Dr. Daels_Strategies to optimize breeding _20150913_114029

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International Conference of the Caspian Horse & 50th
Anniversary of the Rediscovery
Rasht, Iran 7-9 September 2015
Strategies to optimize breeding with small numbers of horses.
Prof. Dr. Peter Daels and Dr. Pouya Dini
Department of Reproduction, Faculty of Veterinary Medicine,
University of Gent, Ghent, Belgium.
Email: Peter.Daels@UGent.be or Pouya.Dini@UGent.be
1. Introduction
Increased mechanization has pushed back the numbers of some old native horse breeds to
levels that are endangering the genetic diversity within the breed and the future survival of the
breed. Several domesticated and wild horse breeds are at present endangered because their
numbers have reached a critical low limit. Through the careful planning of breeding with
diverging bloodlines, application of modern reproductive techniques and rigorous selection, some
breeds, such as the Friesian Horse, have been able to rebuild a sufficiently broad and diverse
genetic pool to safeguard the future of the breed.
In this review, we want to illustrate that there is currently a wide variety of artificial
reproductive techniques that can be used to help assure the genetic divergence and the
preservation of valuable bloodlines. These techniques vary widely in their technical complexity
and expense but relatively simple techniques such as the carefully planned use of insemination
with transported, cooled or frozen semen and the strategic use of embryo transfer can have a great
impact on the use of genetically valuable stallions and mares. Other more sophisticated
techniques can be applied in specific situations for the preservation and/or re-introduction of rare
bloodlines within a breed. These techniques include also the use of frozen semen, the storage of
frozen epididymal sperm after castration, embryo transfer, transport of cooled-fresh embryos,
storage of frozen embryos, pre-implantation gender determination, ICSI and the preservation of
cells for future cloning freezing. The use of artificial insemination and freezing of semen can
greatly increase the number of different stallions available for breeding and improve the available
choices for targeted breeding. On the other hand, it has been demonstrated that embryo transfer
can be a valuable tool to increase the number of offspring of genetically valuable mares without
compromising the fertility or athletic performance of that mare.
Although the horse was probably the first animal to experience and benefit from artificial
insemination, it took a long time to introduce the application of embryo transfer and other oocyte
and embryo-related modern breeding technologies. Improvements in semen extenders and
cryoprotectants have resulted in an increase in the transport and insemination of cooled and
frozen stallion semen, and parallel improvements in ovulation induction and synchrony,
exogenous gonadotrophic stimulation of multiple ovulations and simplified, more efficient
methods for non-surgical transfer of embryos to recipient mares have contributed to the
application of embryo transfer to all breeds and athletic types of horses worldwide. Cloning holds

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enormous promise not only for the Sporthorse industry to re-create champion horses but can also
be used in a strategy to ensure genetic divergence by re-introducing or multiplying animals of
genetic value.
2. Artificial Insemination
Artificial insemination (AI) in horses can be done with
semen collected on the studfarm for immediate use, but
maximum utilization of this technology is achieved when
mares are bred at considerable distances from where the
stallion stands. To achieve this, semen can be transported
at 4°C in a cooling container and stored for 12-36 hours
before it is used for insemination. The semen can also be
frozen and maintained in liquid nitrogen for several weeks
or years. Three factors must be considered in order to
achieve acceptable fertility with cooled, transported
semen: (1) the inherent good fertility of the mare, (2) the quality or fertility of the cooled,
transported semen, and (3) the reproductive management of the mare before and after
insemination. Proper application of artificial insemination (AI) in an equine breeding program
can dramatically improve operating efficiency and increase the availability of sires to the general
public. Very few breeds do not allow the use of AI, the largest exception being the Thoroughbred
(Jockey Club). Artificial insemination offers numerous advantages over natural breeding, for
instance: division of one ejaculate over several mares (5-20 insemination doses/ejaculate), the
transport of cooled semen to other locations where mares are housed and frozen storage of
semen. In addition, the potential for injury to the stallion and mare are decreased as compared to
live cover and the risk for the transmission of
venereal diseases can be greatly reduced by
avoiding direct contact between the mare and
stallion and by adding antibiotics to the semen.
Some disadvantages are inherent to AI programs.
The success of AI requires knowledge on the part
of the stallion manager because ejaculated semen
is very susceptible to environmental effects
(temperature, light, pH, etc…). Improper semen
collection, handling, processing, and insemination
technique can lower pregnancy rates. Therefore,
proper training of persons involved in the semen collection process is essential but fairly simple
to accomplish. The proper techniques, management and expected results using insemination with
fresh semen and cooled, transported semen are presented in another lecture at this meeting. In
the context of breeding with a limited number of horses that are spread out geographically, it is
important to realize the potential of artificial insemination when it is combined with techniques
for transportation and preservation. The use of cooled, transported semen in Europe has allowed

