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Proceedings of The Pre-Congress of The 16th Italian Association of Equine Veterinarians Congress
Proceedings of The Pre-Congress of The 16th Italian Association of Equine Veterinarians Congress
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Claire Card
DVM PhD diplomate ACT, Dept LACS, Western College of Veterinary Medicine,
University of Saskatchewan, Saskatoon, Sk S7N 5B4 Canada
[email protected]
HYSTEROSCOPIC INSEMINATION
4
Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
Published in IVIS with the permission of SIVE Close window to return to IVIS
Figure 2 - Hysteroscopic view of the tip of a uterine horn. Figure 3 - Hysteroscopic view of an insemination cathe-
ter near the uterotubal opening.
location of the endoscope before insemination. retracted to just cranial to the internal cervix os
Following the insemination the catheter is and redirected. Picking up the uterine horn
withdrawn, the air flow is turned off, and the through the rectum is an easy way to confirm
biopsy port opened to allow the air to escape. your location. The location of the pipette may
Transrectal palpation may be used to confirm be confirmed by feel or by ultrasound. Tips to
the location of the endoscope and to eliminate perform the procedure include elevating the
pneumovagina, and massage is used to evacu- horns of a mare with a dependent uterine loca-
ate the air from the uterus. A variation of the tion, or manipulating the broad ligament to fa-
procedure is to guide the endoscope rectally cilitate passage of the pipette. A few mares re-
then insufflate only the end of the horn by quire sedation for the procedure, most mares do
pressing down, or around the uterine horn. not. Sperm are then deposited at the tip of the
It is important to develop a standard protocol uterine horn. Multiple straws may be intro-
to rinse and clean the equipment following duced using a burred stylet that allows with-
each procedure to keep the endoscope func- drawal of the spent straw. Closure and elevation
tional and to reduce the potential for mechan- of the uterine horn after deep horn insemination
ical transfer of pathogens between patients. has been reported as a means of coating the end
Iatrogenic infection is a risk of the procedure. of the uterine horn with sperm7,8.
for insemination. The most efficient way of cause of lower pregnancy rates. Breeding tri-
using low dose insemination is when the se- als in commercial settings using prepared se-
men is sold or packaged in a way that the dose men allow an examination of ranges of insem-
supplied may be divided and still obtain the ination dosages but lack controls20.
same pregnancy rate.
put, or in the situation where the stallion is de- gelded, terminally ill, or deceased. The sperm
ceased and there is no opportunity to replenish are often flushed out of the ampulla/vas defer-
the stored amount. Injuries or disease that de- ens and collected from the tail of the epi-
crease the number of sperm produced influ- didymides12. The number of mature sperm ob-
ence the number of mares a stallion may breed. tained from this method is low. Processing
Examples of conditions that will interfere or through density gradients may assist with im-
render a stallion sub or infertile include: testic- proving the quality of the semen10,24,25. Insem-
ular torsion or inguinal hernia requiring unilat- ination with epididymal semen has been
eral castration, testicular tumours, breeding in- shown to result in pregnancies.
juries, or chronic diseases such as kidney dis-
ease, laminitis, or peritonitis. In general these
stallions are of low fertility and may or may SEX SELECTED SPERM
not be assisted using low dose insemination.
Advanced age and associated health problems A cell sorting machine called a flow cytome-
such as chronic degenerative joint disease, or ter has been adapted to separate live fluores-
chronic obstructive pulmonary disease, are al- cently sperm by their sex chromosome content
so a problem for some stallions who are from many different species, including horses.
booked to a large number of mares9. In stal- The x and y sperm each have a different fluo-
lions with other health problems correction of rescent marker and the sperm are sorted into 3
the problem may result in a stallion converting pools: x chromosome bearing, y chromosome
from partial ejaculation to full ejaculation, bearing, and undetermined25. The process of
making low dose insemination unnecessary. sperm sex- selection is inefficient and only a
small percentage of the total sperm sorted are
available for insemination, with the current
COSTLY SEMEN technology only 2.5 million sperm/instrument
per hour may to be sorted requiring low dose
Frozen semen is often sold by the breeding insemination techniques. A dose of 25 million
dose without a guarantee of fertility. In these total sex-selected sperm is often used. The
cases when the frozen semen is very expensive, reason an owner would pursue the use of sex
but of good quality, the insemination dose may selected sperm is because there is an econom-
be sufficient to inseminate more than one mare. ic or sentimental or benefit to having either a
This may be achieved by dividing the insemi- filly or colt. Many horse breeders feel that cer-
nation dose or by application of semen with tain traits are carried in the male or female
low dose insemination technology. Alvarenga line. This may be due to the fact that the mito-
published a report where he obtained the same chondrial DNA which control the metabolism
pregnancy rate using 2 straws of frozen semen of the individual are inherited almost entirely
delivered through a hysteroscopic approach, as from the mare. Cloning experiments where
when 8 straws were used with conventional oocyte donors were different than the identical
AI15. This type of approach increases reproduc- nuclear donors revealed underlying differ-
tive efficiency. However knowledge of the stal- ences in metabolism that was believed to be
lion’s fertility is a requirement for reducing the related to DNA methylation and mitochondri-
supplied dose of semen. al DNA differences in the offspring.
