Animal Reproduction Science 146 (2014) 34–41

Contents lists available at ScienceDirect

Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

Review article

Sex determination in horses—Current status and

future perspectives
Christine Aurich ∗ , Jana Schneider
Centre for Artificial Insemination and Embryo Transfer, Vetmeduni Vienna, Vienna, Austria

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

Article history: In the equine species, sex determination of the conceptus is of growing interest for the
Received 30 September 2013 breeding industry. In horses, the sex ratio of the offspring depends on changes in body
Received in revised form 23 January 2014 condition of the mother at conception and under natural conditions may thus markedly
Accepted 25 January 2014
deviate from an expected 1:1 ratio. Insemination with sex-sorted spermatozoa allows a
Available online 6 February 2014
pronounced shift of the sex ratio but at present pregnancy rates are low and vary consider-
ably under field conditions. In equine embryo transfer programmes, sex determination in
Keywords: embryos before transfer via genetic methods is a promising approach with high reliability.
Horse In ongoing pregnancies, fetal sex can be determined in utero by transrectal or transabdo-
Sex determination minal ultrasound between days 57 and 220 after ovulation, but experience is required to
Embryo achieve satisfying accuracy. Recently, genetic sexing via identification of circulating cell-
Fetus free fetal DNA in the maternal circulation has been successfully performed in the last three
Environment months of pregnancy. Development of this technique may also allow fetal sex determina-
tion at earlier stages of pregnancy. Further research is required to allow for techniques that
enable sex determination in equine embryos as well as in ongoing pregnancies under field
conditions.
© 2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
1.1. Embryo transfer as an application of sex determination in horse breeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
1.2. Aims of the review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2. Manipulation of the sex ratio in offspring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.1. Nutritional status of the mother . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.2. Time of mating or insemination in relation to ovulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.3. Insemination with sexed semen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3. Sex determination in early equine embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.1. Detection of sex-specific antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.2. Detection of sex-specific DNA sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.3. Detection of sex-specific RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
4. Determination of fetal sex in utero . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
4.1. Ultrasonographic identification and localization of the genital tubercle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
4.2. Ultrasonographic identification of fetal primary sex organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

∗ Corresponding author Tel.: +43 1 250776400; fax: +43 1 250775491.

E-mail address: christine.aurich@vetmeduni.ac.at (C. Aurich).

0378-4320/$ – see front matter © 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.anireprosci.2014.01.014
 C. Aurich, J. Schneider / Animal Reproduction Science 146 (2014) 34–41 35

4.3. Analysis of fetal sex in cells collected from the placenta or fetal fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
4.4. Analysis of fetal sex in fetal material collected from the maternal circulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

1. Introduction are much higher. Thus, in Germany 501 and in France 816
embryo transfers were registered for Warmblood mares
In many farm animal species, the preference of a specific in 2011 (German Equestrian Federation, 2012; French
sex is obvious and mainly due to commercial implications, National Studs, 2012). This illustrates that the IETS data
e.g. economic benefit of producing female calves in dairy may in general underestimate equine embryo transfer
cattle or male calves in beef cattle. In contrast, in horses, activities. Altogether, the interest in embryo transfer
the choice between a desired and non-desired sex is often has increased also in European sport horse breeding
based on individual considerations. In Polo sports there programmes (Aurich and Aurich, 2006). One specific aim
is a higher demand for the female sex (Panarace et al., is the production of offspring from successful mares in
2013), while in Thoroughbred racing or classical riding parallel to performance in equestrian sports. A possibility
competitions the male sex is preferred. At Thoroughbred to predict the sex of the embryo before transfer might
auctions, higher prices are paid for male than for female further stimulate the use of this biotechnology.
yearlings (Chezum and Wimmer, 1997). At the Aachen Embryo production in horses with regard to the num-
International horse shown (CHIO) in Germany only 24.1% ber of transferrable embryos produced per mare is limited
(1641 out of 6801) of the horses started in the final because even under experimental conditions superovula-
dressage and show jumping competitions between 1928 tion rates are low and at present no drugs for induction
and 2010 were female (Aurich and Spicker unpublished). of multiple follicle ovulations are commercially available
In western riding, females are preferred as cutting horses, (Roser and Meyers-Brown, 2012). Thus, embryo collection
but males are more desired as reining horses. In yearling is usually performed in non-manipulated oestrous cycles
Quarter horses bred for racing, prices paid for fillies exceed with a mean embryo recovery rate of at least 50% (Squires
those paid for colts and geldings (Lansford et al., 1998). et al., 1999; Koblischke et al., 2010). In an individual mare,
Possibilities to determine the fetal sex may thus influence embryo collection can be performed in successive oestrous
the mare owner’s choice of the sire for this specific mare for cycles eventually leading to production of several foals per
the upcoming breeding season, the decision to sell a mare season (Aurich et al., 2011).
pregnant with a fetus of the undesired sex or the value of
the mare if she is being sold with a fetus of the preferred sex. 1.2. Aims of the review

