Embryo Transfer Technology - Embryo Transfer Technology of embryo-assisted reproductive technology
It is also called ovum /embryo transfer. It is the collection of the fertilized ovum from the donor before its attachment/nidation with the uterus and transfers into the surrogate/recipient mother for completion of the gestation period.
The cow from which we get the embryo is called a donor. The cow to which we transfer the embryo is called the recipient. In AI, semen is inseminated in different cows; the genetic potential of males is distributed which is the same for all cows but different cows have different potentials. In embryo transfer, the genetic potential of females is distributed.
Every cow produces one egg at the time of estrus. Donor cows are induced to produce more than one egg at the time of estrus. These eggs are inseminated and then these fertilized eggs/embryos are taken from the donor. At the same time recipient cows, having low genetic potential, are also prepared and one embryo is placed in each cow. In this way, more than one calf can be obtained from one donor.
Advantages of Embryo Transfer:
·
It improves the genetic potential
·
It increases the productivity
·
It increases the economic benefit
·
It increases the disease resistance
·
It increases the no. of calves in a lifetime
·
It reduces the generation interval
·
It increases the selection intensity
· Import and export are easier because it is done in the form of the embryo in which transportation is easier, genetics is diversified, and the cost is decreased.
Steps of Embryo Transfer
·
Selection of donor
·
Selection of the recipient
·
Synchronization of donor and recipient
·
Superovulation of donor
·
Insemination of donor
·
Collection of embryos
·
Evaluation of embryos
· Transfer or storage of embryos.
Selection of Donor
·
It should be from a known fertile bloodline i.e. we must have a pedigree record
·
It should have calved once earlier so heifer as a donor is not
required.
· Donors should have known calving history or easy calving history.
·
It should be a regular cyclic animal.
·
It should be a disease-free animal e.g. T.B, brucellosis
·
It should have been vaccinated
·
It should be high production and reproduction wise normal
Super Ovulation
Production of more than one egg by the use of hormones or drugs is called superovulation. PMSG and FSH are the hormones used for superovulation. FSH is short-acting and PMSG is long-acting. Repeated FSH injections have to be given, while PMGS is given at once. On day 10 of the estrous cycle, CL is present. So give 5 mg of FSH in the morning and 5 mg in the evening. Similarly, FSH is given 5 mg morning and 5 mg evening on day 11, 5 mg morning and 5 mg evening along with PGF2α on day 12, and 5 mg morning and 5 mg evening on day 13. In this way, FSH is given for four days two times a day intramuscularly. On day 14, the donor will be in heat and there will be ovulation of more than one egg.
Synchronization
The reproductive cycle of the donor and recipient should be at the same level. We have 100 non-pregnant cows and we have to bring them in heat in a small given period. Synchronization means bringing the events together. We synchronize all these cows. Out of 100, on palpating 20 are already in estrus, 20 are in proestrus that may come in the heat after two days, 15 are in post estrus they are just in ovulation, and 30 are in diestrus. Other 10-15 are in anestrous. The function of PGF2α is to regress the CL. This purpose can be used anywhere.
d1 d6 d11 d14 d17
Estrus:
∙----------∙----------∙------∙------∙
CL
PGF2α Heat
d1
d3
d9 d11
d14 d17
Proestrus: ∙----∙------------∙----∙------∙------∙
Heat
CL PGF2α Heat
d1 d4
d11 d14 d15
Metestrus: ∙------∙--------------∙------∙--∙
CL
PGF2α Heat
d1 d3 d9 d11 d14 d20
Diestrus: ∙----∙------------∙----∙------∙------------∙
PGF2α Heat CL PGF2α
Heat
·
Animal in estrus does not have any CL. 6 days later there will be
CL. This CL will persist for 11-17 days.
·
Proestrous animal comes in estrus after one or two days. They do
not have CL. So no response of PGF2α on day third. On day 9, CL will
present which remains for day 20.
·
Metestrus animal was in estrus two days earlier. On day four CL
will be formed which stays in the ovary till day 15.
·
In diestrus animal has functional CL. Give PG on the first day, on
day 3 they come in heat. They will develop CL on the ovary on day 9. 6 days will be
taken for CL to develop. On day 20 CL will persist.
· Anestroue does not respond to anyone.
Insemination of Donor
Inseminate donor in standing heat. Inseminate two or three times after twelve hours and each time go for a double dose.
Collection of Embryo
Until
1975, embryos were collected surgically. After anaesthesia (general or local),
enter the uterus and collect embryos. It was very difficult to collect from
donors at the farm. The cost of collection was too much. Another problem was, that some
damage may lead to future adhesion in the reproductive tract. People tried to
collect without surgery.
The non-surgical method is preferable now because there is no damage
or very little damage that provides repeatability for the donor to donate
embryos and made collection at the farm level possible. A disadvantage of this method
is that we can only collect the embryo when it enters the uterus. But with
surgery, we can collect embryos while they are in the oviduct.
The procedure of Collection:
·
Place the donor in crushes, and wash the perennial region. Evacuate
rectum from feces and evaluate no. of CL on both ovaries, it will tell the no.
of embryos. Older cows suck air in the rectum and uterus, so very old cows are not
used but if they are good we can use them. Apply a bally band that would create positive pressure instead of negative pressure.
