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Assisted
Reproductive Technologies for Horses:
Embryo
Transfer
By Dr. Simon Robinson
Embryo
transfer can extend the breeding life of old mares and produce foals from
mares with a history of abortion. There are, however, a number of factors
to consider before using ET to produce a foal from your champion mare.
The first successful embryo transfers in horses were performed over 25
years ago. However, it has only been in recent years that embryo transfer
has become widely accepted as a means of producing foals from a variety
of breeds. In the United States and Europe, the use and availability of
embryo transfer services has increased greatly throughout the last decade.
Reasons
for using Embryo Transfer
To the wider horse world, there are a number of reasons to use embryo
transfer to produce foals from top class mares. To many performance mare
owners, the most appealing aspect of embryo transfer is that they can
produce foals from their mares without disrupting their training and competition
schedules. Other advantages include producing foals from older mares who
are no longer able to carry their own foals; obtaining pregnancies from
late foaling mares, allowing them to be bred early in the next season;
and producing more than one foal from elite mares in a year. However,
not all breed societies allow the use of embryo transfer in all of these
situations.
Breed
Regulations: What are we permitted to do?
Before using embryo transfer to produce a foal from your mare, it is wise
to consult your breed registry regarding the use of this technique. The
Arabian Horse Society of Australia Limited have only recently adopted
a new set of regulations regarding the use of' embryo transfer. The regulations
are quite clear but demand a considerable amount of paperwork, which is
essential if the foal is to be registered with the Society. Essentially
the new regulations allow breeders to produce a single, foal per year
by embryo transfer, but only from mares which are unable to carry their
ovn foal. This needs to be verified in writing by it veterinarian, and
approval to perform the procedure needs to be sought BEFORE you start.
A further requirement is that the donor mare and stallion be blood-typed
prior to the mare being served or inseminated and the foal needs to be
blood-typed immediately after birth. This blood-typing is likely to become
obsolete when the simpler and less expensive method of flair follicle
DNA testing becomes available. The visual identification of the recipient
(surrogate) mare needs to be carefully recorded at the time the embryo
transfer is performed and this needs to be verified by a veterinarian
when the foal is blood-typed. Although it is ultimately the responsibility
of the mare owner to satisfy all the requirements of the Society, veterinarians
who perform embryo transfers should be able to guide and assist to ensure
that the resulting foal may be registered.
So
is your mare a candidate for Embryo Transfer?
Ask yourself these questions:
- Is your mare empty when you wanted her in foal?
- Is your mare old? · Has she ever aborted or slipped a foal?
- Does your mare take all season to get in foal….and then lose it?
- Has she ever had an infection or difficult foaling which may have
scarred her uterus?
If you answered yes to any of these questions, embryo transfer is likely
to be a solution to producing your next foal.
Embryo transfer involves the collection of an embryo from a donor mare
and transfer of that embryo into a suitable recipient mare. The donor
mare is bred normally using fresh, chilled or frozen semen. Seven days
after she ovulates, the embryo, which is less than half a millimetre in
diameter, is flushed out and implanted into a recipient mare. An important
point is that the resulting foal is the product of the genetics of the
stallion and donor mare. The genes of the recipient or surrogate mare
have no influence on the foal she carries, she merely provides an ideal
environment for the developing foal the grow.
Synchronised Cycling
The first step an embryo transfer program is to synchronise the reproductive
cycles of the donor and recipient mares. It is critical that the recipient
mare is at the same stage in her cycle as the donor mare. This ensures
that the recipient mare's uterus is ready to accept and nurture the transplanted
embryo. This sounds simple enough, but success is dependent on considerable
veterinary input and experience. The ovaries and reproductive tracts of
the donor and recipient mares will need to be examined using an ultrasound
scanner. From this examination, the veterinarian can establish what stage
of the cycle each mare is at. To improve the chances of having the mares
synchronised, it is better, to have at least two recipient mares for each
donor mare. Furthermore, to assist in achieving accurate synchronisation
of the donor and recipient mares the veterinarian may also use hormones
such as Regumate and prostaglandin to control the mares' reproductive
cycles, and human chorionic gonadotrophin (hCG; Chorulon) or GnRH (Ovuplant)
to induce ovulation. Further examinations are necessary to determine the
correct time to breed or inseminate the donor mare, and the day of ovulation.
Ideally, the recipient mare must ovulate on the same day or up to 48 hours
after the donor mare.
It is important to know the day of ovulation as this dictates the day
on which the mare will be flushed to recover the embryo. In the mare,
the embryo doesn't enter the uterus, from where it can be recovered, until
around day 6 after ovulation. Once the embryo is in the uterus it undergoes
a rapid period of' expansion, with its size increasing more than tenfold
in three days.
Therefore, if the donor mare is flushed too early, it is likely that the
embryo will not yet be in the uterus, and if she is flushed too late,
the embryo may well be too large to handle and transfer without causing
damage. Synchronisation and timing are critical for successful embryo
transfer.