1.1. Embryo transfer as an application of sex The present review aims to give an overview on the
determination in horse breeding possibilities to either manipulate the sex ratio in equine
offspring, to determine embryonic sex before transfer or to
Until now sexing in horses is mainly performed by fetal assess fetal sex during later stages of pregnancy. In addition
ultrasonography at about 2 months of pregnancy (Curran to the description of methods that have already been pub-
and Ginther, 1989; Renaudin et al., 1999). Interruption lished for the horse, a perspective is given to approaches
of pregnancy in recipient mares carrying a fetus of the that might gain interest in this species in the near future.
undesired sex is becoming common practice in Argentina
(Aguilar et al., 2012). Methods to allow selection for the 2. Manipulation of the sex ratio in offspring
desired sex before or at a very early stage of pregnancy are
thus preferable. The introduction of genetic sex determi- 2.1. Nutritional status of the mother
nation in embryo transfer programmes is certainly among
the most promising applications of this technique for the The Trivers–Willard hypothesis proposes that where
horse. one sex has more variable reproductive success (i.e. males
Equine embryo transfer has become increasingly in polygynous species), mothers in good condition would
important during the last decade, especially in North be advantaged by producing more of that sex, whereas
and South America. In 2011, worldwide a total of 40,833 mothers in poor condition would be advantaged by pro-
embryo flushes in mares have been performed resulting in ducing more of the reproductively stable sex (Trivers and
29,132 (71%) transferrable embryos. The vast majority of Willard, 1973). In agreement with this hypothesis, in many
embryo collections (28,624) were performed in Argentina mammalian species the theoretically expected 1:1 sex
and Brazil, while in the US another 9916 embryo flushes ratio of offspring is influenced by the environmental con-
in horses were done. In contrast, the official number of ditions present at conception (e.g. Rutberg, 1986; Kojola
embryo collections in Europe is still rather small with 694 and Eloranta, 1989; Wauters et al., 1995; Flint et al., 1997;
flushes being reported to the International Embryo Trans- Krackow and Burgoyne, 1997; Luna-Estrada et al., 2006). It
fer Society (IETS) for 2011 (Stroud, 2012). Interestingly, the is postulated that the external environment is signaled to
numbers reported to the respective national institutions the conceptus by the mother via the uterine environment
36 C. Aurich, J. Schneider / Animal Reproduction Science 146 (2014) 34–41
(Aiken and Ozanne, 2013). Nutritional status of the mother
seems to be of utmost importance for this signal. In several
species, male embryos have a better chance than females
to survive in an enriched uterine environment i.e. under
high glucose concentrations (Avery et al., 1992; Enright
et al., 2001; Gutiérrez-Adan et al., 2001; Larson et al.,
2001; Cameron, 2004). Similarly, in Camargue horses the
sex ratio of foals was female-biased in years following
a breeding season of poor food availability. However, it
was unclear whether this shift resulted from influences
on conception or differences in loss rates between female
and male fetuses (Monard et al., 1997). The results were
supported by investigations in feral horses in New Zealand
(Kaimanawa horses). In this study, mares that gave birth to
a female foal were in poorer condition at conception than
those that produced a male (Cameron et al., 1999). Because
foaling rates did not differ between mares in good and in
bad condition at conception and mare condition at mid- or
late-gestation did not influence foal sex it can be concluded
that deviations in sex ratio of foals born to mares with dif-
ferent body condition at conception are not a result of fetal
loss. Thus, sex ratio variation of offspring is probably influ-
enced close to conception (Cameron et al., 1999). When the
data from that study were re-evaluated and not body con-
dition itself but the change in body condition between the
pre-conception and post-conception periods was analysed,
influences on sex ratio became even more pronounced.
In mares losing body condition at the time of conception,
the percentage of male foals was reduced to 3%, while it
