BBMay

FEATURE - ARTICLE
Advances In Assisted
Reproductive
Techniques
For many years, artificial insemination
using chilled and frozen semen has
been the most widely used assisted
reproductive technique(ART) in the
horse-breeding industry.
Assisted Reproductive Techniques:
- AI Fresh, Chilled and Frozen
- Embryo Transfer
- Ovum Pickup (OPU) - OT, IVF, ICSI
- & embryo sex determination
- Cloning
- Embryo and Oocyte Freezing
Embryo transfer(ET) in cattle has been
well established for well over twenty
years as a way to increase the number
of progeny per year from a genetically
superior animal. The use of embryo
transfer in horses has been much
slower to develop, partly due to a lack
of commercial/economic incentive,
combined with a much lower yield of
embryos. Cattle can be successfully
super-ovulated (given a program of
drugs similar to women having IVF) ,
producing up to 20-30 embryos per
flush, compared with horses where
only one embryo per flush is expected
unless the mare has ovulated from both
ovaries. The most relevant and useful
application of ET is in the production
of off spring from mares still competing
which increases the ability and
likelihood of improving the genetics
of equine athletes. This seems like an
ideal scenario when previously mares
were only bred once they were older,
retired and less fertile, reducing the
chance of producing many offspring.
This is not always as easy as it sounds,
and sometimes owner expectations
exceed what can be achieved in reality.
Exercise, heat and stress can disrupt
ovarian function while frequent joint
injections have been seen to lower the
overall fertility of mares and stallions.
Furthermore, some disruption to
exercise schedule should be expected,
although every effort is made to plan
breeding around the mare’s work
schedule.
The process of ET involves the donor
mare being bred with either fresh,
chilled or frozen semen; fertilization
takes place within the mare’s fallopian
tube and the developing embryo is
present in the mare’s uterus by the
sixth day following fertilization. Once
the embryo is in the uterus it can be
flushed out using special tubing through
a cup with a filter; this is usually done
on day 7-8 after ovulation depending
on mare age, time of year and type
of semen used. The gaps in the filter
are smaller than the smallest embryo,
therefore the cup will catch and
retain the embryo during the flushing
process. The contents of the cup is
examined under a microscope and
the embryo which is approximately
the size of a spec of dust is located,
washed and held in a special media
until the recipient mare is prepared
for transfer. It is ideal to select a
couple of recipients for each donor
mare flush to increase the chances of
perfect synchronicity, and provides us
which a choice so we can select the
best recipient on the day and therefore
increase the chances of the transfer
being successful. Studies show more
success with transfer when recipients
have ovulated two days after the donor
mare. ET is widely available throughout
the UK although a high level of skill is
needed to achieve high transfer and
pregnancy rates.
Embryos can be frozen using a method
called vitrification to be stored
indefinitely for the preservation
of genetics or until recipients are
available for transfer. It involves
transferring a small, day 6 - 6.5
‘morula’ stage embryo through varying
concentrations of antifreeze, before
submerging it in liquid nitrogen. Size
of the embryo is critical for success,
therefore it is important to have a back
up plan (i.e. a recfipient on stand-by)
if the embryo that is flushed is too
big, as this will probably not survive
the freezing and thawing processes.
A decreased pregnancy rate following
thawing and transfer is expected with
every frozen embryo, however this can
be limited by using an experienced
technician. The next step in breeding
technologies beyond ET needed to
provide many advantages over embryo
transfer to warrant the ensuing
research needed for development and
the extra costs involved in making it
commercially viable. Ovum pick-up
(OPU) ticks all the boxes!
Advantages Of Opu/Icsi Over ET
1. Donor mares can have one-off
visits to collection center when it
suits work schedule.
2. OPU can be done outside the
normal breeding season; in fact
autumn and early spring are often
the best times to get a high yield
of oocytes.
3. Infertility issues in the mare
where no embryos are retrieved
using conventional ET e.g.
inability to ovulate/ poor uterine
environment/ cervical tearing do
not affect the OPU process.
4. Embryos can be produced using
semen from stallions with low
fertility or very limited stocks of
semen using ICSI once oocytes are
collected using OPU.
5. Potentially more embryos can
be produced per year (this will
hopefully improve as the OPU/ICSI
processes improve with practice
and research).
6. Embryos are usually frozen after
OPU/ICSI and can therefore be
transferred into recipients when
desired; synchronizing with donor
unnecessary.
OPU is where the oocytes (ova or
eggs) are aspirated directly from the
ovary, either transvaginally or transabdominally
and identified under a
microscope, washed and transferred
into a special medium ready for further
processing. Trans-vaginal OPU is the
most commonly used technique now; a
long rigid ultrasound probe, held within
the vagina against the ovary, acts as
a visual guide for an attached needle.
Once each follicle is visualized the
needle is advanced and the follicle is
repeatedly flushed with a solution using
a pump. 15-20 follicles may be flushed
from the two ovaries during a single
OPU session, yielding anything from
0-20 oocytes. A large dominant
follicle which is close to ovulation
yields a mature oocyte. As previously
mentioned and similarly to ET, only
1-2 mature oocytes are available per
flush if only dominant follicles are
flushed. However, many immature
oocytes can be harvested from
multiple smaller follicles. Furthermore,
immature follicles are much more
stable and easier to transport for
further processing. A donor mare in the
transitional phase in early spring and
late autumn have the ideal ovaries for
harvesting as many oocytes as possible
each flush. OPU can be carried out
every 2-3 weeks. Oocyte transfer(OT)
has been used in the place of ET for
mares with reproductive issues which
results in no embryo production.
46 | BRITISH BREEDER