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Animal Reproduction Science 87 (2005) 93109

The freemartin syndrome: an update


A.M. Padula

Division of Farm Animal Science, Department of Clinical Veterinary Science,


University of Bristol, Langford House, Langford,
Bristol BS40 5DU, UK
Received 20 July 2004; received in revised form 30 September 2004; accepted 30 September 2004
Abstract
The freemartin condition represents the most frequent form of intersexuality found in cattle, and
occasionally other species. This review considers the current state of knowledge of freemartin biol-
ogy, incidence, experimental models, diagnosis, uses for freemartins in cattle herds, occurrence in
non-bovine species, effects on the male, and highlights potential new research areas. Freemartins
arise when vascular connections form between the placentae of developing heterosexual twin foeti,
XX/XY chimerism develops, and ultimately there is masculinisation of the female tubular reproduc-
tive tract to varying degrees. With twinning rates in Holstein cows increasing, there will be greater
economic importance to establish early diagnosis of the freemartin and the detection of the less com-
mon single born freemartin. New diagnostic methods based on the detection of Y-chromosome DNA
segments by polymerase chain reaction (PCR) show improved assay sensitivity and efciency over
karyotyping and clinical examination. The implications for the chimeric male animal born co-twin
to the freemartin are contentious as to whether fertility is affected; if germ cell chimerism does in-
deed occur; and, if there are any real effects on the sex ratio of offspring produced. In beef cattle, the
freemartin carcass has similar characteristics to normal herdmates. Hormonal treatment of freemartins
for use as oestrous detectors has been used to obtain salvage value. The biology of freemartin sheep
has recently been studied in detail, and the condition may be increasing in prevalence with the in-
troduction of high fecundity genes into ocks. Potential new research areas are discussed, such as
detection of foetal DNA in maternal circulation for prenatal diagnosis and investigation of the anti-
tumour properties of M ullerian inhibiting substance (MIS). The freemartin syndrome will always
be a limiting factor in cattle and to a lesser extent in sheep production systems that have the goal

Tel.: +44 117 331 9062; fax: +44 117 928 9582.
E-mail address: andrew.padula@bristol.ac.uk.
0378-4320/$ see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2004.09.008
94 A.M. Padula / Animal Reproduction Science 87 (2005) 93109
to produce multiple reproductively normal female offspring from a single dam without using sex
predetermination.
2004 Elsevier B.V. All rights reserved.
Keywords: Freemartin; Bovine; Chimera; Gender determination; DNA; XX/XY
1. Introduction
The freemartin syndrome represents the most frequent form of intersexuality found in
cattle, and less commonly in other species. Freemartins arise when vascular connections
form between the placentae of developing heterosexual twin foeti, and the result is mas-
culinisation of the female reproductive tract to varying degrees. The literature concerning
the fundamental biology of the freemartin syndrome was last reviewed in detail over 30
years ago (Marcum, 1974) although was initially well described much earlier (Lillie, 1917).
Since then, a number of authors have reviewed specic aspects of the freemartin syndrome
(Jankowski and Ildstad, 1997; Cribiu and Chaffaux, 1990; Kastli, 1979; Long, 1979). The
objective of this review is to collectively consider new studies on the freemartin condition,
with reference where necessary, to earlier original studies avoiding repetition of the basic
biology of the condition that has previously been reviewed.
2. Single born freemartins
The singleton birth of freemartins has rarely been reported in the literature, although they
have been suspected for some time (Makinen, 1974). Multiple single born freemartins were
described in a group of subfertile Friesian heifers that had detectably normal internal repro-
ductive tracts, of which 5 of 12 were XX/XY chimeras and from single births (Wijeratne
et al., 1977). It was suspected that these freemartins were the result of in utero death of
a male co-twin. In a study where transfer of multiple embryos resulted in the subsequent
birth of only single calves, 1 of the 22 female calves born had an XX/XY karyotype, but an
almost normal internal reproductive tract (Kadokawa et al., 1995). Again it was suspected
that a male twin had developed in utero in early pregnancy, sufcient to inuence sexual
development, then was lost, resulting in a single born freemartin. There are two case reports
of instances where a freemartin was born co-twin to a grossly abnormal calf (Meinecke
et al., 2003; Cavalieri and Farin, 1999).