increased up to 80% when mares gained weight at concep-
tion (Cameron and Linklater, 2007). These results provide
strong support for the Trivers–Willard hypothesis in the
horse. They also demonstrate that changes in body condi-
tion at conception are more predictive of fetal sex than the
body condition itself (Cameron and Linklater, 2007). This
suggestion is in agreement with the finding that changes
in maternal glucose levels mediate sex ratio of offspring
(Cameron, 2004; Gutiérrez-Adan et al., 2006; Cameron
et al., 2008). Similarly, under in vitro conditions, bovine
female embryos exposed to high glucose concentrations
fail to develop into expanded blastocysts (Larson et al.,
2001). Similar studies have not yet been performed in the
horse. However, a considerably higher gene expression of
IGF-1 in female than in male equine embryos at least until
day 12 after ovulation (Beckelmann et al., 2013) may reflect
a mechanism to counteract excess death of female con-
ceptuses under detrimental conditions e.g. when exposed
to high glucose concentrations. IGF-1 has been shown
to support the survival of early embryos because of its
anti-apoptotic properties (Herrler et al., 1998; Fabian et al.,
2004; Block et al., 2007). Increased IGF-1 gene expression
may thus be an advantage in female embryos (Beckelmann
et al., 2013). This is in agreement with the hypothesis that
female pregnancies have a better placental adaptation to
adverse conditions than male (Aiken and Ozanne, 2013).
In conclusion, it is suggested that manipulating the envi-
ronmental conditions of mares at conception or during
early pregnancy influences the chance of embryos from one
sex to survive and can thus bias the sex ratio of offspring.
It is important to note that this may also hold true for any
in vitro environment to which embryos are exposed under
the conditions of biotechnologies such as embryo transfer
or preservation.
2.2. Time of mating or insemination in relation to
ovulation
Sex ratio of offspring is often believed to be influenced
by the time of mating or insemination in relation to the
time ovulation. Such a sex bias could be related to influ-
ences of the reproductive tract on the transport of either X-
or Y-chromosome bearing spermatozoa, preferential selec-
tion of sperm at fertilization or sex-specific embryonic
losses. There are numerous publications on this topic with
conflicting results. In cattle, any effect of time of insemina-
tion on sex ratio of offspring if present at all seems to be
small (Rorie, 1999). For horses, to the best of our knowl-
edge, no controlled studies exist but experience does not
support the hypothesis (Samper et al., 2012a).
2.3. Insemination with sexed semen
A pronounced shift in sex ratio of the offspring to the
desired sex already at conception has been realised by the
insemination of horses with semen sorted by high-speed
flow cytometric separation of X and Y-chromosome-
bearing spermatozoa (Samper et al., 2012a, 2012b). The
accuracy of this procedure is relatively high with a purity
of spermatozoa of the desired sex of >90%. Despite this
fact, the use of sex-sorted semen in horse breeding is still
limited by low sorting rates and detrimental effects of
the sorting process on sperm function (Buchanan et al.,
2000; Samper et al., 2012a, 2012b). Despite an enormous
improvement of sorting rates, the process remains inef-
ficient for the production of standard AI doses (250 to
500 million sperm per AI) required in the horse (Samper
et al., 2012a). Thus insemination in horses with sexed
semen usually has to be performed with semen doses
much lower than the standard AI dose. To enhance the
chance of conception semen is deposited directly on the
uterine papilla. With this technique, pregnancy rates with
fresh sex-sorted semen average between 10% and 50% (e.g.
Buchanan et al., 2000; Lindsey et al., 2002; Clulow et al.,
2008; Panarace et al., 2013). Fertility after insemination of
sex-sorted semen from individual stallions varies between
0% and 100% (Panarace et al., 2013). At present the use of
sex-sorted semen in horse breeding is further limited by
the fact that cooling and freezing of semen after the sorting
procedure is detrimental to fertility (Samper et al., 2012a,
2012b).
3. Sex determination in early equine embryos
3.1. Detection of sex-specific antigen
In early embryos of different species sex determina-
tion has been realised via identification of the male specific
