Journal of Equine Veterinary Science 32 (2012) 387-389

Journal of Equine Veterinary Science

journal homepage: www.j-evs.com

Review Article

The Use of Sex-Sorted Stallion Semen in Embryo Transfer Programs

Juan C. Samper DVM, MSc, PhD, Dipl. ACT a , Lee Morris BVSc, DVSc, Dipl. ACT b,
Tracy A. Plough DVM a
a
JCS Veterinary Reproductive Services Ltd., Langley, British Columbia, Canada
b
Equibreed, Cambridge, New Zealand

a r t i c l e i n f o a b s t r a c t

Article history: Sorting stallion semen into two separate populations of enriched X- or Y-bearing sperm
Received 7 May 2012 can be done successfully. For this, stallion semen can be shipped to a sorting facility, but
Accepted 22 May 2012 the mare must be in close to the sorting laboratory. Fertility rates when using 20-40
million sperm are an acceptable 60% per insemination. The procedure can be imple-
mented in embryo transfer programs, with no deleterious effect on the pregnancy rate or
Keywords:
embryonic death.
Stallion
Ó 2012 Elsevier Inc. All rights reserved.
Sex-sorted Sperm
Artificial insemination

1. Introduction personal communication, 2010). Polo breeders and the Polo

The selection of the gender of the offspring before
conception has commercial and economic implications in
many species. Dairy farmers find an economic benefit of
producing female calves, whereas beef producers would
prefer male calves. In some deer species, males are highly
sought because of their antlers; in swine, female piglets are
preferred because of castration issues. In these species, it is
not difficult to determine the difference in the economic
value between the desired and nondesired gender.
However, in horses, the difference between the desired and
nondesired gender is a much more subjective issue. Still,
the horse owner often wants to choose the gender of the
offspring for different reasons, such as a replacement
broodmare, a potential stallion from a specific line, stallions
that apparently produce better fillies than colts or vice
versa, or industries where a specific gender is perceived to
perform better than the other.
One breed of horse where the difference in price of the
female is higher than the male is the Polo horse (F Riera,
players have a greater demand for fillies because of their
perception that fillies are more precocious and easier to
train. However, it is clear, although for subjective reasons,
that most horse breeders when having the opportunity to
choose a specific gender would do so as an added bonus.
However, because of the subjectivity of the choice and the
inefficiency of separating X- and Y-bearing sperm with the
current technology, mare owners might try once or maybe
twice, but if faced with the possibility of leaving the mare
open for the year, they opt for breeding the mare with
conventional semen, as the residual economic value of the
nondesired offspring has the potential of being much
higher than in other species. Another challenge that sex-
sorted semen faces is the lack of support from some
breed registries, for example, Standardbred stud book in
Australia prohibits the registration of foals produced by
sex-sorted sperm.
2. Sex Determination

Currently, there are more than 100 patents claiming to

Corresponding author at: Juan C. Samper, DVM, MSc, PhD, Dipl. ACT,
JCS Veterinary Reproductive Services Ltd., 2943 216 St. Langley, British
Columbia V2Z2E6, Canada.
E-mail address: jsamper@telus.net (J.C. Samper).
have technology to significantly alter the sex ratio of the
born offspring in several species. However, most proce-
dures are only anecdotal and have not withstood scientific
challenge over time.

