Many breed organizations now allow the use of liquid transported
semen. Interest among mare and stallion owners in this
technology is increasing due to advantages gained by
breeding with transported semen. Unfortunately, not all
stallions produce sperm that survive the cooling process.
An evaluation of the viability of a stallion's sperm
after cooling is an important quality control measure.
Stallions should be tested prior to the breeding season
to determine if the sperm motility will be acceptable
when mares are bred with cooled transported semen.
Management of the mare should be precise and intense in
order to reduce expenses and maximize fertility
associated with breeding with cooled, transported semen.
Semen must be ordered so that it arrives at a proper time
for insemination, preferably within 12-24 hours prior to
ovulation. Following are recommendations for testing stallion semen for ability to survive cooling, processing and shipping of semen, and management
of the mare for breeding with cooled transported semen.
Not all of the breed registries allow registration of foals produced by
artificial insemination and, of those that do, not all
permit the use of preserved or transported semen. Several
breed organizations within the United States sanction the
use of liquid transported semen. To ensure full compliance with the
breed registry involved, the specific breed registry
should be contacted prior to becoming involved in
breeding with transported semen so all requirements are
met to permit registration of any foals produced.
Fertility achieved
Fertility trials using cool-stored
equine semen have yielded pregnancy rates varying from 0%
to 70% per cycle, emphasizing that there is great
variability among stallions in the ability of their sperm
to maintain fertilizing capacity during cooling/storage.
Breeding with semen cooled for 48 hours at 5degC usually
reduces pregnancy rates to approximately half that
achieved with fresh semen. Since the pregnancy rate per
cycle achieved by breeding with semen cooled for 24 hours
at 5degC typically averages 60 to 70%, one should expect
near normal pregnancy rates when semen is used for
breeding after short term (œ24 hours) storage,
provided semen quality is good following this cooling
period, and mare management has been optimal.
Factors influencing fertility
Numerous factors influence pregnancy
rates achieved when mares are bred with cooled stallion
semen. Some of the factors identified as influencing
fertility of cooled stallion semen in controlled
experiments include the number of sperm inseminated,
frequency of insemination, concentration of sperm in
extender, type of extender and antibiotic(s) utilized,
cooling rate of extended sperm, storage duration and
storage temperature, stallion variability in response of
sperm to cooling, and inherent fertility of the stallion
and mare. If quality of fresh stallion semen is poor, or
if fertility achieved by breeding with fresh semen is
poor, it is highly unlikely that successful results can
be obtained by breeding with cooled transported semen.
Other factors which undoubtedly impact fertility using
this technology include whether or not the semen is
collected and handled properly, the raw semen is promptly
and accurately diluted in a suitable prewarmed extender,
the semen is properly packaged for passive cooling and
shipment, the insemination is properly timed in relation
to ovulation, and proper technique is utilized during
insemination. Sperm are very sensitive to many
environmental factors, including temperature, light,
physical trauma, and a variety of chemicals. Any factor
that impacts the ability of sperm to resist
environmentally-induced damage will adversely affect
fertility achieved when using cooled transported semen
for breeding.
Testing a stallion's ability of semen to survive cooling
We recommend that each stallion, for
which breeding with cooled transported semen is planned,
be tested prior to the breeding season to determine if
the sperm will survive the cooling process. The
practitioner is referred to the Society for
Theriogenology manual on examination of the stallion for
breeding soundness for procedural considerations (Journal
of the society for Theriogenology. Volume IX.
Theriogenology and the Equine: Part II . The Stallion;
August, 1983). Collection of several ejaculates may be
required to stabilize extragonadal sperm reserves and
improve semen quality, thereby maximizing chances sperm
will survive the cooling process. If sperm quality is
acceptable after 24 hours of cooling, it is reasonable to
assume the stallion's semen will survive the cooling
process and the stallion can be recommended for breeding
with cooled transported semen. Equally important, an
estimation can be made of the total number of sperm that
will need to be processed in order to provide an
insemination dose of cooled semen that should maximize
pregnancy rates.
Preservation of semen begins with the
actual collection process. Accurate assessment of semen
quality relies heavily on proper semen collection
techniques. Ejaculated semen is very susceptible to
environmental influences, so mishandling semen samples
before evaluation negates their value for representing
the ability of a stallion's sperm to survive the cooling
process. Semen collection techniques and procedures
utilized to ensure that sperm quality is optimized should
be meticulously followed when collecting stallion semen.