H–Y antigen by fluorescing antibodies. In equine embryos
this method resulted in an overall accuracy of 82% when
compared to results obtained by karyotyping (Wood et al.,
1988). The immunological approach is beneficial because
of its non-invasiveness. However, the method has not been
 C. Aurich, J. Schneider / Animal Reproduction Science 146 (2014) 34–41
used in large-scale equine embryo transfer so far. Similar
methods for sex determination in embryos were realized
for several other mammalian species, including mouse, rat,
pig, sheep, goat and cow (Anderson, 1987; Utsumi et al.,
1993; Ramalho et al., 2004; Gardón et al., 2004). In bovine
embryos, determination of embryo sex was verified via
induction of developmental arrest of male embryos dur-
ing transit from the late morula to the blastocyst stage
by incubation in medium containing high titer H–Y anti-
sera (Utsumi et al., 1993; Ramalho et al., 2004). Embryos
that underwent sexing were transferred to recipients and
achieved pregnancy rates of approximately 70%. The over-
all accuracy of sex determination was 81% (Ramalho et al.,
2004). It is important to note that the method does not
prove successful in embryos that have already initiated
blastocoel formation because this will increase false nega-
tive results (Ramalho et al., 2004). Because in equines the
embryo enters the uterus at the late morula or early blas-
tocyst stage (Battut et al., 1997) only a small percentage
of embryos recovered from the uterus would be applica-
ble for this method. The collection of earlier embryos with
non-invasive methods is at present not routinely possible
in the horse.
3.2. Detection of sex-specific DNA sequences
Sex determination in cattle embryos before transfer is
routinely and successfully achieved by DNA amplification
using polymerase chain reaction (PCR; Aasen and Medrano,
1990; Peura et al., 1991). Early bovine embryos are sexed
with nested PCR using DNA extracted from as few as two
to four blastomeres (Kirkpatrick and Monson, 1993). How-
ever, equine embryos were much more sensitive to cell
biopsy than those from other species because successful
development in the uterine environment is dependent on
an intact acellular capsule (Skidmore et al., 1989; Stout
et al., 2005). This capsule develops between the trophoblast
and the zona pellucida and surrounds the equine embryo
until approximately day 21 of pregnancy (Flood, 1975;
Flood et al., 1982; Betteridge et al., 1982). Early attempts
for genetic sexing of cell material recovered from equine
embryos via microblade dissection and subsequent trans-
fer of the embryos resulted in pregnancy rates as low as
21% (Huhtinen et al., 1997). Recently, successful transfer
of equine embryos after biopsy has been reported (Choi
et al., 2010). Viability was maintained not only in small
embryos collected on day 6 where the capsule has not yet
completely formed but also in larger embryos collected
on days 7 to 8 with confluent capsules. Most probably
a smaller degree of capsule disruption is the explana-
tion for the success. The authors suggest that the small
size of the capsular defect did either not affect the abil-
ity of the embryo to develop in the uterus or was even
repaired.
At present PCR aimed to determine embryo sex is mainly
performed with specific genes located on the Y chro-
mosome. During evolution these genes have undergone
a variety of changes such as degeneration, duplication,
translocation, inversion and loss of gene content (Graves,
1998; Skaletsky et al., 2003). Comparative mapping of these
genes between species is rarely useful. Some exceptions of
37
Y-chromosome-specific genes include the zinc finger genes
(ZFX and ZFY) which show a high degree of conservation
in the bovine, porcine and equine species (Poloumeinko,
2004). ZFX and ZFY have also successfully been used for
equine embryo sexing (Peippo et al., 1995; Huhtinen et al.,
1997). Another gene used for embryo sexing also in the
horse is the sex-determining region of the Y chromosome
(SRY; Choi et al., 2010; Jarazo et al., 2012; Beckelmann et al.,
2012). Because ZFY and SRY are present in only one copy
(Hirota et al., 2001) single cell PCR identification may be dif-
ficult. Genes present in multiple copies could enhance the
sensitivity of PCR for sex determination (Choi et al., 2010).
In the study by Choi et al. (2010), sex determination was