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breeders to choose stallions that are either at a long travel distance from the mare or are in active
competition. This has enabled breeders to match genetic superior mares and stallions and thus
make progress in genetic selection.
3. Use of transported, cooled semen
It is well established that semen from
normal fertile stallions can be stored at 4°C for
24h without a significant loss of fertility. To
optimize the results of cooled-stored semen, two
factors need to be respected. Immediately after
semen collection, the semen needs to be diluted
with a skim-milk based diluent for protection
against the detrimental effects of cooling. Several
very effective semen extenders are now on the
market for the preparation of cooled semen. The
cooling curve of the semen from 37°C to 4°C
needs to be controlled and the ideal cooling curve
is not linear but rather is divided in three temperature zones for optimal fertility: 37°C to 19°C
rapid cooling; 19°C to 9°C slow cooling (-0.05°C/min) and 9°C to 4°C rapid cooling. Specially
designed transport boxes for equine semen are available and offer the best cooling rate. These
cooling boxes are relatively inexpensive, effective and can be used multiple times. One must
keep in mind that not all stallions have semen that can be cooled without loss of fertility. In some
stallions, the choice of an adapted semen extender can increase the fertility but other stallions
have semen that does not tolerate cooling for unknown reasons. A wide range of different semen
extenders is commercially available and it is worthwhile to test several extenders when testing
the fertility of cooled semen for a particular stallion. In isolated cases, removal of the seminal
plasma by centrifugation and dilution of the spermatozoa in an appropriate extender may increase
the longevity during cooled storage in problem stallions.
Insemination with cooled semen is relatively simple
as the cooled semen can be inseminated directly into the
uterus of the mare without pre-warming. Cooled-stored
semen from a stallion with normal fertility is considered to
have a fertile lifespan in the mare of about 48h. Thus mares
inseminated with cooled, transported semen need to be
inseminated only once if ovulation occurs within 48h after
ovulation. Under routine management of the mare and with
the use of ovulation-inducing agents (such as hCG or GnRH)
this is easily accomplished in the majority of mares.
4. Use of frozen semen

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While artificial insemination and cooled storage offer the capability to
increase the number of mares that can be bred to one stallion and also
increases the geographic area wherein the stallion can be used to breed
mares, it remains limited in time to a maximum of 36-48h between
semen collection and insemination. Cryopreservation (freezing) of
equine semen offers the added advantage that the storage time is
unlimited and thus the opportunity for transportation is also unlimited.
Freezing semen can be applied to transport semen over larger distances,
to store for a long time, or to construct a gene bank of stallions that are
genetically important for the breed either because of their particular
phenotype or because of their genetic diversity. Because of the
unlimited storage capability, frozen semen also allows for better
protection of disease transmission, because necessary tests can be
carried out while the semen is being stored. This last point makes export of frozen semen
possible whereas that is practically not possible for cooled, transported semen.
With all the benefits of frozen semen
come also several disadvantages. The
preparation of frozen semen is more time
consuming, requires more training, more
expensive equipment for freezing and a system
for storage that necessitates the regular
availability of liquid nitrogen. In addition, unlike
semen from the bull, stallion semen does not
tolerate very well cryopreservation and a
significant drop in the number of viable and
fertile spermatozoa during the freezing and
thawing procedure is commonplace for the stallion. Whereas bulls have been selected for the
freezability of the semen, this is not the case for stallions. This has resulted not only in a
significant loss of viable spermatozoa during the freezing procedure but also results in some
stallions having semen that simply cannot be frozen. The reasons for this total loss in viability
and fertility after freezing and thawing for some stallions is still an enigma. As the freezability of
individual stallions cannot be predicted based on other parameters this signifies that each stallion
needs to be tested individually for the freezability of his semen. Another limiting factor that is
associated with the use of frozen semen is the limited survival time after thawing. For most
normal stallions, spermatozoa will remain viable and fertile after insemination of the mare for a
period of about 12h (maximum 24h). Because of
this limited survival time after thawing,
frozen/thawed semen must be inseminated within
6-12h before ovulation. Alternatively, mares can
be inseminated immediately after ovulation but
because of the limited survival of the unfertilized
oocyte, post-ovulatory insemination must be

