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Proceedings of the Pre-Congress of the

16th Italian Association of Equine
Veterinarians Congress

Carrara, Italy – 2010

Next SIVE Meeting:

Feb. 4-6, 2011 – Montesilvano, Pescara, Italy

Reprinted in the IVIS website with the permission of the

Italian Association of Equine Veterinarians – SIVE

http://www.ivis.org
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Low Dose Insemination Techniques

in Mares

Claire Card
DVM PhD diplomate ACT, Dept LACS, Western College of Veterinary Medicine,
University of Saskatchewan, Saskatoon, Sk S7N 5B4 Canada
[email protected]

INTRODUCTION There have been reports of new assisted re-

productive techniques, such as low dose in-
Reproductive efficiency in the equine indus- semination using a hysteroscopic approach, or
try is mainly a function of extrinsic (manage- via deep horn insemination. Low dose insem-
ment) and intrinsic factors such as the quality ination technologies, such as hysteroscopic in-
of the mare’s reproductive system, and the semination are relatively new and were first
dose and fertility of the semen. Stallion fertil- reported in 19962,3. This was followed by the
ity is highly variable and therefore the recom- introduction of deep horn insemination in
mended number of sperm per insemination 20014. The pertinent questions about these
dose are generally high, to account for this techniques include: do they help increase re-
variability. Presently very large insemination productive efficiency and profitability of
doses are recommended for use in mares with breeding? What are the low dose assisted re-
100-300 x 106 sperm commonly listed for productive technologies? How are they per-
fresh semen, and up to 500 x 106 progressive- formed? Are they suitable for use in all mares?
ly motile and normal sperm (Society for The-
riogenology), per insemination. Frozen se-
men is often prescribed at 800 million total LOW DOSE INSEMINATION
sperm1. This is problematic because semen, PROCEDURES
particularly frozen semen, is not always avail-
able in large quantities. Overall pregnancy Low dose insemination techniques in the mare
rates are still lower than desirable, and this include hysteroscopic insemination and deep
creates significant economic loss for the horn insemination2,3,5. The concept is that
equine industry. According to the Jockey deposition of the sperm closer to the junction
Club of North America for example only 55% of the uterus and oviduct (called a uterine tube
of all Thoroughbred mares that are bred foal. in the horse) will increase the chance of con-
An evaluation of mares bred versus foals reg- ception with a reduced dose of sperm, and/or
istered in many parts of the world suggests improve the chance of conception in a stallion
that there is a similar level of reproductive ef- with suboptimal fertility. One study reported
ficiency in some breeds. High rates of fertili- that when the sperm in a low volume were de-
ty, such as 90% pregnancy rates, are often posited in the body of the uterus, the sperm
achieved in 60 day breeding seasons with were distribute equally between the uterine
mares and stallions in pasture breeding situa- horns, but when deposited in the uterine tip
tions, indicating that selection for fertility around 80% of the sperm were found in that
may be successful in increasing reproductive horn6. This is one of the rationales for using a
efficiency. low volume, low dose amount of sperm.
3
Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
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HYSTEROSCOPIC INSEMINATION

Estrus mares with a 35 mm follicle are admin-

istered hCG or deslorelin, and 24-30 hrs or
40-46 hrs respectively the procedure is per-
formed. To ready a mare for a hysteroscopic
examination it is best to empty the rectum of
manure and prepare the mare as if for artificial
insemination with the perineum of the mare
cleansed, and the tail wrapped. Mares are usu-
ally sedated intravenously using alpha ago-
nists, such as xylazine combined with butor-
phanol to minimize movement or any discom-
Figure 1 - Hysteroscopic view of the bifurcation of the
fort during the procedure, and to safeguard the
uterine horns.
equipment in the event the mare becomes im-
patient, or anxious resulting in her kicking or
rearing. The mare should be observed while comfortable. Care must be taken while passing
sedated so that she does not inadvertently the endoscope into the uterus to maintain the
close off her airway when her head drops, orientation of the endoscope or the location
such as might occur if she rests her neck on should be confirmed per rectum either at the
the front gate of the stocks. beginning or the end of the procedure2,7.
The endoscopic equipment should be chemi- Once a uterine horn has been entered the direc-
cally sterilized (usually with glutaraldehyde tional turning wheels of the endoscope may be
based products) and then the disinfectant re- locked and the tip of the scope directed using
moved using alcohol and copious amounts of fine manipulation as needed to align the endo-
phosphate buffered saline or physiological scope with the uterotubal papilla. The utero-
saline. The procedure is easiest to perform tubal papilla appears as a small mound that is
with an endoscope with excellent air flow that located dorsally and eccentrically at the end of
can insufflate a rectal sleeve in under 30 sec- the uterine horn. Figure 2 shows a view of a
onds when the airflow is set on high. uterine horn, and Figure 3 a close up view of the
Generally 2 people are required to perform a uterotubal papilla and an insemination catheter.
hysteroscopic examination. A one meter gas- Once the uterotubal papilla is located a sterile
troscope, or videoendoscope is used. A small plastic insemination catheter (2 mm x 200-230
amount of sterile lubricant is applied to the mm)a,b preloaded with the semen, by retro-
sides of the endoscope. An examiner wearing a grade aspiration, is passed through the biopsy
rectal sleeve and sterile glove guards the channel of the endoscope. A single lumen or
equipment through the vagina, and passes it double lumen catheter may be used.a,b The se-
through cervix into the uterus. The cervix is men is deposited directly on the surface of the
then held closed around the endoscope and the uterotubal papilla, followed by air to fully eject
uterus is insufflated with air or gas. Complete the semen, such that a froth is formed. Smaller
uterine insufflation should take less than 20 volumes are an advantage when using this
seconds with fully functional endoscopic technique. If the examiner is uncertain if the
equipment. If the uterus is not readily insuf- endoscope is located the in the correct uterine
flated an external insufflation source such as horn ipsilateral to the dominant follicle, tran-
nitrous, or CO2 way be used. When the uterus srectal palpation may be used to confirm the
is sufficiently insufflated the examiner will see
the bifurcation of the uterine horns (Figure 1), a
MILA International Low dose insemination set. Erlanger,
and then the endoscope is passed into the horn KY USA www.milainternational.com
ipsilateral to the dominant follicle. Air flow is b
Endoscopy support services, Vet aspiration catheters,
adjusted during the procedure to keep the mare Brewster, NY USA www.endoscopy.com

