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It is also called ovum transfer. It is the
collection of fertilized ovum from the donor prior to its
attachment/nidation with uterus and transfer into surrogative/recipient
mother for completion of gestation period.
The cow from which we get the embryo is called as donor. The cow to
which we transfer embryo is called recipient. In AI, semen is
inseminated in different cows; the genetic potential of male is
distributed which is same for all cows but different cows have
different potential. In embryo transfer, genetic potential of female
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 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:
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It improves the genetic potential
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It increases the productivity
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It increases the economic benefit
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It increases the disease resistance
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It increases the no. of calves in life time
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It reduces the generation interval
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It increases the selection intensity
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Import and export is easier because it is done in the form of
embryo in which transportation is easier, genetics is
diversified, and cost is decreased.
Steps of Embryo Transfer
Selection of Donor
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It should be from known fertile blood line i.e. we must have
pedigree record
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It should have calved once earlier so heifer as a donor is not
required.
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Donor should have known calving history or easy calving history.
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It should be a regular cyclic animal.
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It should be a disease free animal e.g. T.B, brucellosis
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It should have been vaccinated
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It should be high producing and reproduction wise normal
Super Ovulation
Production of more than one eggs by the use of hormones or drugs is
called super ovulation. PMSG and FSH are the hormones used for super
ovulation. FSH is short acting and PMSG is long acting. Repeated FSH
injections have to be given, while PMGS is given at once. At day 10
of estrous cycle CL is present. So give 5 mg of FSH at morning and 5
mg at 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,
donor will be in heat and there will be ovulation of more than one
eggs.
Synchronization
The
reproductive cycle of donor and recipient should be at the same
level. We have 100 cows which are non pregnant and we have to bring
them in heat in a small given period. Synchronization means to bring
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 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
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Animal in estrus do not have any CL. 6 days later there will be
CL. This CL will persist for 11-17 days.
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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 remain for day 20.
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Metestrus animal was in estrus two days earlier. On day four CL
will be formed which stays in ovary till day 15.
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In diestrus animal has functional CL. Give PG on first day, on
day 3 they come in heat. They will develop CL on ovary on day 9.
6 days will be taken by CL to develop. On day 20 CL will
persist.
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Anestroue do not respond to any one.
Insemination of Donor
Inseminate donor in standing heat. Inseminate two or three times
after twelve hours and each time go for double dose.
Collection of Embryo
Until 1975, embryos were collected surgically. After anesthesia
(general
or local), entering the uterus and collect embryos. It was very
difficult to collect from donors at farm. The cost of collection was
too much. Another problem was, some damage may lead to future adhesion in
reproduction tract. People tried to collect without surgery.
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 farm level possible. 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 oviduct.
Procedure of
Collection:
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Place the donor in crushes, 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 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 a positive pressure instead of negative pressure.
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Apply
epidural anesthesia to decrease the movements in cow. Collection
is done with the help of catheter. Three kinds of catheters are
used; most commonly used is Foley’s Catheter. It could be two
ways or three ways.
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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 non
surgical collection we have to fix catheter in uterus horn in a
method that embryos cannot pass out of horn into uterus and
portion is blocked completely. There are two methods:
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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.
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Interrupted syringe method:
All equipment is disposable so less chance of contamination
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When catheters are used, they are sterilized either with
ethylene oxide or by color sterilization method. These agents
are harmful for embryos. With ethylene oxide it should be
sterilized one week before collection and put it in air so that
harmful material is decreased. If cold chain is to be used then
use normal saline.
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Embryo collection is done in diestrus when cervix is closed but
still it is penetratable. A steel rod known as cervix dilator is
used. All things sterilize. Pass it slowly that will loosen the
rings of cervix, take out dilator and pass the catheter.
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Catheters are not hard enough because they are of rubber. To
create stiffness in catheter we pass steel rod in it and after
passing we direct it toward one of the horns. Tip of catheter
has opening and an area where there is 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.
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Now fill the uterus from the opening inside the catheter, the
fluid when starts filling in uterus, move the top of horn where
the embryos would be and take out the fluid which contains
embryos. In first attempt of filling fluid it contains 85 % of
embryos. No. of plates are marked. Immediately after the
collection, shift the material in lab and then
search of embryo.
Evaluation
Square shaped Petri plates are used. Search with stereomicroscope at
10X.
Criteria for
Evaluation:
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Continuity of zona pellucida (zona is double layered and its
diameter is 12-15 µm).
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The arrangement of blastomeres in the zona: they should be
arranged in circular fashion and no extension of cells from the
zona.
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Blastomeres should be of uniform size.
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Presence of degenerative area: If degeneration present it will
appear as black area. It is in %age.
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Presence of vacuole.
By following the above mentioned
criteria we can evaluate the embryos in all grade:
Excellent
→
A:
Ideal, spherical, symmetrical with uniform blastomeres, no
degenerative area and no vacuole
formation.
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 upto 25
%
Unfertilized:
There will be no cell and just spots present
For fresh embryo transfer, embryo
upto C grade
can be used. If embryo is to be transferred after freezing then only upto B grade embryo
can be used. The
searching of embryo is done at 10 X but evaluation is done at 50-100
X magnification.
Transfer of Embryo
Washing:
First step before 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. Embryo is put
in each one for five minutes.
Loading of Embryo:
Straw of semen can be used for loading of embryo. Sterilize it with
ultraviolet rays or by ethylene oxide gas. Attach straw with
tuberculin syringe for picking of embryo. Then wash it with pure
water 2-3 times but that water should not touch PVP plug.
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 medium
of 2-3 cm and again column of air and then there is open space.
Transfer:
Embryo transfer gun is used for transfer. Pull plunger of E.T gun
back. Put the straw inside from the side of cotton plug. Then cover
with another straw. But keep in mind that the margin of straw which
is inside the gun and the margin of straw which is outside the gun
should be at same level because if one drop remain may be embryo is
present in it. Then cover it with another outer straw which will
protect the corner from any debris in tract.
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Use 1-2 ml of epidural anesthesia and go for examination of
ovary to check on which ovary the CL is present. Transfer of the
embryo should be transferred into ipsilateral horn (horn
containing CL).
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Ideal site for the deposition or transfer is 5-10 cm above from
bifurcation.
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Firstly you have to straighten the horn
After transfer that recipient should
be kept in observation for the next estrous cycle. If it does not
come in estrous then go ultrasonography for pregnancy confirmation.
Storage of embryo
Short Term Storage:
For 1-2 days, short storage is required if recipient not ready.
Procedure:
5 ml of DPBS, put embryo in it and cover it with aluminium foil and
place it in a beaker and shift the beaker in refrigerator at 4-5
oC.
For longer period of storage
deep freezing is done after loading of straw and then sealed it.
Automatic embryo freezing chambers are available. Cryoprotective agents
is also added. 10 % glycerol may be used.
Seeding:
For straw you have to give the point for formation of ice. Freezing
started from the end other than cotton plug.
Thawing:
Thawing is done at 37-38 0C @ 19-15 second. In semen straw is
directly loaded in AI gun but in the case of embryo the viability of
embryo is checked under microscope before the transfer. For freezing
morula or compact morula stage is the best.
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