The incidence rate of single born bovine freemartin calves is not known. Although
based on recent studies, the percentage of twins that survive in cattle to full term when
one twin dies appears to be very low. One study in beef cows reported a 4.6% incidence
of single births following twin pregnancy diagnosis on ultrasound between 35 and 70 days
of gestation, but suggested that these may have been errors in diagnosis (Echternkamp and
Gregory, 2002). In a separate study in Holstein cows, a 6.2% incidence rate of embryo
reduction and subsequent birth of single calves was reported following twin diagnosis by
transrectal ultrasonography (Lopez-Gatius and Hunter, in press). Based on these and other
studies the following conclusions can be made: (i) 50% of all twin sets are heterosexual;
A.M. Padula / Animal Reproduction Science 87 (2005) 93109 95
(ii) pregnancy loss rate of approximately 25% of all twins diagnosed at 3642 days after
AI (Lopez-Gatius and Hunter, in press); (iii) approximately 5% survival and singleton birth
following embryo reduction of twin sets (Lopez-Gatius and Hunter, in press; Echternkamp
and Gregory, 2002); and (iv) 90% likelihood of freemartin development in the surviving
twin sets (Marcum, 1974). Twin pregnancies that may have existed prior to earliest possible
ultrasound diagnosis (<36 days after AI) may also be susceptible to developing into a single
born freemartin. Thus, the true incidence of the freemartin syndrome is the sum of known
freemartins from heterosexual births plus the single born freemartins.
Echternkamp (2002) suggested that it was not possible for single born freemartins to
occur because when placental circulation is shared (>90% of cattle twins), the death of one
twin will always result in loss of both, hence their lowincidence rate reported, despite a 50%
live twin birth rate in their study (Echternkamp and Gregory, 2002). The ultrasound study
of Lopez-Gatius and Hunter (in press) does not reveal the nature of the placental circulation
in those twin sets that reduced to singletons and subsequently resulted in live births. It is
possible that in the case where placental circulations are joined (as with freemartins), death
of one foetus would release endotoxins sufcient to cause death of the co-twin making it
extremely rare for a single born freemartin to survive to full termhence the extremely low
incidence. This appears to be the case in human pregnancy with twin transfusion syndrome
(Jain and Fisk, 2004). The true prevalence of single born freemartins remains unknown,
until a reliable, large-scale survey is performed.
3. Experimental methods for producing freemartins
One of the problems with studying freemartinismis reproducing the syndrome efciently
to facilitate a meaningful study. Many different models for induction of freemartinism have
been utilized in studies on this condition and a discussion is given below.
Superovulation with or without multiple embryo transfer is an efcient method of creat-
ing large numbers of freemartin calves for experimental purposes. This method of studying
bovine freemartins has been used for describing morphological changes in foetal gonads
during development of heterosexual twins, following slaughter of dams (van der Schoot
et al., 1995; Dominguez et al., 1990). The limitations of the efciency of this method
are dependent on pregnancy rates to embryo transfer, random generation of foetal gender,
and potential for embryo reduction to singleton pregnancies. Advantages include that the
stage of gestation is precisely known, a control group in the form of isosexual twins is
produced, potential to study the effects on sexual development when one twin dies (e.g.
single born freemartin). Multiple pregnancies without embryo transfer were created by in-
duction of superovulation using, 20003000 iu equine chorionic gonadotrophin (eCG) 4
days before oestrus and was used to produce 29 cases of multiple pregnancies with het-
erosexual foetuses and fused chorionic vessels, giving 48 presumptive freemartins (Jost
et al., 1972). Of these, 1 pregnancy had no chorionic fusion, and 12 pregnancies had foe-
tuses of the same sex (Jost et al., 1972). Through the use of dye injection of placental vessels,
the anatomical location of chorionic vascular anastomoses, which are well developed by 39
days of gestation, was described and used to rene the denition of a freemartin foetus for
experimental purposes (Dominguez et al., 1990).