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performed within 12h after ovulation. Consequence of this is that insemination with
frozen/thawed semen requires a more intensive management of the mare. Mares that are in heat
and close to ovulation must be monitored for follicular growth and ovulation every 6-8h or 3-4
times per day to pinpoint the ideal moment for insemination. Failure to do so will often result in
disappointing pregnancy rates following insemination with frozen/thawed semen. The highest
pregnancy results with the smallest amount of frozen semen used are obtained when mares are
examined 3 times per day for ovulation and are inseminated immediately following ovulation.
However, other timed insemination protocols have been developed. The most popular protocol
requires the mare to be inseminated at 30h after induction of ovulation using hCG or GnRH and
again 24h later.
5. Freezing of epididymal semen
Freezing of epididymal semen consists in
the collection of the semen reserves that are
present in the epididymis of the stallion after
castration. This method is used occasionally in
stallions that die unexpectedly, in an effort to
preserve some valuable semen from the animal.
However, in the context of horse breeds that are
facing low numbers and lack of genetic
diversity among the stallions, this method
allows to bank semen from a large population of
young stallions destined to be castrated. This
frozen, epididymal semen can be used at a later
date to help diversify the genetic pool within
the race and as such contribute to the
reconstruction of a solid and diverse genepool. Technically, freezing epididymal semen collected
after a scheduled castration is not very challenging. However, the numbers of spermatozoa
obtained are small and thus the number of insemination doses is limited. For this reason, this type
of stored semen is best used in combination with in vitro fertilization (ICSI), a technique that
requires a minimal number of spermatozoa per fertilization. In the context of the preservation of
rare horse breeds, it may be advisable to store frozen epididymal semen in anticipation of more
efficacious and less expensive ICSI techniques in the future.
6. Embryo Transfer
Embryo Transfer (ET) in horses was started in the 1980s and fully developed to a
practical tool in the 1990s when the transport of cooled embryos became possible. The relative
slow development of embryo transfer in the horse is mainly due to: (1) the lack of a good
superovulation treatment protocol and (2) the relative difficulty to synchronize the donor and
recipient mares. For this reason, until recently it was only possible to recover one embryo per
cycle.

