Preparation of in-vitro derived bovine embryos for transfer into
recipients
R.M. Rivera, J. Block, F.F.
Paula-Lopes, Y.M. Al-Katanani, M. Drost, and P.J. Hansen
Department of Animal Sciences and Large Animal Clinical
Sciences, University of Florida
Selection of recipients | Harvesting
and transfer of embryos | Preparation of
materials for transfer
Setup of work area at transfer site | Loading
embryos into straws | Loading straws into
transfer pipettes
Preparation of cows for transfer | Embryo
transfer procedure
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Recipient
cows should be on day 7 of the estrous cycle (d 0 = estrus). Cows can be selected based on estrus
detection (with or without synchronization) or after synchronization of
ovulation without estrus detection. There are many ways to achieve estrous
synchronization. See Estrous Synchronization - A Reproductive Management Tool
from Select Sires, Estrous
Synchronization of Cattle from Brangus World and Synchronization
Programs from ABS among other sources.
One
way to synchronize recipients is by giving one injection of GnRH (100 mg, i.m.) followed seven days later by an
injection of PGF2" (25 mg/ml) followed by
estrus detection. Cows seen in estrus
(most within 48-96 h after PGF) are scheduled to receive an embryo on day 7 of
the estrous cycle. Transfer without
estrous detection can also be performed by using the OvSynch program typically
used for timed artificial insemination.
This timed embryo transfer (TET) procedure, which is still under
development, may be useful when estrus detection is difficult such as during
heat stress (see Ambrose
et al., 1999 and Al-Katanani
et al., 2002). Cows used for TET
receive an intramuscular injections of 100 mg GnRH (Cystorelin) on day 0
followed by 25 mg PGF2" on day 7 and 100 mg GnRH on day 9. Embryos are transferred 8 days after last GnRH injection (note:
the optimal time for transfer has not been determined experimentally).
Harvesting and Transport
of Embryos
1.
Embryos
produced by in vitro techniques (see Procedures for In Vitro
Production of Bovine Embryos by Rivera et al) are harvested on day 7 or 8 after
fertilization (day 0=day of insemination).
For this purpose, excellent and good quality blastocysts (as described
in the Manual of the International Embryo
Transfer Society) are identified using a dissecting microscope and
transferred into a sterile tube containing embryo transfer
medium (we use HEPES-TALP using HEPES-TL from Biowhittaker but other media probably
work well also). Note: Many types of
tubes will work for transport – we frequently use a microcentrifuge tube with 1
ml of medium and as many embryos as available.
2.
Tubes
are sealed by placing a strip of Parafilm around the cap and loaded in a device
to keep embryos warm during transport.
Note: Our only experience is with a portable
incubator from Minitub (catalog #
19180/0000) that can maintain temperature at 39.0°C and which can operate on
batteries or a car cigarette lighter. This apparatus works great but
undoubtedly other containers will also work.
Preparation of
Materials for Transfer
The
following is prepared on the day of transfer and transported to the embryo
transfer site:
a. Slide
warmer set at 39°C
(if not available use a Styrofoam rack or other piece of Styrofoam as a
platform for Petri dishes containing embryos to keep embryos from cold
surfaces)
b. Bench
paper (not necessary but helpful at creating a clean area to work on)
c. 70%
Ethanol (wipe all areas with ethanol before starting any procedure)
d. Petri-dish to place embryos prior to pick up into transfer straw (Intergrid
plate works very well because
of the demarcations on the plastic)
e. Round
Petri dish to transfer embryos from
the tube were the embryos were transported (60 x 15 Petri dish works very well due to its small size)
f. 1
ml tuberculin syringes
g. Instrument
to handle embryos – many are available as described in the Guide on Use of Instruments for Picking Up
Oocytes and Embryos
h. Water
bath
i.
200 ml
pipet tips (yellow)
j. 0.25
ml French straw
k. Pipettor
set at 70 ml
l.
Embryo transfer rods (we use IMV 21” deep chamber
rods from Agtech cat# F17)
m. Sheaths
for embryo transfer rod (these have sideways openings to reduce contamination
of the uterus – from Agtech; catalog number F18A)
n. Oversleeve
Sanitary Chemise – a plastic sleeve used to cover the sheath and transfer rod
manufactured by IMV (available from Agtech; catalog #F27A)
o. Additional
sterile wrap (Dualpeel
tubing from VWR works well for this step)
for embryo transfer rods
p. Dissecting
microscope
q. Plastic
Pasteur pipets
r. Transfer
medium (HEPES-TALP - ~ 15-30 ml)
Setup of Work
Area at Transfer Site
a)
Prepare a clean area at the farm were the
preparation of straws will take place.