Following collection, semen should be
processed in a careful and efficient manner. Briefly, the
gel is separated from the gel-free fraction of the
ejaculate, and the volume of filtered gel-free semen is
measured. Sperm concentration of the gel-free semen is
determined. The total sperm number in the ejaculate is
calculated by multiplying sperm concentration by volume
of gel-free semen. A small portion of the gel-free semen
is diluted in a suitable prewarmed extender prior to
estimating progressive sperm motility using a microscope
equipped with a warming stage (a phase-contrast
microscope provides optimal viewing of sperm). Warmed
(37oC) nonfat dry skim milk-glucose extender may be used
since it supports sperm motility and does not interfere
with microscopic visualization of the sperm. To
standardize the sperm motility testing protocol, all
semen samples should be diluted to a specific
concentration with extender before analysis. If the
quality of fresh stallion semen is poor, it is highly
unlikely that successful results can be obtained by
breeding with preserved semen.
The remainder of the semen should be
mixed with a prewarmed (37oC) extender within 2-15
minutes after ejaculation in peparation for cooling. A
final concentration of 25-50 million sperm per ml in
extended semen generally maximizes sperm survivability in
vitro. The semen should be diluted with extender at a
ratio of at least 1 part semen to 4 parts extender. If
this dilution results in a sperm concentration < 25
million sperm per ml, the semen may be centrifuged to
concentrate sperm. The semen is first mixed with extender
at a ratio of 1-2 parts extender to 1 part semen, and
then is centrifuged in 50-ml round-bottom centrifuge
tubes at 500 x g for 10 minutes. The supernatant is
removed and the sperm pellet is resuspended in additional
extender. If seminal plasma is removed by centrifugation,
the sperm can be resuspended to a concentration of 50-100
million sperm/ml without adversely affecting motility.
The extended semen must contain a minimum of 5% seminal
plasma to prevent reduction in longevity of sperm
motility. Extended semen should not be left for a
prolonged period in an incubator (37oC) because extensive
cellular death will occur within a few hours if sperm are
maintained at this temperature.
Determination of insemination dose
The most accurate method for
determining the insemination dose required to transport
for breeding is to conduct semen cooling trials for the
individual stallion. The semen is diluted in an
appropriate extender(s) as described above, and the semen
is cooled for 24 hours. The cooled semen sample is gently
remixed after this cooling period, and an aliquot is
warmed to 37oC. Sperm motility is evaluated after 15
minutes of warming, and the percentage progressively
motile sperm obtained is used to calculate the number of
progressively motile sperm so that a minimum of 500
million progressively motile sperm will be present after
24 hours of cooling. For example, if after 24 hours of
cooling the progressive sperm motility is 50%, 1 billion
total sperm would need to be prepared for shipment to
ensure an insemination dose of 500 million progressively
motile sperm was available for breeding the mare.
Insemination volume
The number of normal motile sperm in an
insemination dose appears to be more critical to
fertility than the volume of the inseminate. Although
smaller or larger volumes can be used successfully,
typical insemination volumes for cooled transported
equine semen range from 20 to 120 ml. Many practitioners
prefer to limit insemination volume to œ 60 ml.
Insemination volumes as large as 120 ml of cooled semen
have been found to have no adverse effect on fertility of
pony mares as long as a sufficient number of
progressively motile sperm are placed in the uterus.
Some practitioners believe large
insemination volumes should be avoided when breeding
mares susceptible to uterine infection. Uterine
inflammation commonly follows breeding in the horse, and
it is likely that uterine inflammatory response in such
mares is at least partly due to neutrophil migration into
the uterus as a result of sperm chemotactic stimuli
(sperm themselves have been shown to stimulate an influx
of neutrophils into the uterus). In this regard, it
appears that more concentrated semen produces a greater
influx of neutrophils into the uterus. Therefore, if
chilled semen is properly diluted (i.e., to 25 to 50
million sperm per ml), the inflammatory response may be
lessened.
Broodmare management
To reduce time and expense involved in
breeding a mare with cooled transported semen, the
practitioner should strive to minimize the number of
shipments required for breeding. If pregnancy does not
result from breeding during one estrous cycle, expenses
are multiplied as additional breeding(s) will be
necessary. If breeding during two estrous cycles does not
result in pregnancy, a thorough breeding soundness
examination (if not already performed) may be necessary
to determine if the mare is at fault. If semen quality is
suspect, particularly if it cannot be corrected, breeding
to another stallion may be an option for consideration.