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A great deal of research has been done on the induction of
multiple ovulations in the mare. Mares do not respond to the hormonal
superovulation protocols based on porcine FSH that are commonplace
in cattle ET. Only equine LH and FSH are bioactive in the mare, and it
took a long time to develop recombinant equine LH and FSH.
Recombinant FSH and LH specific to the horse have been developed
and their efficacy has been demonstrated in mares. Also, due to the
very unique anatomy of the mare’s ovary, it will likely never be
possible to induce more than 3-4 simultaneous ovulations in the mare.
This limits the number of embryos that can be produced per cycle
compared to the large number that can be produced in cattle. While this
treatment currently is still expensive and therefor reserved for
exceptional mares, it will no doubt become more commonplace in the
future and be used to produce several embryos per cycle.
Because synchronizing donor and recipient mares remains unpredictable, researchers have
devised methods to transport embryos from the place where they are collected from the donor
mare to highly specialized embryo transfer centers where a large number of recipient mares are
available for implantation of embryos. Because of the large numbers of recipient mares available
in these ET centers the chance that a recipient mare will be available and in synchrony with the
donor mare is very high. This has led to the development of large recipient mare herds in North
and South America and Europa. Currently, likely more than 80% of all embryos that are
transferred are either collected on one of these centers or shipped to these centers for transfer in a
recipient mare at the ET center. The introduction of cooled, transported embryos has greatly
simplified the procedure and reduced the cost of housing recipient mares in small numbers at
many different places. With the current techniques, horse embryos can be cooled to 4°C and
shipped for up to 24h without any loss in viability and resulting in the same pregnancy rates as
those obtained for embryos collected and transferred immediately. Embryos are placed in a
specific embryo holding/transport solution that is commercially available from several suppliers.
The embryo is placed in a sterile tube filled with Embryo Holding Solution which is placed in
turn in a specially designed cooling container (similar to those used for cooled transported
semen).
In the mare, the embryo remains in the oviduct for approximately 6.5 days. Flushing the
mare before this time will not result in embryo recovery because the embryo has not arrived in
the uterus yet. Once the embryo is present in the uterus it will start to increase its diameter very
quickly making it impossible to flush and transfer embryos older than 8 days. Horse embryos
need to be collected on either 7 or 8 days after ovulation depending on the age of the mare and
the type of semen (fresh versus frozen) that is used. The timing of this embryo collection is
critical in the success. When using proper techniques/materials and working in a clean/sterile
manner, the procedure will have minimal effect on the future fertility of the mare. Mares have
been known to be collected 10 or more times in a given season and produce 8-10 embryos in one
year.

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The standard method of embryo collection in
the mare is a non-surgical transcervical uterine
lavage. For this purpose, the mare is first
carefully washed; a sterile silicone large
diameter catheter is introduced through the
vagina and cervix into the uterus. With the aid
of an inflatable balloon located at the tip of the
embryo flushing catheter (Bivona Catheter) the
catheter is held in place and the cervix is closed
off hermetically. The uterus is filled with 0.5
to 1.5 liters of embryo flushing solution and
this is then allowed to flow back out of the
mare through an embryo filter. This procedure
is repeated 3-6 times. After completion of the flushing procedure the embryo is then searched in
the filter with the aid of a binocular microscope. The embryo is subsequently washed in embryo
holding solution and then placed in a 0.25ml or 0.5ml sterile embryo straw. The straw with
embryo is then loaded in a transfer gun (Cassou gun) and carefully passed into the vagina and
very delicately advanced through the closed cervix of the recipient mare until the tip of the
Cassou gun reaches the lumen of the uterus where the embryo is deposited.
Theoretically, the ideal recipient mare has
ovulated 1-2 days later than the donor mare.
More practical approach is to state that recipient
mares should be preferably 6 or 5 days after
ovulation on the day an embryo is transferred
into their uterus. In addition, to a good transfer
technique also the fertility and health of the
donor mare has a very important impact on the
pregnancy results after transfer. When using
young, fertile and healthy recipient mares,
pregnancy rates after embryo transfer can be as
high as 80-85% and embryo mortality rate after
first positive pregnancy diagnosis is less than
10%, resulting in a total pregnancy rate of around
70%. Many researchers have compared pregnancy rate following transfer of fresh embryos and
cooled, transported embryos. Each time they have concluded that there is no difference in
pregnancy rate between the two types of embryos, making cooled transportation of embryos a
viable option.
Transfer of equine embryos can be performed by asurgical or a non-surgical transcervical
approach. Today, nearly all embryos are transferred non-surgically into the uterus of
synchronized recipients. Non-surgical ET has generally been performed using a reusable stainless
steel “insemination gun”. An outer guard is generally placed over the transfer instrument to