Embryo Recovery
On day 7 or 8 after ovulation, the uterus of the donor mare is flushed
to recover the embryo. This is a non-surgical procedure performed while
the mare is standing and usually doesn't even require sedation. Firstly
a soft catheter is inserted through the cervix into the uterus and a balloon
cuff is inflated around the catheter to form a seal around the cervix
to prevent any fluid loss. The uterus is filled with 1-3 litres of a warm
saline and protein solution which is allowed to flow in by gravity. Once
it is full, the uterus is gently massaged through the rectum to ensure
the embryo is released into the fluid. The fluid is then allowed to flow
out of the uterus and passes through a very fine in-line filter which
captures the embryo. This procedure is repeated two or three times unless
the embryo is spotted in the filter after the first flush. Depending upon
the, time of the day the mare is flushed, the embryo may be identified
as a perfect translucent sphere. Once the embryo is recovered, it is washed
and examined using a microscope. The embryo can be held in a 37'C holding
solution for a short period prior to transfer, but should be transplanted
into the recipient mare as soon as possible.
The recovery rate of embryos from mares generally reflects the conception
rate. This is dependant upon the age and reproductive status of the donor
mare, and on the type and quality of semen used to breed the mare. The
recovery rate from older mares and those that are normally difficult to
get in foal is lower than for young healthy mares, which is usually 70-80%.
Similarly, use of frozen or poor quality semen results in a lower recovery
rate than good quality fresh or chilled semen
One of the major commercial drawbacks with embryo transfer in horses is
that the horse cannot be reliably induced to superovulate as is the case
with cattle. Therefore mares are only able to produce one or two embryos
each cycle.
Is
it safe for my mare?
The flushing procedure only takes about 30 minutes and causes little discomfort
to the mare. More importantly, this is the end of the donor mare's involvement
in the embryo transfer program. Large embryo transfer programs in Argentina
and Brazil have shown that repeated flushing of mares to recover embryos
year after year has no effect on the mare's reproductive ability or performance.
And with a little planning, the disruption to a performance schedule by
the embryo transfer program should be minimal.
Embryo Transfer
The embryo may be into the uterus of the recipient mare either surgically
or non-surgically. Non-surgical transfer is the simplest and quickest
method involving the least discomfort to the mare.
For this method the embryo is loaded into a sterile plastic transfer pipette,
similar to an AI pipette. The transfer is performed with the mare standing
and is similar to performing artificial insemination, but requiring considerably
more skill and cleanliness. The mare's vulva is cleaned thoroughly rising
an iodine solution and the transfer pipette is covered in a sterile sheath
which guards the pipette as it passes through the vagina, and is torn
once the pipette is into the cervix.
This ensures that contamination is not carried into the uterus. The veterinarian,
wearing a sterile glove, will carefully introduce the pipette into the
cervix and pass it through to the uterus, causing minimal trauma on the
way through. The embryo is then deposited into one of the uterine horns.
Non-surgical transfer has a 60-70% pregnancy rate, which is 5-10% lower
than surgical transfer. The success is highly dependent on the experience
and skill of the operator and on the anatomy of the recipient mare.
Surgical transfer is most commonly performed with the recipient mare standing
and sedated. A small incision is made into the flank of the mare and the
uterine horn is carefully exteriorised. A small puncture is made through
the wall of the uterus and the embryo is deposited into the uterus using
a fine sterile pipette. The uterus is then replaced the abdomen and the
wound is sutured. The mare usually recovers rapidly and uneventfully.
Once mastered, this method of transfer is quite rapid and results in pregnancy
rates of 70-80%.
Recipient selection and management is the primary determinant of a pregnancy
following embryo transfer. The most suitable recipients are large, young
(4-10 years) mares with a normal reproductive history, and preferably
having had at least one foal. For non-surgical transfer, the temperament
of the mare and the anatomy of the cervix are also very important.
Six days after the transfer, the recipient mare may be examined for pregnancy
using an ultrasound scanner. It is useful to follow up this initial pregnancy
test with further tests at around three weeks and five weeks after transfer.
At these stages, the veterinarian can see a foetal heart beat and establish
that the foetus is growing and developing normally.
What about the costs?
The costs involved in embryo transfer are often a major deterrent. The
cost of embryo transfer varies between practitioners, and depends on the
number of attempts required to obtain a pregnancy. The cost can as little
as $1,000, but an average is $2,000-$2,500 per pregnancy. Embryo transfer
requires considerable expertise, both in the reproductive management of
the mares and in performing the procedures. Other than professional time
and expertise, the main cost associated with an embryo transfer program
is maintaining a group of' suitable recipient mares. Often clients are
able to provide a recipient (or two) for their donor mare. This will significantly
reduce the cost, but the recipients will need to be screened for suitability.
It is not worth using a poor recipient to save money as the chances of
getting a pregnancy are reduced.