The price a pregnant mare brings is influenced
by the sex of the foal she is carrying. Mares
EPIDIDYMAL SPERM that are examined in early pregnancy by ultra-
sound examination to determine the sex of the
Epididymal sperm are a pool of sperm that are fetus, prior to sale, bring a higher price at auc-
in the final stages of maturation or are in stor- tion. Some owners wish to have an offspring
age prior to ejaculation. Epididymal sperm are of a specific sex because they have been un-
generally collected from stallions that are able to achieve the desired sex of the offspring
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Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
Published in IVIS with the permission of SIVE Close window to return to IVIS
with natural mating. Further improvements in has been reported to have a similar pregnancy
sorting and freezing of sex selected sperm is rate to conventionally frozen semen in field
required for wider application. Bovine sex se- trials. In field trials about 92% of the offspring
lected semen is available in 14 countries and born were heifers. This extensive worldwide
TABLE 1
Selecte references on hysteroscopic insemination in mares
Author Year Hysteroscopic insemination dose Preg. rate
Manning et al. 1998 Normal PM Extended
100 mil, body, 33% 4/12,
10 mil body, 17% 2/12,
1 mil hysteroscopy, 22% 2/9,
10 mil hysteroscopy 0% 0/11
Vasquez et al. 1998 3.8 million motile PM 20 µl 30% 3/10
Morris et al. 2000 Percoll separated mil PM in 30 - 50 µl
10.0 60% 6/10,
5.0 75% 6/8,
1.0 64% 16/25,
0.5 29% 4/14,
0.1 22% 2/11,
0.001 10% 1/10
Lindsey et al. 2002 Fresh, 40% 4/10,
Flow sorted fresh 5 mil PM 230 µl and frozen, 38% 6/16
frozen flow sorted in 230 µl 13% 2/15
Lindsey et al. 2002 5 mil tot in 100 µl,
Deep horn non sorted, 0% 0/10,
hysteroscopy non sorted, 50% 5/10,
hysteroscopy sorted 25%, 5/20
Koene et al. 2002 motile 6 mil 0.2 ml, 5.9%,
12 mil 0.4 ml, 17.1%,
80 mil 1.6 ml 25.3%
Morris et al. 2003 Frozen body 14 mil PM 500 µl, 8/12 67%,
hyster ipsilateral 14 mil PM 500 µl, 64% 9/14,
hyster ipsilateral 3.1mil PM 100 µl, 47%16/34,
body 3.2 mil 100 µl, 14% 2/14,
hyster contralateral 3.0 mil 100 µl 8% 1/12
Gomes G.M. et al. 2003 Frozen semen percoll separated UTO 66% (10/15),
percoll selected body 40% (6/15),
non - selected in the body 20% (3/15)
Alvarenga M.A., 2002 Frozen thawed
Leao K.M. 10 mill percoll separated UTO, 33% 4/12,
10 mill non separated UTO, 33% 4/12,
400 million non-separated uterine body 0% 0/12
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Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
Published in IVIS with the permission of SIVE Close window to return to IVIS
TABLE 2
Selected References on Deep Horn Insemination
Author Year Experiment Preg. rate
Buchanan et al. 2000 Body 500 mil, 90% 18/20,
deep horn: 25mil 20cc, 57% 12/21,
5 mil 1 cc, 30% 3/10,
5 mil 0.2 cc 40% 4/10
Woods et al. 2000 2 stallions good vs poor
Good body 25 million, 63% 9/16,
Good horn 25 million 56% 10/18,
Poor Body 25 million, 29% 4/14,
Poor horn 25 million 29% 4/14
Brinsko et al. 2003 5 mil tot cooled hys vs deep horn 67% 12/18,
56%, 10/18
Reger et al. 2003 200 mil tot frozen body vs horn 50% 10/20,
at 24, 40 hrs 20% 5/20
Nie, et al. 2003 25 mil tot deep horn GWS, Sephadex, 50% 15/30,
Percoll separation 43%, 13/30,
neat, normal stallions 33% 10/30
Samper et al. 2005 50 - 150 mill sperm deep horn or hysteroscopic 547% 98/191,
46% 217/473
Samper et al. 2008 Frozen semen 88 stallions, ave. 1 – 2 straws
deep horn 43% 1279/2544
hysteroscopic 45% 1265/2544
network may be used in the future for the sale that caused their fertility to decline (founder or
of sex selected semen from other species. In- colic for example). The types of subfertility
deed many mare owners express a preference that are helped by low dose insemination tech-
for a foal of a specific sex when they are hav- niques remain to be defined. The most likely
ing their mare bred. stallions to be helped are those with low num-
bers of otherwise normal motile sperm. Stal-
SUBFERTILITY lions with severe motility and morphology is-
sues may not benefit from low dose insemina-
There are many causes of subfertility in stal- tion. Enrichment techniques to isolate the most