0737-0806/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.jevs.2012.05.056
388 J.C. Samper et al. / Journal of Equine Veterinary Science 32 (2012) 387-389
It is possible to have a deviation of sex ratios without
separating the sperm carrying the X or Y chromosome.
Often, a breeder will have a streak of several males born in
a row or a year when a horse breeder will have all fillies.
Breeders desiring a specific gender often believe that
male or Y-bearing sperm “swim faster but live shorter” in
the female tract compared with X-bearing sperm. There-
fore, timing of insemination with respect to ovulation
should alter the proportion of males and females born.
Recently, Sanchez et al. (unpublished observation) recor-
ded a 49.4%:50.6% male to female ratio in 433 births
regardless of whether mares had conceived when bred pre-
or postovulation with fresh or frozen semen. The results
from this observation clearly dismiss the concept that sex
ratio can be changed based on the relative proximity of
breeding to ovulation.
There are two methods that are commercially available
for determining the gender of the offspring in the embry-
onic stage. After fertilization, the embryo is biopsied and
fluorescence in situ hybridization techniques are used to
amplify the signal for the presence or absence of the Y
chromosome [1-4]. Although this technique is reliable, it
requires sophisticated laboratory equipment and is done at
a much greater expense to the owner. The advantages of
this technique are obviously the possibility of detecting the
presence of genes that could result in the expression of
different diseases in the offspring [2]. The other technique
is based on the separation of sperm bearing the X and the Y
chromosome.
Except for a few species, the difference in DNA between
X- and Y-bearing sperm is between 2% and 4%, and in the
stallion it is approximately between 3.4% and 3.7% [5]. To
date, the only reproducible way of separating X- and Y-
bearing sperm based on DNA content from an ejaculate or
a semen sample is by the use of a high-speed flow
cytometer with a cell sorter, a technique developed by
Johnson et al. [6] and improved by Rens et al. [7]. This
technique, which relies on the uptake of the permeable
fluorescent DNA dye Hoechst 33342 (Sigma Chemical Co,
St Lois MO 63178) by the sperm, results in purities of
>90%, with the proportion of born live offspring consistent
with the purity of the sperm inseminated. During the
sorting process, three populations of sperm are harvested:
X- and Y-bearing sperm and a third population of non-
sortable and dead sperm. After the sperm are separated,
the sample to be used is reanalyzed to confirm the purity
of the desired sperm population. Purity of the samples can
be arbitrarily changed by changing the gates or the zones
R1 and R2 in the sorter. However, when a higher purity is
desired, the sorting speed is decreased. Purities that are
generated for commercial use with stallion semen are
between 90% and 95%. The requirement for single sperm
interrogation limits the number of sperm sorted per
second using this technology. With the most modern
technology, 70,000-80,000 sperm/sec (F DeGraaf, personal
communication, 2012) can be analyzed, resulting in 20%-
25% females and 15%-20% male sperm sorted. This results
in an average of 40-68 million sperm sorted per hour.
Despite the significant improvements, the process remains
inefficient in the production of sperm for standard AI
doses for different species [8]. To achieve a dose of 250-
500 million live sperm at the present sorting rates, it
would take between 5 and 10 hours, which is nonviable
economically. To make the procedure an economically
viable process maximizing the use of the sorter as well as
the production of doses for the horse, it is imperative to
significantly reduce the number of sperm in the insemi-
nation dose. This requires changes in the insemination
technique, as well as in the timing of insemination
compared with standard artificial insemination
procedures.
3. Fertility of Sex-Sorted Stallion Semen
The first report on fertility using fresh sex-sorted sperm
in the horse was published in 2000 by Buchanan et al. [9]
who inseminated mares with 25 million sperm, obtaining
fertility rates ranging between 30% and 50%. However,
these experiments used a limited number of stallions.
Lindsay et al. [10] reported a nonsignificant difference in
the fertility of fresh sorted or nonsorted spermatozoa
hysteroscopically inseminating 5 million sperm. However,
there was a significant reduction in the fertility of frozen-
thawed sex-sorted sperm compared with the nonsorted
frozen sperm [11,12]. In another study by Lindsay et al.
[10,11], mares inseminated with 5 million fresh unsorted
sperm from the same ejaculates resulted in similar results
than those of the fresh sex-sorted inseminations. Subse-