A temperature controlled room works best but any draft-free area can be
sufficient.
b)
Wipe all surfaces with 70% ethanol.
c)
Place bench paper (if available) on area where the
embryo manipulations will take place.
d)
Set slide warmer at 39°C (or set up
Styrofoam rplatform)
e) Place
round and Intergrid Petri-dishes on
slide warmer
f)
Set water-bath set at 38.5°C - Alternatively,
one could use the portable incubator to the tube containing transfer medium
warm. If a portable incubator or a
water-bath is not available, another alternative is to place the drops of medium into an Intergrid plate that is on
top of the slide warmer. Place the lid on the plate to prevent
evaporation. In about 5-10 minutes, the 70 ml drops of medium should be
warmed up and ready for use.
g) Transfer
embryos from the tube in which they were transported into a round Petri dish with a plastic Pasteur
pipet.
Loading Embryos into
Straws (see Figure 1)
a)
Locate, count and evaluate embryos for quality.
b)
Insert 200 ml
yellow tip onto the 1 ml syringe as shown (see Figure 1).
c)
On a Petri-dish (i.e. Intergrid plate) place 3
microdrops of 70 ml
holding medium side by side (A).
d)
Place one blastocyst into the middle drop (B).
e)
Insert the cotton plug side of a 0.25 ml French
straw into the wide end of the pipet tip (C-D).
f)
Aspirate one empty drop (E).
g)
Aspirate air to create a ~0.5 cm column (E)
h)
Aspirate the microdrop containing embryo (make sure
that the embryo is aspirated by observing under the microscope; F)
i)
Aspirate air to create a ~0.5 cm column (F)
j)
Aspirate the remaining empty drop until the cotton
plug is wet (G).
k) Remove
transfer straw from syringe and place on slide warmer until use (H).
Figure 1. Loading
embryos into straws.
Loading Straws into
Transfer Pipettes (see Figure 2)
a)
Open bag containing sterile transfer rod (A)
(use rod designed for 0.25 cc straws)
b)
Pull the plunger from the back of the transfer rod
(B)
c)
Insert the embryo containing French straw into the
transfer rod cotton side first (do this by holding the straw from the side
of the cotton plug to minimize contamination of the straw). If resistance is encountered stop
immediately and check if the plunger if pulled back (C).
d)
Move blue ring towards the center of the gun. Place a disposable blue sheath over the
transfer gun without touching the front end.
Push the sheath all the way to the back of the rod (you may need to
apply some pressure). Move blue ring to
the back of the rod to fasten the sheath in place (D).
e)
Place a chemise over the gun without touching the
front end (E).
f)
Place loaded rod into a protective bag (F).
g)
Transport to the site of transfer in a horizontal
position.
h)
Remove rod from protective bag and keep plastic bag
clean to reuse for next transfer
i)
After completion of transfer, bring transfer rod
back to the preparation area and wipe with ethanol. Do this by performing a single movement from the tip of the rod
(cleanest area) to the back of the rod (dirtiest area).
j)
Wait until ethanol has evaporated completely.
Figure 2. Loading
straws into embryo transfer pipettes
Preparation of Cows for Transfer
a)
Restrain the cow
b)
Palpate recipient cows for the presence of the
corpus luteum (CL) – if no CL present, do not use the recipient.
c)
With chalk mark the side were the CL is present
d)
Shave hair on the tailhead
e)
Prepare tailhead for epidural by cleansing with
betadine scrub followed by alcohol (70% ethanol)
f) Give
an epidural by injecting 5 ml of lidocaine into the epidural space.
When the tail is relaxed, the recipient
is ready for the embryo to be transferred.
In general, transfer is
performed as for AI (see Artificial Insemination Technique by Michael O’Connor. Special modifications and concerns are
listed
below
a) Care must be taken to avoid contamination
with feces. Clean the vulva thoroughly
before inserting the transfer pipette.
In addition, the vulvar lips should be opened before insertion of the
pipette. This can be accomplished by
the technician (by pushing the arm in the rectum downwards and back slightly)
or by an assistant (by grabbing the vulvar lips and pulling backward).
b) The tip of the
transfer pipette is placed approximately one-third of the way up the uterine
horn ipsilateral to the corpus luteum.
The embryo is then gently expelled from the pipette.
A contribution from the International
Consortium for Dairy Heat Stress funded by a grant from the USDA Initiative for
Future Agricultural and Food Systems
(USDA-CSREES #2001-52101-11318)
this page was last
updated 11-25-02
all original material for
this website © Rocio Rivera, Peter J. Hansen et al. 2000-2002
Links to commercial sites do
not constitute endorsement by the authors or the University of Florida
