Successful breeding of
horses is not always as easy as presenting a receptive mare to a
fertile stallion. There are many things that can go awry with
the reproductive process. Broodmares that were once fertile and
produced many excellent foals might one day face the realities
of repetitive foaling injuries, chronic uterine infections, or
simply old age. Stallions also can experience a decrease in
sperm number and quality as the years pass. Pain associated with
arthritic hocks or chronic back problems can interfere with a
stallion's ability to mate. When a mare or stallion develops any
kind of problem that interferes with its ability to perform
reproductively, it could be time to investigate the options of
assisted reproductive technology (ART). These are the techniques
that offer the hope of producing foals from mares or stallions
that might no longer be able to pass along their genes to future
generations any other way.
The Sub-fertile Stallion
Research has shown that maximum pregnancy
rates are achieved when a stallion's ejaculate is of sufficient
quality to allow a mare to be inseminated with 500 million
progressively motile sperm every other day during the five to
seven days of heat.1 Problems arise when semen
evaluation reveals either insufficient numbers or decreased
quality of sperm, with more abnormal sperm or fewer sperm that
can swim vigorously in one direction (progressively motile).
Therefore, methods have been developed to make the best use of
limited numbers of good-quality sperm.
Fertility With Fewer Sperm
Deep Uterine and Hysteroscopic
Insemination--As the name implies,
deep uterine insemination involves artificial insemination of a
mare deep within the uterus, inside the uterine horn adjacent to
the ovary with a pre-ovulatory follicle identified via
ultrasound. Only five to 25 million progressively motile sperm
are required for this method, because the long journey from
uterine body to uterine horn, during which many sperm divert
from the path to the egg and are lost, has already been
completed.
Sperm are taken even farther in the female
tract during hysteroscopic insemination, during which sperm are
deposited at the uterotubal junction, or the very edge of the
oviduct. Here, the sperm need only travel a short distance to
meet the egg or oocyte within the oviduct. Only one to 10
million progressively motile sperm are required.
Gamete Intrafallopian Transfer (GIFT)--GIFT is a newer technique in horses that involves the transfer of
both sperm and oocytes directly into a recipient mare's oviduct.
Obviously, the sperm needn't travel far, but must have the
ability to penetrate and fertilize an oocyte. A mere
200,000-500,000 progressively motile sperm are required.
Pregnancy rates ranging from 27-82% have been reported.2
Intracytoplasmic Sperm Injection
(ICSI)--When a subfertile stallion is
known to be the problem in a breeding pair and various
insemination procedures as well as GIFT have failed, there is
another ART technique that has produced several foals and
requires only minimal numbers of sperm.3 During
intracytoplasmic sperm injection (ICSI), a single sperm is
injected into a mature oocyte (one ready for fertilization).
Injected oocytes are then cultured in the laboratory until newly
formed embryos can be transferred to recipient mares. The ICSI
technique can provide a means of obtaining foals from stallions
with very low sperm numbers or poor-quality sperm.
Understanding Ovulation
It is important to understand the processes
of a normal estrus, or heat, in order to understand what can go
wrong with ovulation and interfere with fertility. Mares are
seasonally polyestrus, starting to cycle each spring as daylight
lengthens and continuing to ovulate approximately every three
weeks until fall, when the days get shorter. After a transition
period in the spring, the first ovulation is marked by the
appearance of a dominant follicle on one ovary, reaching a size
of approximately 35 mm. This follicle secretes estrogens, which
signal the start of an estrus period lasting five to seven days.
The dominant follicle continues to grow during estrus, reaching
a maximum diameter of about 45 mm. Toward the end of estrus,
there is a surge of luteinizing hormone (LH), which stimulates
ovulation. The empty follicle forms the corpus luteum (CL),
which secretes progesterone to support a new pregnancy that
might be developing. If no pregnancy occurs, 14-16 days after
ovulation the CL is destroyed in a process called luteolysis,
and the cycle begins again.
The Sub-fertile Mare
Unlike the subfertile stallion, identifying
a subfertile mare is not as easy as doing a semen evaluation.
Most subfertile mares have a history of occasional reproductive
underperformance or outright failure. The veterinarian will thus
perform a thorough history and physical examination to try to
diagnose the problem. What is the mare's age? We know that
fertility decreases after age 13 and drops precipitously after
age 20.4 Is she in good health? Is there evidence of
Cushing's disease or poor nutrition?
The mare's reproductive history is also
very important. If she has foaled in the past, she might have
stretching or scarring that has allowed pneumovagina, or air in
the vagina, to develop. This can promote bacterial entry into
the cervix and uterus. Examination with rectal palpation and
ultrasound might reveal interior tears, adhesions, scars, and
retained fluid that could be interfering with successful
breeding.