lions. Stallions that have lower than 30% per vigorous sperm from subfertile stallions and
cycle pregnancy rate are not usually commer- the removal of potentially toxic seminal plas-
cially viable. When considering using a sub- ma needs to be further investigated to deter-
fertile stallion for breeding the mare owner mine if it will increase pregnancy rates11.
should be aware that some causes of subfertil-
ity are heritable. There are stallions that are ge-
netically inferior in terms of semen quality and OTHER MANAGEMENT
will pass this characteristic along. The subfer- PROCEDURES HAVE FAILED
tile stallions that are best selected are ones that
had a previously successful breeding career There are cases where conventional breeding
and some untoward event has been identified management has failed to produce acceptable
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Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
Published in IVIS with the permission of SIVE Close window to return to IVIS
pregnancy rates from certain stallions. In ther from the hysteroscopic procedures, or
these cases the number of sperm used with deep horn insemination, will interfere with
low dose insemination techniques needs to be fertility. Initially it was believed that the re-
tailored to each individual stallion. Generally duce dose of semen may have an advantage
improved breeding management, conventional with subfertile mares, as sperm was reported
artificial insemination, is tried first and is then to be the main cuase of uterine inflammation,
followed by deep horn insemination or hys- however the nature of the procedure is inva-
teroscopic insemination9,11. sive and it carries a risk of contamination.
Mares that are subfertile are not good candi-
dates for the procedure28,29 and may require
PREPARATION OF THE SPERM post-breeding treatment for uterine inflamma-
tion or delayed uterine clearance. Mares with
There is some debate as to whether the removal double ovulations and preovulatory follicles
of seminal plasma, or processing the sperm to on both ovaries should have semen deposited
enhance the number of fertile sperm is benefi- in each uterine horn near or on the uterine
cial for low dose insemination. Nie et al., 2003 tubal junction because the small volume of the
evaluated the use of 3 processing techniques: inseminate is unlikely to allow easy move-
percoll, sephadex glass wool, and nothing26. ment of the sperm between the uterine horns.
Percoll is a substance that selects sperm based Post procedure monitoring is useful in detect-
on their density and tends to remove sperm ing uterine inflammation. Donor mares in em-
with abnormal head shapes or bent midpieces; bryo transfer programs have also been insem-
while sephadex glass wool, is a system that re- inated with deep horn techniques.30
moves membrane damaged sperm because of
their abnormal membrane charges, rather than
by a molecular exclusion principle. These au- SUMMARY
thors showed that with normal stallions the ad-
ditional processing steps did not improve fertil- Hysteroscopic insemination of the uterine
ity. They were using 25 million total sperm10. tube and deep horn insemination are non-sur-
The benefit to processing may only be realized gical assisted reproduction techniques that are
when fewer sperm are inseminated or when used to breed fertile mares with a reduced
there are higher percentages of damaged or ab- dose of semen such as: sexed fresh semen,
normal sperm. Fertile frozen semen is usually sexed frozen semen, or expensive/rare cryop-
inseminated without further processing when reserved semen. The hysteroscopic delivery or
using either deep horn or hysteroscopic insem- deep horn insemination technique may be
ination15,27. Fixed time insemination manage- used for insemination of low numbers of
ment systems are sometimes used. sperm in small volumes. Some stallions with
low sperm output or that are subfertile may
benefit from using this technology.
MARE SELECTION
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Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
Published in IVIS with the permission of SIVE Close window to return to IVIS
11
Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010