96 A.M. Padula / Animal Reproduction Science 87 (2005) 93109
The United States Department of Agriculture began an experiment over 20 years ago to
investigate the potential of twinning in beef cattle to improve farm protability. Continued
selection pressure has increased the proportion of cows giving birth to twins from around
1% to more than 50% in the year 2000 (Echternkamp and Gregory, 2002). This has led to
a unique setting in which to investigate the growth and biology of freemartins from natu-
rally occurring cases. Triplets occurred at a rate of around 1.85% diagnosed by transrectal
ultrasonography at 3675 days gestation, but a live birth rate of only 0.60%, allowing some
study of freemartinism in bovine triplets (Echternkamp and Gregory, 2002).
Twinning rates appear to be increasing in Holstein cows, and this may be further increased
by the use of ovulation synchronisation programs (Fricke and Wiltbank, 1999). One study
reported double ovulations at 14.1% and live born twin sets at 5.2% (Fricke and Wiltbank,
1999), and based on these gures, sufcient numbers of freemartins could be obtained for
experimental purposes from within a single large dairy herd.
Because the condition of freemartinism is the result of vascular connections between
male and female foetuses, the surgical creation of articial anastomoses was investigated
as a method of creating ovine freemartins (Marcum et al., 1985). The technique involved
creating surgical stulas between the dorsomedial walls of the uterus of days 2840 ovine
pregnancies such that the chorioallantoic membranes were in contact. However, the tech-
nique described was unable to produce any ewes with functional connections and was not
recommended.
The presence or absence of testosterone during critical periods of development has
been used to produce masculinisation of female foetuses, although not truly a model for
freemartinism because of failure to produce XX/XY chimerism. Testosterone treatment of
pregnant bitches results in the formation of a masculinised canine female reproductive tract
following birth, aspects of which may not become obvious until puberty (Johnston et al.,
2001). In sheep, prenatal androgen exposure resulted in failure to have regular oestrous
cycles and conceive (from Aldrich et al., 1996). Although in cattle, similar treatment from
days 60 20 of gestation did not appear to affect reproductive performance and subsequent
lactation (Reiling et al., 1995).
One approach has been to use endogenous M ullerian inhibitory substance (MIS) to
suppress development of tubular reproductive tract structures in utero. Asuitable method of
achieving this has been by the creation of transgenic mice that produce MISduring gestation,
and this results in the non-formation of tubular internal reproductive tracts in pups. In male
mice foetuses born to mothers that express high levels of MIS, some feminsation of the
tract occurs (Behringer, 1995).
4. Diagnosis
The challenge remains to develop a rapid, sensitive, specic and inexpensive test for
freemartinism that can be used from a young age or even during gestation. A freemartin
may be suspected based on known history of having been born co-twin to a male, but single
born freemartins would be missed and only 8095% of females born co-twin to a male are
freemartins (Marcum, 1974). A summary of the various diagnostic methods used for the
freemartin syndrome is shown in Table 1.
A.M. Padula / Animal Reproduction Science 87 (2005) 93109 97
Table 1
Summary of diagnostic methods described for the diagnosis of bovine freemartins
Method Diagnostic criteria References
Clinical
Vaginal length Normal female calf 1315 cm;
freemartin calves up to 30 days old 5 to
8 cm length. Normal adult female 30 cm;
freemartin adult 810 cm
Khan and Foley (1994); Zhang et al. (1994);
Long (1990); Miyake et al. (1980); Greene
et al. (1979); Kastli (1974); Marcum (1974)
Internal tract Varies from near normal to almost
entirely masculine. Absence of
identiable cervix
Long (1990); Kastli (1974); Marcum (1974)
Karyotyping Identication of at least one Y
chromosome on a karyotype
Zhang et al. (1994); Dunn et al. (1981)
Blood grouping Demonstration of partial haemolysis of
red blood cells
Long (1990); Stormont (1982); Mayr and
Hager (1978); Marcum (1974)
DNA analysis
AMX/Y Identication of a 217 bp fragment from
Y-chromosome
Ennis et al. (1999); Ennis and Gallagher
(1994)
BOV97M Identication of a single 157 bp fragment
from Y-chromosome
Ennis et al. (1999); Oh et al. (1997); Miller
and Koopman (1990)
BRY Identication of fragment from
Y-chromosome, size dependent upon
primer
Cavalieri and Farin (1999); Ennis et al.
