the estrous cycles of the donor and recipient are within 24 hours of
each other.
b. In practice, the recipient should exhibit estrus from 24 hours before to 12
hours after the donor was in estrus.
c. Embryos are typically transferred on day 7 of the estrus cycle.
Procedure for ET
Select donors and surrogates
1. Superovulation in Donor
a. Development of multiple oocytes in ovaries is stimulated by injecting donor cow
twice daily with FSH for a period of four days. Treating with FSH allows for the
recruitment, growth, and development of multiple ovarian follicles. The number of
2
follicles that develop is highly variable and is influenced by animal age, breed,
lactation status, nutritional status, season and stage of the estrous cycle when FSH
treatment is initiated.
b. The donor cow is treated with FSH between day 9 and 14 of the estrous cycle (day 0 =
day of estrus). High levels of FSH are administered twice daily for 4 or 5 days (NB-
donor at diestrus so that a high dose of FSH is needed to cause follicle development in
the presence of progesterone produced by a corpus luteum).
c. Two or three days after beginning the FSH treatment, PGF 2α is administered to both
the:
a. Donor to suppress development of corpola lutea or lyse existing CL
b. Selected recipients to synchronize estrous 2 to 3 days later.
d. At least 10 recipients should be in the same stage of the estrous cycle as the donor
cow. Therefore 14-18 recipients are synchronized for each donor.
2. Insemination of donor
Donor cows usually exhibit estrus within two days after the last day of FSH administration.
When in standing estrus or shortly thereafter, the superovulated donor is:
a. Bred with frozen semen of one or more choice bulls (Parentage can be
determined by blood type).
b. Usually multiple inseminations at 12, 24 and 36 hours after the onset of estrus
are recommended using more than one vial or ampule of high-quality semen per
insemination.
3. Expected fertilization
About 10 to 20 oocytes develop to maturity but on average, 5 to 12 embryos (blastocysts)
develop and are flushed from donor cows and are transferred to recipients or frozen for storage.
4. Flushing: Non-Surgical Embryo Recovery
Most embryos are collected by a non-surgical process between day 6 – 8 (estrus = day 0) of
the estrous cycle with day 7 being the most common.
e. A specially designed Foley catheter is used for the flushing procedure. The Foley is a
2-way catheter that has 2 channels: one for inflation of a balloon at the end of the
catheter and a second for the inflow and outflow of flushing medium. Flushing is
accomplished by
a. Inserting a catheter with an inflatable balloon into the donor’s uterus and
washing a limited area with continuous or intermittent flushes of 30-200 ml
saline solution or other suitable culture media.
b. Each uterine horn is flushed separately.
c. The embryos are flushed out with the saline solution, which is collected.
d. When an embryo is found, it is picked and transferred to fluid containing
bovine serum.
a. The flushed embryos are encased in zona pellucida I.e. are not hatched- and are
identified using a microscope
Flushing Process
After restraining the donor:-
3
a. Epidural anesthesia is administered to relax hind limb muscles during the flushing
process
b. The Foley catheter is inserted through the vagina, cervix and into one of the uterine
horns.
c. The balloon is inflated to close the anterior opening of the horn to prevent fluid from
leaking out of the uterus as flushing media is added to the uterine horn to recover the
embryos.
d. A small amount (100-300 m L) of saline solution is “flushed” into the uterus. The fluid is
then recovered from the uterus and the process is repeated on the opposite horn.
e. The flushing media recovered from the uterus is filtered (75 μm pore size) to assist in
separating embryos from the flushing media. Once the embryos are recovered they are
evaluated for stage of development and graded for quality.
f. The embryos are further processed e.g. by sexing of embryonic cells and (or) splitting
the embryo in half to make twins.
g. Embryos are either transferred immediately into a recipient or frozen for storage.
5. Embryo transfer to recipients
Recovered embryos are transferred in two ways
1) Same day transfer- Embryos are transferred into recipients on the same day they
are collected from the donor.
- Transferring fresh embryos results in greater pregnancy rates, but requires a great
coordination to get recipients and donors on the same day of the estrous cycle when
flushing is done.
2) Transfer of Frozen Embryos – After thawing, the embryos are transferred to recipients
7 days after displaying estrus.
- Pregnancy rates for frozen embryos are slightly lower than fresh, but the process has
numerous advantages including:
o Frozen embryos offer important logistical and economic advantages. Many
cows can be flushed in a day, which facilitates freezing of embryos.
o Frozen embryos can be imported and/or exported easily.
4
Freezing
1) Cryoprotectants like glycerol and ethylene glycol are used to dehydrate embryonic
cells and protect the cells from damage in freezing and thawing processes.
a. Ethylene glycol is the cryoprotectant of choice in cattle since embryos are
frozen in a single step and also thawed in a single step and transferred into a
recipient- the embryos are known as direct transfer (DT) embryos.
2) Embryos are:-
a. Equilibrated for 5 to 10 minutes in cryoprotectant freezing medium
b. Loaded and stored in a 0.25cc polyvinyl straw.
c. Frozen in a step-wise procedure in a special freezing equipment viz embryos are:
i. Placed in a freezing unit at -6°C for 5 minutes.
ii. “Seeded” i.e. picked with forceps cooled in liquid nitrogen to facilitate
uniform freezing.
iii. Held at -6 to -7°C for another 5 minutes.
iv. Then cooled at 0.5°C/minute to -32°C.
v. Held at -32°C for 15 minutes then straws are plunged into liquid nitrogen
(-196°C).
Process of transferring embryos
The ovaries of recipient are palpated to determine location of the CL (right vs. left) and:-
a. Epidural anesthesia is administered to relax the muscles in the pelvic area.
b. In direct transfer from donor to recipient, an embryo is drawn into an A.I.
pipette and deposited in the middle third of the uterine horn on the side
bearing a CL
c. Frozen embryo is thawed in a warm water bath (36-37°C) for not more than
30 sec, placed in a specially designed transfer gun and covered with a sterile
sheath. The gun is passed into the uterine horn on the same side as the CL and the
embryo is deposited 1/3 the way up the uterine horn.
d. The embryos are transferred on day 7 of the estrous cycle.
e. Pregnancy rates are highest when recipients are in heat 24 hours before to 12
hours after the donor was in estrus.
Pregnancy Rates Resulting from Embryo Transfer
When done by experienced personnel, the
a. Transfer of fresh embryos yields pregnancy rates of 70 to 80%
b. While transferring frozen embryos yields pregnancy rates of 50 to 60%.
5
Superovulated ovary
Inflated Foley’s catheter
Double tubes of Foley’s catheter
6
Flushed Blastocysts at day 7 after insemination