performed successfully by duplex PCR of the male specific
SRY-gene. PCR amplification was possible in 20 out of 24
samples. Nine of the pregnancies were allowed to be car-
ried to term. In all of them, sex determination of the embryo
before transfer proved to be accurate (Choi et al., 2010).
The technique requires sophisticated equipment as well
as skilled personnel and may thus not be applicable under
field conditions or in regions where it is only sporadically
requested. However, even overnight shipment of embryos
before collection of cellular biopsies did not impair preg-
nancy results and suggests that such a procedure could
be offered by specialized central laboratories (Choi et al.,
2010).
3.3. Detection of sex-specific RNA
Female somatic cells are equipped with two X chro-
mosomes and could thus theoretically produce twice as
much X-specific transcripts as male cells (Epstein et al.,
1978). However, to avoid over-expression, one X chromo-
some is inactivated to compensate for the extra dosage
of X-linked genes in females. X-inactivation requires a
specific RNA called X-inactivated specific transcript (Xist)
RNA. Xist is a long non-coding RNA that inactivates one
X chromosome by coating it in each female cell (Rastan,
1994; Penny et al., 1996). Based on the knowledge on
X-chromosome inactivation, an approach for sex determi-
nation in equine embryos was published in Romagnano
et al. (1987). Sex determination was performed by C-
banding while visualization of R-bands after incorporation
of 5-bromodeoxyuridine allowed identification of inactive
X chromosomes. The cytogenetic approach also enabled the
detection of X chromosome activity within individual cells.
Sexing was possible in 77% of embryos, but uneven incorpo-
ration of 5-bromodeoxyuridine may have biased the results
(Romagnano et al., 1987). Inactive X chromosomes were
detectable from day 7.5 after ovulation onwards with first
positive signals occurring in the trophoblast. The authors
concluded that exploitation of X-linked factors for differen-
tiation between male and female embryos would need to be
directed at conceptuses collected before day 7 (Romagnano
et al., 1987). In the horse, embryo collection from the uterus
is not possible before day 6 but still results in low recov-
ery rates because the majority of embryos enter the uterus
on day 6.5 or 7 after ovulation (Battut et al., 1997). More
importantly, the technique requires embryo fixation and is
thus not applicable for sexing of embryos with the aim to
produce foals.
38 C. Aurich, J. Schneider / Animal Reproduction Science 146 (2014) 34–41
Recently, determination of Xist RNA has been pub-
lished to allow sex determination of whole female equine
embryos (Beckelmann et al., 2012). With an error rate
of 10% by qualitative PCR and an even higher accuracy
by quantitative PCR, results of the study are thus com-
parable to those of routine sex determination in bovine
embryos (e.g. Hasler et al., 2002). Embryos collected from
day 8 of pregnancy onwards allowed collection of sufficient
amounts of RNA. The amount of DNA might be a limiting
factor for reliable results of PCR at very early stages of
pregnancy. Xist may also be an interesting candidate for
sex determination in the horse with specific fluorescence
in situ hybridization (FISH) probes. Fixed biopsies of bovine
embryos have been analysed by FISH (Lee et al., 2004;
Cenariu et al., 2008) and a method for in situ hybridization
of RNA in living cells does exist (Paillasson et al., 1997).
Establishing similar techniques for introducing Y and X-
chromosomal-specific probes into living embryos could
enable sex determination by FISH before transfer. However,
it is still unclear whether such a technique is applicable for
the equine embryo due to the existence of the acellular
capsule.
4. Determination of fetal sex in utero
4.1. Ultrasonographic identification and localization of
the genital tubercle
In the horse, fetal sex can be determined by transrectal
ultrasonographic identification and localization of the gen-
ital tubercle. At day 55 of pregnancy, the genital tubercle is
located between the fetal hind limbs at an approximately
equal distance from the tail and the umbilical cord. Sub-
sequently it migrates towards the tail in the female and
the umbilical cord in the male which allows sex deter-
mination. The optimal time is between days 59 and 68 of