4
Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
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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.

DEEP HORN INSEMINATION UNDER WHAT CIRCUMSTANCES

DO THESE PROCEDURES INCREASE
Deep horn insemination involves passing a THE EFFICIENCY OR
flexible AI pipettec,d through the cervix using a PROFITABILITY OF BREEDING?
vaginal approach and into the uterine body just
ahead of the cervix. The pipette is then imme- Low dose insemination techniques may have
diately direct toward the desired uterine horn. application when: there is a limited amount of
The inseminator’s hand is introduced into the semen, the semen is very costly, there is low
rectum and the end of the pipette identified in sperm production, epididymal sperm are used,
the uterine horn, or if in an correct location it is sex-selected sperm are used, the stallion is
subfertile, or other management procedures
c
have failed to produce acceptable fertility9-12.
Universal Insemination pipette and Gun, Minitube, Vero-
na, WI USA www.minitube.com
Sex-sorted semen is now available for mares
d
IMV Equine IUI catheter and stylet, Maple Grove, MN and requires a low dose insemination tech-
USAwww.imv-technologies.com nique10,11,13 as 25 million sperm are often used
5
Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010
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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.

Hysteroscopic insemination has been reported CHOICE OF LOW DOSE TECHNIQUE

in horses as a method used to reduce the number
of sperm cells required for conception Pregnan- The balance of the reports in the literature that
cies have been achieved using the hysteroscopic compare pregnancy rates between deep horn and
technique with 1% (5 million) of the conven- hysteroscopic insemination were similar7,21,22
tional insemination dose (500 million sperm)14. when fertile stallions were used. There are a few
Most authors report that this technique shows an reports showing higher pregnancy rates with
advantage over conventional artificial insemina- hysteroscopic insemination compared with con-
tion (AI) when the sperm number is below 50 ventional AI9,11. Ultra low dose (1000 sperm)
million sperm and the volume is low2,5. If the pregnancies have only been reported with hys-
sperm number is greater than 50 million motile teroscopic insemination23 or surgical intratubal
sperm cells it is likely that conventional artificial inseminations. Hysteroscopic insemination does
insemination (AI) will achieve similar results. however require expensive equipment and facil-
There is also an advantage when more sperm are ities to maintain the endoscope. Deep horn in-
supplied in a breeding dose of frozen semen, semination requires only special insemination
and hysteroscopic delivery allows a reduction in catheters. Both techniques require an advanced
the number of sperm (and straws used) per in- skill level and training to perform.
semination15. At the time that the hysteroscopic
insemination method was developed the flow
cytometer was adapted to separate live fluores- INSEMINATION DOSE
cently labelled equine sperm by their sex chro-
mosome content. Hysteroscopic insemination The Society for Theriogenology recommends
has been used to deliver sex - selected sperm. 500 million normal and motile sperm cells for
This technique allows fresh, cooled, and frozen a conventional intrauterine insemination.
- thawed sex - selected sperm to be used to Most stallions achieve good fertility with 100
achieve pregnancies13,16,17. Currently this tech- million motile fresh sperm cells. There are
nique has been applied to use sex-sorted semen however some stallions that achieve excellent
in commercial settings.18 fertility with as few as 5 million sperm cells
per insemination dose deposited in the uterine
body. All stallions in theory have a fertility
DEEP HORN INSEMINATION curve that is dose dependent, and by incre-
mentally increasing the number of sperm fer-
The advantage to this technique over hys- tility increases until a plateau is reached. Most
teroscopy is that it does not require expensive authors conclude that for the lowest dosages
equipment to perform8,19. One of the difficul- of sperm that hysterscopic insemination is
ties in assessing the usefulness of the tech- more likely to result in pregnancy when com-
nique is that there is little scientific agreement pared to deep horn insemination5.
on the dose of motile, or motile and normal
sperm needed for conception in the mare
when using fresh, cooled, or frozen sperm. In LIMITED AMOUNTS OF SEMEN
fact the huge variability in stallion fertility ac-
counts for this variation. Uterine injury result- Limited amounts of semen are available from
ing in bleeding during the deep horn insemi- stallions with excessive book sizes, or from
nation procedure has been identified as a stallions that have below average sperm out-
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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
7
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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

8
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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
9
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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

The hysteroscopic procedure should take un- REFERENCES

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Published in IVIS with the permission of SIVE Close window to return to IVIS

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Proceedings of the Annual Meeting of the Italian Association of Equine Veterinarians, Carrara, Italy 2010