To avoid the expense of repeat breedings, mare owners
should try to select a stallion for breeding with
transported cooled semen that has been proven to achieve
good pregnancy rates with cooled semen. If the stallion
has not been used previously for breeding with cooled
semen, they should attempt to utilize a stallion that is
known to have acceptable semen quality after being cooled
to optimize fertility. However, this information may not
always be available, particularly if a stallion's semen
is being offered for cooled transported breeding for the
first time.
The practitioner should play a primary
role in maintaining quality control (semen quality,
insemination dose, timing of breeding, etc.) when horses
are bred with transported cooled semen. To be successful,
stallion owners/managers, mare owners, and veterinarians
involved in stallion and mare management must work
together as a team to optimize the chance of conception
from each breeding.
Prebreeding preparation of the mare
Prior to the onset of the breeding
season, the mare should be examined to detect
reproductive abnormalities. Underlying problems that
might contribute to infertility, such as poor body
condition or endometritis, should be corrected.
Prediction or detection of ovulation
When the mare is in estrus, the cervix
becomes progressively more relaxed and the uterus becomes
softer and more edematous as follicle size increases and
ovulation approaches. Once a follicule reaches 35-50 mm
in diameter, the ovulatory follicle(s) soften(s)
detectably on palpation per rectum. Using transrectal
ultrasonography, the cross-sectional size and shape of
follicles and the echogenicity of the follicular fluid
can be used to aid in prediction of ovulation. Within 24
hours prior to ovulation, the shape of most follicles
tends to change from spherical to a conical or
"pear" shape, and the follicular wall may
become "scalloped" or thickened in appearance.
The apex of the conical follicle will be located at the
ovulation fossa. The echogenicity of the follicle changes
from a predominantly anechoic appearance to a
heterogenous echotexture as ovulation occurs and luteal
development begins. The developing corpus luteum often
contains a small anechoic center resulting from retained
follicular fluid or blood.
Hormores to control ovulation
When cryopreserved semen is used for
breeding, or when breeding to certain subfertile
stallions, the fertilizable lifespan of sperm may be
reduced, requiring breeding closer to the time of
ovulation. To optimize fertility, hormones can be
adminstered to increase the probability that ovulation
will occur near the time of breeding with cooled semen.
Onset of estrus can be controlled in cycling mares by
administration of progestogens and/or prostaglandins.
Human horionic Gonadotropin (hCG) or a
GnRH analog can be administered to reliably induce
ovulation. Administration of hCG (1500-3300 units, i.v.,
or i.m.) when a 3 35 mm in diameter follicle is present
in mares in estrus will induce ovulation in a majority of
mares within 36-48 hours. If the day of estrus is
unknown, and the follicle is larger than 35 mm in
diameter, the interval to ovulation is less precise and
may occur earlier than expected after hCG administration.
Some practitioners believe hCG will more predictably
induce ovulation at a precise time if endometrial edema
(as detected by ultrasound examination) is maximal at the
time of administration.
The stallion manager should be
consulted to make sure semen will be available for
breeding when the mare is expected to be in estrus. Human
chorionic gonadotropin (hCG) can also be used to improve
synchrony between arrival of shipped semen and time of
ovulation. When a mare is in estrus, daily or even twice
daily examinations are performed to monitor size of
developing follicles. Typically, once follicular diameter
reaches 35 mm, hCG may be administered after it is
confirmed that transported semen can be obtained and will
arrive the next day. The semen generally arrives 24 to 36
hours later in time for breeding just prior to ovulation.
Timing and frequency
Since viability of stallion semen may
be shortened by cooling, many practitioners recommend
breeding as near to ovulation as is feasible to optimize
pregnancy rates. While satisfactory pregnancy rates have
been reported in mares inseminated after ovulation with
transported cooled semen, critical study of the optimal
time for breeding in relation to ovulation with cooled
semen has not been done.
Breeding the mare with cooled semen
When the cooled semen arrives for
insemination of the mare, current recommendations are to:
- Prepare the mare for breeding. The
mare should be adequately restrained with her
tail wrapped and held to the side. The perineal
area is thoroughly scrubbed and rinsed, paying
particular attention to the vulva. Any dirt or
fecal material within the caudal vestibule should
be removed during the washing process to prevent
contamination of the anterior reproductive tract
during insemination. Two to three scrubs with a
non-irritating soap (e.g., Ivory Soap) or a
surgical scrub are recommended, followed by
thorough rinsing to eliminate residual soap that
may be spermicidal or irritating to mucous
membranes. The perineal and vulvar area should be
thoroughly dried prior to breeding.