gestation (Curran and Ginther, 1989; Curran, 1992; Bucca,
2005). Success rates of the technique vary in a wide range
and depend much upon expertise of the examiner (Holder,
2000; Mari et al., 2002; Bucca, 2005). Sex determination in
equine fetuses via transrectal ultrasound is thus not com-
mon practice, but in some geographical areas with large
broodmare populations the demand on fetal sex determi-
nation via transrectal ultrasound is high. In Kentucky, from
more than 2500 sex determinations performed over an
eight-year period by an examiner with appropriate exper-
tise and experience only two proved to be wrong (Holder,
2000).
4.2. Ultrasonographic identification of fetal primary sex
organs
After day 70 of gestation the fetus becomes more and
more inaccessible to transrectal ultrasound due to its
position deep in the mare’s enlarging uterus. From day
100 onwards visualisation of the fetus by transabdomi-
nal ultrasound is possible. Combination of transrectal and
transabdominal ultrasound allows visual examination of
the entire fetus with reliable identification of the fully
developed fetal primary sex organs. Between approxi-
mately days 100 and 220 of pregnancy the accuracy of
this technique is high (Bucca, 2005), but subsequently it
is increasingly difficult to identify anatomical structures
like penis and prepuce in male or mammary glands and
teats in female fetuses (Renaudin et al., 1999). Recently it
has been published that 3D ultrasonography of equine fetal
sex organs also allows identification of the genital tuber-
cle between days 63 and 76 as well as detailed imaging of
the genital organs between days 90 and 150 of pregnancy
(Kotoyori et al., 2012).
4.3. Analysis of fetal sex in cells collected from the
placenta or fetal fluids
To the best of the authors’ knowledge, determination
of fetal sex in cells collected via amniocentesis from fetal
fluids has never been reported for the horse. In cattle,
this technique was applied already in Bongso and Basrur
(1975). During this time, sex determination was performed
via chromosome analysis. The technique was improved by
the introduction of PCR methods that allowed sexing of
bovine fetal cells collected by amniocentesis via determi-
nation of sex-specific DNA. This method was first published
in Kamimura et al. (1997) and Makondo et al. (1997). The
practicability of the technique is limited because the col-
lection of fetal fluid is challenging. However, in most cases,
fetal fluid was successfully collected at the first needle pen-
etration (29 out of 33 pregnancies), but in other animals
repeated needle penetrations were necessary to aspirate
fetal fluid. In total, 5 out of 35 animals aborted within 1
week after aspiration of amniotic fluid (Kamimura et al.,
1997). Obviously, expertise and experience decreased the
risk of abortion (Makondo et al., 1997) which in horses is
expected to be higher than in cattle.
4.4. Analysis of fetal sex in fetal material collected from
the maternal circulation
A new promising method for fetal sexing in the horse
is the identification of circulating cell-free fetal DNA
(ccffDNA) in maternal blood (De Leon et al., 2012). The
risk of this technique for mother and fetus is minimal as
it requires only collection of maternal blood. The absence
of Y-chromosome sequences in maternal plasma implies
that the fetus is female; however, this may also be the con-
sequence of undetectable concentrations of ccffDNA in the
presence of male fetuses. When sex determination was per-
formed in 20 Thoroughbred mares in the final 3 months of
pregnancy, overall accuracy was 85% (De Leon et al., 2012).
The significance of this or a similar method would be even
greater if it could be implemented at an earlier stage of
pregnancy. At present it is unknown whether fetal cells
from the chorionic girdle happen to enter the maternal cir-
culation at the time of invasion of the endometrium. This
takes place as early as day 37 of pregnancy (Allen, 1975).
Equine chorionic girdle cells are extremely invasive and
behave similar as cancer cells. The possibility of intrusion
into blood vessels can thus not be excluded and would form
a basis for a very early presence of fetal DNA in maternal
blood.
Identification of male fetal cells in maternal blood could
also be possible by fluorescent activated cell sorting or
 C. Aurich, J. Schneider / Animal Reproduction Science 146 (2014) 34–41 39