When added to the service fee embryo transfer can be a relatively expensive
method of creating a foal. However, the costs need to be weighed up against
the benefits of breeding from elite mares while they are still competing
and from older proven mares. If we are to improve the quality of our horses,
the cost of producing quality foals has to be seen as an investment in
the future. Moreover, for many mare owners, the costs of embryo transfer
are low compared to the expense of buying another advanced horse to compete
with while breeding from another.
Embryo Storage and Transport
The collection of embryos at one venue and shipment to a centre for transfer
has become quite popular in the United States. In this situation, the
mare remains at home and is examined, inseminated and flushed by a local
veterinarian who has been trained in embryo collection. The embryo is
then placed in a special culture medium and cooled to 5'C in a refrigerated
Equitaner. The embryo is transported to the central laboratory within
24 hours of collection for transfer to a recipient. Such a system is ideal
in a country the size of Australia, but is limited as the number of veterinarians
experienced in embryo collection and transfer is low.
Although it is possible to successfully freeze equine embryos, it is uncommon.
This is because the pregnancy rates achieved following transfer of these
embryos tends to be quite disappointing. Small day 6 embryos are most
tolerant of freezing and thawing, but the recovery of these embryos is
low. Considerable research is being conducted to improve the methods for
freezing larger embryos.
A further impediment to the widespread freezing and transport of embryos
is that many breed societies only permit embryo transfer involving freshly
collected embryos. With improvement of freezing methods, breed societies
should reconsider their position as there are certainly a member of breeds
in Australia that would benefit greatly from the introduction of frozen
embryos collected from top performing mares in Europe and North America.
New Technologies
There have been some recent developments in assisted reproduction for
horses which broaden our ability to produce foals from sub- or infertile
mares and stallions. Multiple eggs can be harvested from the ovaries of
mares using a needle guided by ultrasound. These eggs call then be harvested
in an incubator and fertilised in vitro (in the laboratory) using a technique
called ICSI - IntraCytoplasmic Sperm Injection. ICSI refers to the injection
of a single sperm into an egg to achieve fertilisation. ICSI was developed
in Belgium about 10 years ago for the treatment of male-factor infertility
in humans, and is now the technique of choice in human infertility or
IVF clinics where the male partner is unable to produce large numbers
of good quality sperm. ICSI itself is a fairly simple procedure once mastered.
It requires considerable skill and experience, and a set of very precise
micromanipulators. Once the egg is fertilised it is cultured for a short
period and then transplanted into the oviduct of a recipient mare using
a surgical procedure.
Throughout the world, only a handful of foals have been produced using
ICSI and at present there are only two centres in Australia which offer
this technique. This situation is unlikely to change in the short term
due to the skill and expertise required to successfully perform all steps
in the procedure and the cost of the equipment required. However, for
the horse, ICSI offers a means of producing a foal in cases where there
are limiting numbers of normal motile sperm (from subfertile stallions
or from poorly frozen semen) or from mares which fail to conceive due
to blockages in the tube which carries the egg into the uterus.
Embryo transfer offers a perfect solution to owners of competition mares
who are debating whether to breed or carry on competing. The improvement
of many breeds is impaired because there are too few competition mares
being used for breeding, and often those mares that have had long and
successful careers are too old to maximise their breeding potential. With
such a large proportion of the breed's genetic input coming from overseas,
the Arabian Horse Society should consider taking the next step of allowing
embryo transfer from healthy competition mares.
For further information on embryo transfer, please contact Dr Simon Robinson
at Alabar bloodstock by phone on
(03) 5859 2201, fax (03) 5859 2206 or by Email : alabar@alabar.com.au
HORSE
REPRODUCTION AND EMBRYO TRANSFER GLOSSARY
Donor
Mare: Mare from which an embryo is recovered for transfer into
a surrogate (recipient) mare.
Embryo Transfer:
The procedure in which an embryo is recovered from one mare seven
days after ovulation and transferred into another mare.
ICSI (IntraCytoplasmic Sperm Injection):
Injection of a single sperm into an egg to produce an embryo. This is
a form of in vitro fertilisation.
Ovulation: Release of the mature egg from the ovary. The egg is transported
to the oviduct or fallopian tube where it is fertilised and develops into
an embryo. Ovulation can be induced in horses by injectiong Chorulon (hCG:
human chorionic gonadotrophin) or by inserting an Ovuplant (GnRH) implant.
Ovum Pick-up:
Harvesting of eggs (ova) directly from the ovaries using a needle guided
by ultrasound. These eggs may be cultured and fertilised in vitro.
Recipient Mare: surrogate mare into
which an embryo is transferred. The recipient will carry the foal and
nurture it as its own after birth. The recipient has no genetic influence
on the foal.
Ultrasound Scanner: A machine with
a transducer which emits and receives sound waves. It is used to visualise
the reproductive tract and ovaries via the rectum.
Uterus: The
part of the female reproductive tract into which semen is deposited during
AI and in which the pregnancy forms.
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