A complete examination will require samples
for uterine culture (to identify any bacteria present) and
cytology (evaluation of cells such as inflammatory cells that
might be present) as well as uterine biopsy specimens to examine
the endometrial lining. The uterus must be capable of supporting
the placenta and the developing fetus.
In terms of treatment, less serious
problems can be corrected or managed with remedies such as
careful selection of a highly fertile stallion, insemination
close to the time of ovulation, and perhaps the timed use of
oxytocin after insemination to help clear fluids from the uterus
and prevent infection. More serious problems might require ART.
Younger mares often have time on their
side. In other words, many of the problems causing subfertility
in mares six to 10 and even up to 15 years of age are related to
the tubular genitalia (vulva, vagina, cervix, etc.) and
anatomical changes of aging that cause the reproductive tract to
shift in position, allowing fluids and contaminants to interfere
with breeding. Luckily, many of these problems can be managed
conservatively. Even when more severe problems develop, younger
mares are usually better candidates for ART because fertility is
maintained in the ovary and oviduct, where many ART techniques
are aimed. Unfortunately, the same is not true for the older
mare.
Problems in the Older Mare
When a mare reaches 20 years or more, the
decrease in fertility experienced is directly related to
structural and functional changes in her reproductive tract.
Owners are sometimes confused when a productive broodmare
reaches her 20s and suddenly can no longer conceive, even after
delivering many foals in the last few years. But these
reproductive changes are due solely to aging and are independent
of past reproductive performance.
For example, the oviduct is critical during
the first five to six days of pregnancy. It collects the oocyte
from the follicle and sperm from the uterus, and provides the
environment for fertilization and early embryonic development.
Yet there is evidence that collection of the oocyte from the
follicle is less effective in older mares, disturbing the rhythm
of the entire process.5
The ovary also suffers from aging. During
the ovulatory season, older mares have been shown to have
sporadic ovulations instead of regular ovulations at 21-day
intervals. Research has shown that during these sporadic cycles,
aging mares begin a process where the ovaries fail to grow large
follicles, perhaps due to depleted reserves of oocytes. In one
study, only 50% of mares aged greater than 20 years had three or
more sequential ovulations during one season.6 Once
cycles become sporadic like this, it is typically only a few
years before reproductive senescence, or the end of a mare's
reproductive life, becomes a reality.
Embryo Viability--Problems with oocyte and ovary viability have prompted the question
of whether aged mares are reasonable candidates for embryo
transfer. For example, aged mares have had embryos removed for
transfer to younger recipient mares. Unfortunately, in general,
fewer embryos are collected from older mares during each
attempt, and these embryos tend to have more morphologic
(structural) abnormalities. Research has also shown that these
embryos tend to be delayed in their development, producing fewer
pregnancies and more embryonic loss.7
Efforts are underway to determine if
waiting an additional day before flushing the embryos from the
uterus allows time for aged mares' embryos to compensate for
delayed development. Even so, it is likely that there are still
defects in embryos collected from older mares because of
problems with the aged oocytes.
Oocyte Viability--There is growing evidence that oocyte defects might be of great
concern for subfertility in aged mares. These defects haven't
been specifically identified, but they are considered intrinsic
and age-related. Research has shown that when the same stallion
is used, oocytes from young mares (six to 10 years old) are
significantly more likely to develop into embryonic vesicles by
Day 12 (92%) than those from mares older than 20 years (31%), as
evidenced by ultrasound examination.4
Another study reported pregnancy rates of
54-83% for oocytes from young mares (less than 13 years old) and
27-40% for oocytes from aged mares (older than 20 years). These
older oocytes were often transferred into subfertile recipient
mares in a commercial program, yet still obtained about 30%
pregnancy or higher.8 These data again suggest that
older oocytes have lower fertility. However, even a 30%
pregnancy rate offers hope, and suggests that transfer of
oocytes from older mares into healthy young recipients could
hold promise.
ART Update for Sub-fertile Mares
New techniques are constantly evolving in
the field of assisted reproduction. Many times, the practical
applications arise from laboratory techniques developed for
experimental purposes.
Oocyte Transfer--Compared to embryo transfer, which requires a donor mare to
conceive and support an embryo for the first five to eight days
of pregnancy, oocyte transfer requires only the growth of a
pre-ovulatory follicle with a viable oocyte. Once transferred to
a recipient mare, fertilization and embryonic and fetal
development all take place within the recipient mare. Although
data suggest that mares greater than 20 years of age have
oocytes with decreased fertility, resulting in perhaps 30%
pregnancy rate, this might be one of the best options for
obtaining foals from aged mares.
Salvaging Oocytes--In this country, there is a limited source of ovaries from mares
(such as slaughterhouses) to collect oocytes from follicles for
experimentation. Therefore, when ovaries become available from a
mare through donation after euthanasia or some other means, the
immature oocytes can be collected, matured in culture, and
transferred to recipients. This technique is now being applied
more practically to salvage oocytes from ovaries of valuable
mares that die suddenly or must be euthanatized.