Table 1
Present methods applicable for sex determination in the equine conceptus with the aim to produce living foals.

Time of pregnancy Method Reliability (%) Present limitations References

Conception
Day 6 to 8 of pregnancy
(blastocyst stage)
Day 59 to 68 of pregnancy
Day 100 to 220 of pregnancy
Day 90 to 150 of pregnancy
Final 3 months of pregnancy
High-speed flow cytometric
separation of X and
Y-chromosome-bearing
spermatozoa (“semen sorting”)
prior to insemination
Genetic sexing of cell material
collected by embryo biopsy
Transrectal ultrasonographic
identification and localization
of the genital tubercle
Identification of the fetal
primary genital organs by
transabdominal ultrasound
3D ultrasonography of equine
fetal sex organs
Identification of circulating
cell-free fetal DNA in maternal
blood
>90 Low sorting rates Buchanan et al. (2000),
detrimental effects of sorting Clulow et al. (2008), Samper
process on sperm function et al. (2012a,b), Panarace
et al. (2013)
>90 Sophisticated equipment Choi et al. (2010)
skilled personnel
>99 Sufficient experience of the Curran and Ginther (1989),
examiner Curran (1992), Bucca (2005),
Holder (2000)
>90 Sufficient experience of the Renaudin et al. (1999), Bucca
examiner (2005)
No information Sophisticated equipment Kotoyori et al. (2012)
85 Unclear, whether fetal cells De Leon et al. (2012)
remain in the circulation
beyond parturition

immunomagnetic cell sorting which are the most common
methods for cell separation in non-invasive prenatal diag-
nosis in humans (Kavanagh et al., 2010). A similar approach
is the identification of XX/XY-karyotypic chimerism in cat-
tle. In suspected animals, FISH has been demonstrated as
a rapid and reliable procedure for identification of bovine
freemartinism in uncultured blood cells (Sohn et al., 2007).
However, whether fetal cells remain in the maternal cir-
culation beyond parturition and thus during subsequent
pregnancies has not been investigated in the horse so far.
This would limit the approach of the detection of male fetal
cells to mares that have not experienced a pregnancy with
a male conceptus.
5. Conclusion
Although a number of possibilities for determination of
the sex of the conceptus in horses exist (Table 1), the major-
ity of these approaches have high demands on equipment,
expertise and experience. Further research is required to
develop techniques that allow sex determination in equine
embryos as well as in ongoing pregnancies under field con-
ditions.
Conflict of interest
The authors do not report any conflict of interest.
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