All manipulations involving the use of live animals should employ aseptic
procedures, not only for the animal's safety, but also to avoid careless
techniques being developed by the operator. Every exercise should be
conducted as if it were real.
UTERINE FLUSHES
- Use non-pregnant cull buffaloes.
- Practise passing pipettes alternately into left and right uterine horns.
- Practise passing large Foley catheters into each uterine horn and feel
the distension of the balloon while it is being inflated.
- With the aid of a Foley catheter flush the uterine lumen with 1 litre
of sterile saline solution and save the effluent in a graduated cylinder.
After 12 animals, 90 percent or more of the fluid should be recovered
each time which should be clear, i.e. free from blood.
OOCYTE RECOVERY
- Obtain 20–30 bubaline or bovine ovaries from a slaughterhouse.
- Aspirate the contents of each grossly visible follicle with a 12-ml
syringe and a 22-gauge needle. Enter the follicle via the ovarian
stroma from underneath rather than pricking and collapsing the follicles
at the external surface.
- Vortex the follicular fluid vigorously with 1-percent heparin solution
to dislodge the cumulus cells. Alternatively, 10–25 percent seminal
plasma may be used instead of heparin.
- Pour the vortexed follicular fluid through a (used) embryo filter and
rinse with physiological saline until clear.
- Place the searching fluid in a grid searching dish (100 x 100 mm)
and search for oocytes, the diameter of which (160 μ) is the same as
that of the embryo up to the expanding blastocyst stage. (See procedure
for embryo searching, Embryo handling and evaluation.)
- Transfer the oocytes to physiological saline in a small (35 x 10 mm)
holding dish and add 10 percent of serum for ease of handling.
TRANSFER
- Load ten oocytes into a 0.25-ml straw by first aspirating a 10-mm
column of medium, next leaving a 5-mm column of air and then
aspirating ten oocytes.
- Place the 0.25-ml straw into an AI syringe or transfer device and
transfer all ten oocytes to a predetermined horn of a practice animal.
“EMBRYO” RECOVERY
- Using standard flushing techniques, practice recovery of the ten
oocytes from the practice animal with the aid of an embryo filter.
DRESS REHEARSAL
- Now you are ready to superovulate and breed a number of practice
animals to exercise your skills in actual sequence.
- The exercises may be made more difficult by using heifers with a
smaller cervix.
- Spontaneously ovulating animals may be used to practise recovery
of single embryos. This offers the advantages of all-or-none recovery,
and avoids the expense of the superovulatory treatment (FSH).