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minimize contamination of the uterus. The embryo can be deposited in the uterine body or in one
of the uterine horns. The complexity of ET is relatively low compared to more advanced
techniques, since embryos are transferred non-surgically into the uterus of recipients. The
difficulty of the procedure lies in the technical aspects of embryo searching and proper embryo
handling, the transfer procedure that must be performed in a sterile and atraumatic manner, and
coordination of the separate components that affect success rates, such as donor mare
management, recipient mare quality, management and synchronization, and transfer
technique/skills. Success of ET is calculated based on two variables: embryo recovery rate and
pregnancy rate after transfer of the embryo. Mean embryo recovery rate per cycle from single
ovulating mares in commercial ET programs should be approximately 65%, with higher embryo
collection rates for younger mares. In addition, pregnancy rate after non-surgical transfer of
embryos of excellent quality (Grade 1) should average between 80 and 90% at first pregnancy
diagnoses. Early embryonic loss rate is similar to normal pregnant mares and averages around
10%. Clinically, ET can be simplified by shipping the recovered embryo to a commercial ET
center, avoiding concerns regarding availability and synchrony of recipient mares and transfer of
the embryo. In addition, the process of cooling and shipping the embryos does not affect
pregnancy rates compared to embryos transferred immediately after collection. Common reasons
for using ET in a breeding program include: (1) obtaining foals from mares that are still active in
competition, (2) obtaining multiple foals from individual mares each year, (3) obtaining foals
from mares with fertility problems, (4) obtaining foals from young mares less than 3 yrs old and
(5) mares that cannot carry a pregnancy or give birth normally for reasons other than reproductive
health problems. Most subfertile mares referred to an ET program are older mares with poor
reproductive histories and unable to produce a foal by natural mating or AI. For ET to be
successful, the donor mare needs to meet several requirements. Ovaries must grow a follicle and
ovulate a healthy oocyte. Oviduct must be able to transport the gamete, support fertilization and
transport embryo to the uterus. The uterus must provide an adequate environment to support
embryo development until collection. The cervix must be patent and able to function properly
during early pregnancy. Mares with abnormalities that prevent conception or maintenance of the
early embryo, such as susceptible to post-breeding endometritis, irreparable cervical lacerations,
or uterine/oviductal scarring after dystocia, are not good candidates for ET. In these cases, mares
should be considered candidates for more advanced techniques such as ICSI
7. Intracytoplasmic sperm injection (ICSI)
Standard in vitro fertilization (IVF) is a technique that consists in placing an oocyte that is
recovered from a donor mare and spermatozoa together in the laboratory and stimulating
fertilization of the oocyte in vitro. This technique is commonly used in cattle and humans and is
relatively simple. For reasons that we still do not understand, the standard IVF does not function
in the horse. It appears that the equine spermatozoa are unable to penetrate and fertilize the
oocyte in vitro. Only two foals have ever been produced by this procedure in the early 1990s.
However, ICSI, a form of IVF, has provided a method to achieve fertilization in vitro in horses.
With this technique a single sperm is injected directly into the equine oocyte using
microscopically small glass needles. This technique is currently used with success in specialized

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centers in Europe, North and South America. Countless foals have been born in de past decade.
The technique offers two major advantages: (1) it allows producing embryos from mares that
would otherwise not be able to produce anymore and (2) it allows producing embryos with very
few spermatozoa and/or immotile spermatozoa. This last point is important when dealing with
stallions with fertility problems and stallions for which very little semen is available, for example
deceased stallions with limited frozen semen reserves.
Oocytes used for ICSI can be obtained ex vivo by aspirating the preovulatory follicle of a
mare. This oocyte is destined to ovulate very soon and is “mature”, ready to be fertilized in vivo.
Initially, only mature oocytes from preovulatory follicles were used for ICSI in horses. However,
today, “immature” oocytes collected from small immature follicles, are also used for ICSI. These
immature oocytes must be placed in maturation for 28-40hrs before they become competent and
can be fertilized using ICSI. This in vitro maturation of oocytes is an added step in the procedure
that has required some research to develop. The added advantage of using immature oocytes
from small follicles is that one can collect several oocytes per ovum pick up session, whereas
before one could only use the oocyte from the single preovulatory follicle.
One of the main advantages of ICSI is that it only requires a very small number of sperm
and, therefore, can be used to produce offspring from subfertile stallions with marginal-quality or
low numbers of sperm. When using frozen semen for ICSI, a small section of one frozen straw
can be thawed and used to produce multiple embryos, maximizing the use of valuable and rare
frozen semen.
Embryos fertilized by ICSI can be immediately
transferred into the oviduct of recipient mares by flank
surgery on the standing mare, but are preferably
cultured for 7–8 days to reach the blastocyst stage.
Blastocyst stage embryos can then be transferred non-
surgically using standard ET techniques into the uterus
of a recipient mare. Alternatively, embryos can be
cryopreserved and stored before transfer. The ICSI

