Embryo Transfer Technology In The Cow
Application
Over the years, techniques associated with embryo transfer (ET) have had
many uses, especially in research. The widespread use of this technology in
animal breeding schemes, however, is relatively recent. Genetic engineering
and related technologies will only increase its utilisation. A few of the more
common uses of ET technology in animal production follow.
Genetic Improvement
Genetic progress is considered to be slower through the use of ET than it
is using conventional artificial insemination (AI), especially on a national
herd basis. However, with increased selection intensity and shortened gestation
intervals, i.e. transferring female offspring, genetic gain can be made on a
within basis. In monotocous animals like the cow, the production of about six
offspring per donor could double selection intensity and the rate of response
to genetic selection for traits such as growth that can be measured in
both sexes. ET is now commonly used to produce AI sires from the very best proven
cows and bulls available.
Planned Matings
By far the most common use of ET in animal production programs is the proliferation
of so-called desirable genotypes. AI has permitted the widespread dissemination
of a male’s genetic potential. ET provides the opportunity of disseminating
the genetics of proven elite females. ET also permits the development of herds
of genetically valuable females, most of which may be sibs if not full sibs.
As AI has let to the very valuable bull, now ET has resulted in the very valuable
female. Many breeders have identified individual females whose offspring are
most saleable and used them exclusively in ET. ET has also been used to rapidly
expand a limited gene pool.
Disease Control
Infectious diseases in the bovine species seems unlikely to be transmitted
by the embryo. Consequently, it has been suggested that ET be used to salvage
genetics in the face of a disease outbreak. However, much research has yet to
be done on embryo-virus interactions before this technique can be done with
complete confidence.
Import & Export
The intercontinental transport of a live animal may cost $2,000 or more, whereas
an entire herd can be transported, in the form of frozen embryos, for
less than the price of a single plane fare. This may be the single most important
potential application of ET. Additional benefits of the export of embryos over
that of live animals includes a wider genetic base from which to select, the
retention of genetics within the exporting country and adaptation. This is particularly
true of tropical and subtropical climates where the embryo would have the opportunity
to adapt both in and then on a recipient indigenous to the area.
There are several potential problems which must be overcome in order to make
the international movement of embryos commonplace. Firstly, this use is dependent
on the successful freezing of embryos. Secondly, the inadvertent introduction
of disease into a herd and/or country with or within the embryo presents some
very difficult regulatory problems. Well defined methods of collection, handling
and washing embryos must also be followed to ensure that disease transmission
is avoided. Finally, the international movement of embryos is heavily dependent
on technology transfer as personnel within the importing country must be able
to successfully thaw and transfer embryos.
DONOR SELECTION
Every breeder will have his own reasons which are more often economic than
genetic for wanting to do ET on a given animal. As optimal results will reduce
costs, making the procedure much more economical, donor selection may involve
a previous history of success in ET. In addition, it has been suggested that
the potential donor animal be at its prime reproductive age, that it has a previous
history of a high level of fertility and that it has demonstrated superiority
in traits of economic selection. Strict selection criteria will not only ensure
genetic superiority, but should also ensure a high level of success thereby
making the
procedure more economical.
EMBRYO TRANSFER IN THE COW
ET technology has been most extensively applied in the bovine species. Not
only is there an economic incentive, but the cow is well suited to complete
utilisation of non surgical techniques making the procedure more cost-effective.
Finally, the cow is easy to work with making cattle excellent experimental animals.
Although it is possible to collect single embryos from the cow with good success
and through the use of prostaglandin (PGF) to do so every 10 days, most cows
are superovulated prior to embryo collection. Recipients are treated with PGF
12-18 hours prior to the donor cow so the estrus will be synchronous between
donor and recipients. As multiple ovulations have been shown to take place over
as long as 24 hours, multiple inseminations are usually done.
The technique of embryo collection involves the passage of a cuffed rubber catheter through the cervix and into one of the uterine horns on days 6 to 8 after estrus. Embryos are then collected by a closed continuous or interrupted flow system, or by an interrupter syringe technique. Embryos are located with a stereoscopic microscope after settling and syphoning or aspiration, or after filtering through a plankton filter.
Although embryos are usually transplanted as soon as possible after collection, it is possible to culture embryos for several hours at 35-37 degrees C. If transfers are to be done soon after collection, embryos may be maintained at room temperature. It is also possible to cool bovine embryos and to maintain them in the refrigerator for 2-3 days. As a final alternative, embryos may be frozen for use at a later date.
Embryos are normally cultured in the same or a similar medium to that in which they are collected. Embryos are classified and evaluated by morphological examination at 50-100X magnification. The overall diameter of the bovine embryo is 150-190 um including a zona pellucid thickness of 12-15 um. Generally, the embryo is described as to its stage of development and quality. The best predictor of an embryos viability is its stage of development relative to what it should be on a given day after ovulation. Embryos of good and excellent quality and at the developmental stages of late morula to blastocysts yield the best pregnancy rates. It is advisable to select the stage of embryo for the synchrony of the recipient. Transfers of embryos in the cow can be made with good success only if the preceding estrus in the donor and recipient occurred within 2 days of each other.
Alternatively, recipients must be synchronous with the stage of development of embryos that had been frozen previously. Most recipients are chemically synchronised regardless of whether embryo transfers are to be done at an ET centre or “on farm”. Until recently, most embryo transfers in the cow were done surgically, whereas, presently most are done using non surgical methods, increasing the utilisation of this technology in cattle breeding schemes because of even less elaborate requirements.
Success Rates
With existing technology, an average for each donor cow superovulated would
be 8 to 10 ova collected, 6 to 7 embryos transplanted and 3 to 4 pregnancies
resulting. It must be emphasized that very few donor cows are average. (Willsbro
donor cows have averaged 16 viable embryos per flush).
Pregnancy rates are generally around 60% with fresh embryos and range from
30% to 40% with frozen embryos. (Willsbro pregnancy rates average 67% from
both fresh and frozen embryos).
Long Term Preservation of Embryos by Freezing
Successful embryo freezing has many applications in ET programs. Firstly, recipient management is improved and made more cost effective. In addition, season of calving can be controlled, even though embryo collection and freezing may take place year round. Embryo freezing also allows progeny and performance tests of sibs to be conducted more rapidly and efficiently. Further, full sibs or identical sibs can be frozen until the genetic worth of those transferred can be established. Finally, embryo freezing is necessary for international movement of embryos because it eliminates critical timing and allows disease testing while the embryos are held in quarantine.
Basic Principles
The freezing of a living cell constitutes a complex physiochemical process
of heat and water transport between the cell and its surrounding medium. There
exists an optimum cooling rate for each cell type. It is dependent on the size
of a cell, its surface to volume ratio, its permeability to water, and the temperature
coefficient of that permeability. To avoid intracellular freezing, embryos must
be cooled at 1 degree C/min, or slower. However, too low a rate of cooling can
also damage cells.
The required thawing rate depends on the freezing regime used. The most recent
technological improvement allows embryos to be thawed as simply as semen. Embryos
are normally stored in liquid nitrogen at -196 degrees C. Consequently, storage
times of 200 years or so are unlikely to produce any detectable reduction in
the survival of frozen embryos or cause genetic change.
With the advancement of embryo sexing and splitting technology, there is great
potential in animal production schemes. It is unlikely that anyone would have
predicted ten years ago that embryo transfer technology would have evolved to
where it is today.
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