- Open the shipping container and
confirm the identity of the stallion using the
accompanying form. Remove the chilled semen,
gently mix it, aspirate the semen into a syringe,
and attach an insemination pipette. Sterile
nontoxic disposable equipment is recommended for
AI procedures. Syringes with non-spermicidal,
plastic plungers (Air-tite, Vineland, NJ) are
preferable for AI since rubber plungers may
possess spermicidal properties.
- Inseminate the semen into the
uterus of the mare. To inseminate the mare, a
sterile shoulder-length plastic sleeve is first
placed over the arm used for insemination. The
tip of a 20 to 22-inch insemination pipette is
then positioned in the cupped hand and a small
amount of sterile, non-spermicidal lubricant is
applied to the back of the hand. The covered hand
and insemination pipette are passed into the
cranial vaginal vault where the index finger
identifies and penetrates the cervix. The
insemination pipette is then advanced through the
cervix to the mid-body of the uterus. A syringe
containing extended semen is attached to the
insemination pipette and the semen is slowly
deposited into the uterine lumen. An alternate,
but equally satisfactory, method of insemination
is to pass the insemination pipette through the
cervix into the body of the uterus using a
lighted speculum preplaced in the vagina.
- A small aliquot of the extended
semen should be kept and warmed to 37oC, and
sperm motility should be assessed and recorded
for quality control purposes. If sperm motility
is poor, inquiries can be made in an effort to
determine if this was an unexpected problem. It
is sometimes helpful to prepare a specimen for
assessment of sperm morphology. A high percentage
of morphologically abnormal sperm would be
expected to yield a low precentage of
progressively motile sperm following cooling. If
sperm motility is consistently poor and the mare
fails to conceive on repeated breedings, it is
possible the stallion is incapable of producing
sperm which survive the cooling/transportation
process. Alternatively, use of a different semen
extender or dilution ratio may prove beneficial
to semen harvested from that particular stallion.
Overseas workers often recommend
warming the cooled semen to body temperature prior to
insemination of the mare. In the U.S., the cooled semen
is usually inseminated directly into the uterus as soon
as possible after opening the shipping container. Since
no detrimental effects on pregnancy rate have yet been
determined by inseminating mares with cooled semen, at
present we see no reason to prewarm the cooled semen
prior to insemination unless a cream-gel extender is
used. This extender forms a gel at refrigerated
temperature, so it must be warmed prior to insemination.
The packaged sample can be placed in an incubator or
waterbath preset at 37-38oC. It is important to avoid
contamination of the extended semen with water when
warming the sample in a waterbath.
Double inseminations
It has become common practice for many
stallion owners/managers to prepare two bags
(insemination doses) of extended semen for shipment - one
to be used for an initial insemination upon arrival, and
one to be held for insemination again the next day. For
many stallions, the longer the semen is held at
refrigerated temperature, the poorer sperm motility
becomes. However, if semen is to be held for breeding
again the next day, precautions should be taken to
maintain the cooled semen at 4-6oC until the time of the
second insemination. Once a shipping container is opened
for the first breeding, temperature of the semen left in
the container is likely to rise above 10oC, causing
premature sperm death. The remaining semen dose can be
immediately placed in an insulated box within a
refigerator (4-6oC) - until it is to be opened for
breeding the next day. This would be especially critical
for semen cooled in some "disposable" type
shipping containers which may only maintain semen at <
10oC for 27.5 to 33.5 hours.
Postbreeding treatment for mares with intrauterine fluid accumulation
If a given mare needs treatment after
breeding that includes evacuation of uterine fluid or
debris, we recommend either postbreeding uterine lavage
or oxytocin treatment. Treatment as early as 4 hours
after breeding does not appear to affect fertility.
Treatment can also be performed after ovulation is
detected. Intrauterine treatment more than 2 days after
ovulation should be avoided since it may impair CL
function, resulting in an early return to estrus with
subsequent loss of pregnancy if the breeding was
successful.
Examination for pregnancy
We recommend the mare bred with
transported cooled semen be examined for pregnancy using
transrectal ultrasound at 14-15 days postovulation. This
enables early detection of a pregnancy that is normal for
stage of development, allows early detection of twins
before they become fixed in the uterus, and provides time
for rebreeding during the same estrus if the mare failed
to become pregnant.
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