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procedure is an extremely specialized technique that requires expensive instrumentation and an
experienced technician to perform the sperm injection into the oocyte. To date, the clinical use of
ICSI in horses has been limited to specialized centers due to the cost of the procedure and the low
success rates of oocyte collection and maturation. Recently, high oocyte collection rates (58%)
and oocyte maturation rates (66%) have been reported, resulting in an average of 10 oocytes
being collected per ovum pick up session in their commercial ICSI program. In well-established
centers, blastocyst development rates of 25–35% can be consistently achieved, with
approximately 50% pregnancy rates after non-surgical transfer of the in vitro produced embryos.
Future improvements in protocols for in vitro maturation of oocytes, as well as embryo culture,
will allow ICSI to be more cost-effective, increasing its use in equine practice.
8. Cryopreservation/freezing embryos
Freezing embryos before transferring to a recipient mother is
commonplace in cattle and humans. However, in the horse freezing
of embryos has been problematic until very recently. Equine
embryos arrive in the uterus fairly late in their development and
have often reached the early or even expanded blastocyst stage.
This has two consequences: (1) the embryo is now filled with fluid
in the center (blastocoel) and it is surrounded by a thin, stiff, semi-
permeable layer of glycoproteins, named “The Capsule”. Both these
characteristics render the equine embryo difficult to freeze. The
presence of fluid in the central cavity represents a great danger for
extracellular ice crystal formation, which is devastating for the
surrounding embryonic cells. The exact role of the embryonic
capsule in the horse embryo is still unclear but presumably it
provides structural strength to the spherical embryo. The major
problem in the context of freezing embryos is that the capsule is
almost impermeable to the cryoprotectant substances that are so
needed to protect the embryonic cells during the freezing process. Many attempts have been
made to make the capsule more permeable to the classic cryoprotectants but all have failed.
In recent years, two approaches have been used with
success. The first consists at collecting the embryo at 6.5
to 7 days after ovulation. At this stage, the embryo is still
in the morula stage and it is very small, does not have a
central fluid-filled cavity and has not developed a tight
capsule. Using vitrification, ultra-rapid freezing, horse
embryos have been frozen/thawed successfully and
commercial freezing kits are now on the market. The
procedure is still complicated and very delicate but with
adequate training and understanding of the critical steps it
can generate acceptable results. However, even under the best circumstances the pregnancy

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results using frozen/thawed embryos at the morula stage are still 10-20% below those obtained
with fresh embryos. In more recent years, a different approach has been introduced which
consists at collapsing blastocyst stage embryos before vitrification. It appears that removal of the
blastocoel fluid using an ICSI type needle and micromanipulator it is possible to remove the
largest part of the fluid. This greatly facilitates the vitrification of these larger embryos. It is
expected that in future years this method will be further developed and will result in acceptable
pregnancy rates.
9. Summary
In conclusion, many factors determine the choice of artificial reproductive techniques that
are used in a breeding program. Reasons to resort to more advanced techniques include the cause
of subfertility, the value of the animal, and the owner’s willingness to spend/invest considerable
time and money attempting to obtain offspring from their mares. The veterinarian has an
important role in educating the owner regarding risks and success rates of the technique to be
used, in order to avoid client disappointment. When advanced techniques are indicated to
improve the breeding efficiency of a breed or to circumvent certain fertility problems in
individual mares, the assistance of specialized centers should be integrated in order to maximize
the probability of success.

Dr. Daels_Strategies to optimize breeding _20150913_114029

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