The ovaries can be surgically removed and
shipped to a facility at Colorado State University for oocyte
recovery and recipient transfer. Three foals have been born
since the technique was first attempted in 1999.4
Research continues to improve the protocol for transfer of
ovaries and maturation of immature oocytes, some of which was
presented at the 2004 American Association of Equine
Practitioners convention. For more information, see the AAEP
Wrap-Up next month.
Cryopreservation and Vitrification--The cryopreservation (cold storage) of oocytes or young embryos is
problematic, since there is a high surface-to-volume ratio of
these cells. The relatively slow process of traditional freezing
can disrupt the cellular structure, rendering it unable to
develop once thawed. Yet the use of frozen equine embryos has
resulted in successful pregnancies, and the equine embryo
structure plays an important role in that success. For example,
the equine embryo is unique in that it forms a type of
membranous protein capsule around itself that prevents
cryoprotectant from penetrating and water from leaving.9
Smaller embryos (less than 300 micrometers) have been frozen
most successfully, yet many breed registries won't register a
foal that is produced this way.
Vitrification is a process of rapid
freezing, and it has allowed oocytes to be successfully
preserved for later transfer. In 2001, the first two foals were
born from vitrified oocytes.10 One of the more
practical aspects of vitrification is that it allows for direct
transfer of frozen oocytes into a recipient, since the thaw is
so rapid. In addition, immature oocytes that are vitrified can
not only survive vitrification as well as mature oocytes, but
can continue the maturation process afterward with good results.3
Future Directions
There are some things that are still out of
reach for subfertile horses, even in the age of ART. Success
with in vitro fertilization (IVF) for example, long a
hope of the equine industry, has simply not materialized.
Superovulation (stimulating multiple follicles to ovulate at
once) in mares has been problematic. But embryo transfer
continues to improve and is in wide use. Oocyte transfer is now
providing the hope of producing foals from older mares.
Insemination techniques are becoming more refined and allow
greatly decreased numbers of sperm to achieve pregnancy.
The techniques of GIFT and ICSI are
successfully achieving pregnancies. Cloning has been achieved in
the equid. As with many other species, multiple laboratories are
working to produce pregnancies from cloned equine embryos. A
clone is simply an identical twin, so if this technique becomes
a reality, particularly valuable horses might be candidates for
cloning. Regardless, ART continues to advance, improving upon
older techniques and adapting newer ones to improve reproductive
success in horses.
REFERENCES:
- Householder, D.D.; Pickett, B.W.;
Voss, J.L.; et al. Effect of extender, number of
spermatozoa, and hCG on equine fertility. Journal of
Equine Veterinary Science, 1: 9-13, 1981.
- Coutinho da Silva, M.A.; Carnevale,
E.M.; Maclellan, L.J.; et al. Use of fresh, cooled and
frozen semen during gamete intrafallopian transfer in mares.
Theriogenology, 58: 763-766, 2002.
- Squires, E.L.; Carnevale, E.M.; McCue,
P.M.; et al. Embryo technologies in the horse.
Theriogenology, 59: 151-170, 2003.
- Carnevale, E.M.; Ginther, O.J.
Defective oocytes as a cause of subfertility in old mares.
Biology of Reproduction Monograph, 1: 209-214, 1995.
- Carnevale, E.M.; Griffin, P.G.;
Ginther, O.J. Age-associated subfertility before entry of
embryos into the uterus in mares. Equine Veterinary
Journal, Supplement, 15: 31-35, 1993.
- Carnevale, E.M.; Bergfelt, D.R.;
Ginther, O.J. Follicular activity and concentrations of FSH
and LH associated with senescence in mares. Animal
Reproductive Science, 35: 231-246, 1994.
- Vogelsang, S.G.; Vogelsang, M.M.
Influence of donor parity and age on the success of
commercial equine embryo transfer. Equine Veterinary
Journal Supplement, 3: 71-72, 1989.
- Carnevale, E.M.; Squires, E.L.;
Maclellan, L.J.; et al. Use of oocyte transfer in a
commercial breeding program for mares with various
abnormalities. Journal of the American Veterinary Medical
Association, 218: 87-91, 2001.
- Seidel Jr., G.E. Veterinary Clinics
of North America, Equine Practice: Diagnostic Techniques in
Assisted Reproductive Technology. 12(1): 85-101,
Cryopreservation of equine embryos. Ed. EL Squires. W.B.
Saunders Co., Philadelphia, 1996.
- Maclellan, L.J.; Carnevale, E.M.;
Coutinho da Silva, M.A.; et al. Pregnancies from vitrified
equine oocytes collected from superstimulated and
non-stimulated mares. Theriogenology, 58: 911-919,
2002.