quently, Lindsay et al. [10] stored sperm for 18 hours at 5 C
or 15 C before sorting. In their experiment, they reported
a 72% 16-day pregnancy rate after hysteroscopic insemi-
nation with 20 million sex-sorted sperm [13]. In 2004,
a trial done in Argentina by Panarace et al. [14] reported
acceptable fertility rates from 16-day pregnancy rates (13/
24 [54%]) and embryo recovery rates (11/18 [61%]) using 40
million sex-sorted sperm packed in a 0.5-mL straw. Other
investigators (Clulow et al., 2007 [12]) have reported low-
fertility pregnancy rates when using sex-sorted frozen
stallion sperm using a limited number of stallions. Owing to
the limited number of stallions used, the inability to cryo-
preserve, and the logistics involved in the sex-sorting of
stallion sperm, this technology has not been implemented
commercially on a wide scale in the equine industry.
4. Sexed Semen in Embryo Transfer Programs
During the breeding seasons of 2009-2010 in the
southern and northern hemispheres, fresh sex-sorted
semen was incorporated into commercial embryo transfer
programs. Twelve different stallions were used to breed
mares that were embryo transfer donors. One hundred
seventy-three inseminations resulted in the recovery of
109 embryos (63%). All mares were bred only once per cycle
with insemination doses ranging between 18 and 80
million sex-sorted sperm. All inseminations were per-
formed by rectally guiding a flexible pipette to the tip of the
uterine horn ipsilateral to the ovary having the preovula-
tory follicle. Timing of insemination was within 12 hours
before or 4 hours postovulation. The requested gender was
20 males and 153 females. Average pregnancy rate was
60.4% with a range between 50% and 75%. Of interest
was that the fertility rate of the mares that were insemi-
nated with the highest doses was only 50%. However, all
stallions that were brought in for sorting their sperm
 J.C. Samper et al. / Journal of Equine Veterinary Science 32 (2012) 387-389
produced pregnancies. The correct gender was produced in
100% (13/13) of the males and 97.4% (76/78) of the females.
No increase in embryonic death after 14 days was detected,
and all foals born were determined to be normal.
Unfortunately, fertility of sex-sorted frozen-thawed
stallion semen has not produced satisfactory results.
Fertility rates have ranged between 0% and 16.6% when
inseminating between 5 and 20 million sperm by hys-
teroscopic insemination or rectally guided insemination
[11,15,16]. In a more recent study, Gibb et al. [17] were able
to achieve a fertility rate of 27% after inseminating mares
with frozen sex-sorted sperm by hysteroscopic insemi-
nation. Unfortunately, there was a higher than normal
incidence of early embryonic death. Although sperm
parameters such as morphology, motility, and viability are
similar between sex-sorted and nonsorted frozen-thawed
semen, the fertility rates when used are significantly
different. The reason for this is unclear, and much research
is needed in this area to determine whether the
mechanical or biochemical effects of sex-sorting are
responsible for the reduction in fertility of cryopreserved
sex-sorted semen.
5. Assisted Reproductive Technologies and Sex-Sorted
Sperm
A major breakthrough with sex-sorted semen would be
the possibility of producing embryos by intracytoplasmic
sperm injection. Cleavage rates reported to date are of less
than 10%, after oocyte sperm injection. In 2010, using two
different stallions to inject transvaginal aspirated oocytes at
two different laboratories resulted in no embryos.
6. Commercial Use of Sex-Sorted Stallion Semen
The full impact of this technology will not be reached
until a system is developed to cool and freeze sex-sorted
stallion semen. However, with the current technology, the
semen can be sent to the laboratory where the sorting
equipment and the mares are located. To be able to increase
the availability of sorting for more stallions, mobile labo-
ratories have been designed. Currently, one or two
conventional breeding doses of 500 million can produce
one sex-sorted breeding dose and with an expected fertility
rate of more than 60% per cycle when mares of normal
fertility are inseminated.
The effects of the sorting procedure on the sperm
membranes, mitochondria, or DNA are presently unknown.
Once these questions are answered with dedicated
389
personnel, preservation protocols before and after sorting
will be able to be designed. The equine industry will need
to adapt to the technology, and the company providing the
service will need to adapt to the equine industry. Extrap-
olating current procedures proved to be successful in the
bovine to the horse will not serve well the equine industry
and might preclude the widespread use of this technology.
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