· Apply
epidural anesthesia to decrease the movements in cows. The collection is done with
the help of a catheter. Three kinds of catheters are used; the most commonly used is
Foley’s Catheter. It could be two ways or three ways.
·
It is easily available and inexpensive also. It is soft in
nature. In surgery, we clamp the uterus horns and throw fluid inside and suck it
again which will contain embryos. In the non-surgical collection, we have to fix the catheter in the uterus horn in a method that embryos cannot pass out of the horn into the uterus and the portion is blocked completely. There are two methods:
Continuous Flow Method:
There is less loss of fluid but there are a lot of tubes that we have to handle. It may cause contamination.
Interrupted syringe method:
All equipment is disposable so less chance of contamination
·
When catheters are used, they are sterilized either with
ethylene oxide or by color sterilization method. These agents are harmful to
embryos. With ethylene oxide, it should be sterilized one week before collection
and put in the air so that harmful material is decreased. If a cold chain is to be
used then use normal saline.
·
Embryo collection is done in diestrus when the cervix is closed but
still, it is penetrable. A steel rod known as a cervix dilator is used. All
things sterilize. Pass it slowly which will loosen the rings of the cervix, take out the dilator and pass the catheter.
·
Catheters are not hard enough because they are of rubber. To
create stiffness in the catheter we pass a steel rod in it and after passing we
direct it toward one of the horns. The tip of the catheter has an opening and an area
where there is a balloon i.e. deflator. Enter N.S so that balloon is flatted.
When the balloon is bigger enough to fill the lumen of the uterus the catheter
is fixed.
·
Now fill the uterus from the opening inside the catheter, the
fluid when starts filling in the uterus, move the top of the horn where the embryos
would be and take out the fluid which contains embryos. In the first attempt of
filling fluid, it contains 85 % of embryos. No. plates are marked.
Immediately after the collection, shift the material in the lab and then search for the embryo.
Evaluation
Square-shaped Petri plates are used. Search with a stereomicroscope at 10X.
Criteria
for Evaluation:
·
Continuity of zona pellucida (zona is double layered and its
diameter is 12-15 µm).
·
The arrangement of blastomeres in the zona: they should be
arranged in a circular fashion and with no extension of cells from the zona.
·
Blastomeres should be of uniform size.
·
Presence of degenerative area: If degeneration is present it will
appear as a black area. It is in %age.
·
Presence of vacuole.
By following the above mentioned criteria we can evaluate the embryos in all grades:
Excellent → A: Ideal, spherical, symmetrical with uniform blastome%
Good → B: Few
extrusion of blastomeres, irregular shape, one or two vacuoles
Fair → C: Extruded
blastomeres, vacuole formation with 19 % degenerative area
Poor → D: Numerous
extruded blastomeres, more variation in size, more vacuoles, degeneration up to 25
Unfertilized: There
will be no cell and just spots present
For
fresh embryo transfer, embryos up to C grade can be used. If the embryo is to be
transferred after freezing then only up to B grade embryo can be used. The
searching of the embryo is done at 10 X but the evaluation is done at 50-100 X
magnification.
Transfer of Embryo
Washing:
The first step before the transfer is washing. It is necessary because when we collect
uterine material, it also contains mucous, blood, etc. 5 ml embryo washing
medium is taken in three Petri dishes. The embryo is put in each one for five
minutes.
The straw of semen can be used for loading the embryo. Sterilize it
with ultraviolet rays or ethylene oxide gas. Attach straw with tuberculin
syringe for picking of the embryo. Then wash it with pure water 2-3 times but that
water should not touch the PVP plug.
An embryo transfer gun is used for transfer. Pull the plunger of the E.T gun back. Put the
straw inside from the side of the cotton plug. Then cover with another straw. But
keep in mind that the margin of straw is inside the gun and the margin of
straw which is outside the gun should be at the same level because if one drop
remain may be an embryo is present in it. Then cover it with another outer straw
which will protect the corner from any debris in the tract.
Loading of Embryo: Suck the embryo washing medium 2-3 cm followed by air column and then second medium column larger than first one i.e. 2.5-3.5 cm having embryo. Again a column of air. Then again column of the medium of 2-3 cm and again column of air and then there is open space.
Transfer:
· Use 1-2 ml of epidural anesthesia and go for an examination of the ovary to check on which ovary the CL is present. The transfer of embryo should be transferred into an ipsilateral horn (horn containing CL).
· Ideal site for the deposition or transfer is 5-10 cm above the bifurcation.
· Firstly you have to straighten the horn
After transfer that recipient should be kept in observation for the next oestrous cycle. If it does not come in oestrous then go ultrasonography for pregnancy confirmation.
Storage of embryo
Short Term Storage:
For 1-2 days, short storage is required if the recipient is not ready.
Procedure:
5 ml of DPBS, put the embryo in it and cover it with aluminum foil and place it in a beaker and shift the beaker to the refrigerator at 4-5 C. For a longer period of storage deep freezing is done after loading of straw and then sealing it. Automatic embryo freezing chambers are available. Cryoprotective agents are also added. 10 % glycerol may be used.
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