(1999); Oh et al. (1997); Olsaker et al. (1993)
ZFX/Y Identication of male specic DNA
fragments following enzyme digestion
Schellander et al. (1992)
SRY Identication of male specic fragment Meinecke et al. (2003); Zeleny et al. (2002);
Utsumi and Iritani (1993)
btDYZ-1 Identication of male specic fragment
on gel electrophoresis
Pessa-Morikawa et al. (2004); Fujishiro et al.
(1995)
FISH Fluorescence following hybridisation of
primer directly to DNA
Meinecke et al. (2003); Zeleny et al. (2002)
H-Y antigen Identication of antigen by immuno-
chemistry
Carlon (1982b); Wachtel et al. (1980); Ohno
et al. (1976)
Hormone assay
Progesterone Non-specic low concentrations Saba et al. (1975); Johnson et al. (1970)
Oestradiol Low oestradiol levels, although not
specic for the freemartin condition
Cavalieri and Farin (1999); Satoh et al.
(1997); Shore and Shemesh (1981); Randel
et al. (1971)
Testosterone Similar levels to normal females, no
increase following hCG stimulation
Cavalieri and Farin (1999); Greene et al.
(1979); Saba et al. (1975)
Gonadotrophins Non-specic attenuated or reduced LH
surge following oestradiol or GnRH
injection
Cunningham et al. (1977); Saba et al. (1976)
MIS Radioimmunoassay in newborn calves
up to 2 weeks old, high levels
(>700 ng/ml). Females <120 ng/ml
Rota et al. (2002)
98 A.M. Padula / Animal Reproduction Science 87 (2005) 93109
4.1. Clinical examination
A consistent clinical nding in young calves has been the presence of a short, blind
ending vagina. This may be readily measured by insertion of a broad blunt ending probe
into the vagina to assess the length noting that care must be taken to avoid catching on the
hymen of normal calves and an incorrect diagnosis (Long, 1990). In normal calves less than
1 month old, the vagina is 135 cm long, but in the case of freemartins it is usually only
58 cm long (Khan and Foley, 1994; Zhang et al., 1994; Long, 1990; Miyake et al., 1980;
Marcum, 1974). In the adult cow, normal vaginal length is approximately 30 cm, while in
the mature freemartin it is only 810 cm. The lack of denable cervix may also be used
clinically as a diagnostic aide when examined using a vaginoscope (Kastli, 1974).
The presence of long and coarse vulval hair, similar to that seen around the prepuce of
male animals is another clinical nding that has been suggested as indicative of freemartin-
ism. However, there appears to be considerable variability in the consistency of this char-
acteristic, making it unreliable for external diagnosis (Gregory et al., 1996; Long, 1990).
Other external changes reported in a study of over 463 malefemale twins over a 5 year
period included an increased ano-vulval distance, occasional enlarged clitoris, and at least
one animal had a scrotal pouch (Gregory et al., 1996). In cases where the clitoris is greatly
enlarged (a case of a 26 cm long clitoris in a Brown Swiss freemartin is recorded), urine
may spurt upward when the animal urinates (McEntee, 1990).
The mature animal may initially present with a history of failure to show oestrous be-
haviour or be non-pregnant after running with a bull. Externally, evidence of secondary
sex characteristics has been in some older freemartins in beef cattle, with thickened necks
and curly hair characteristic of intact males (Gregory et al., 1996). Some anecdotal reports
suggest that freemartins are taller than female herdmates due to excessive long bone growth,
although some researchers have found the opposite (Marcum, 1974). Internally, the repro-
ductive tract has been reported to vary from near normal to almost entirely masculine with
testicle-like structures in place of ovaries (Marcum, 1974). The absence of a cervix is a
consistent nding in freemartins (Long, 1990).
4.2. Karyotyping
Laboratory diagnosis of freemartinism has traditionally been performed by karyotyping
cultured lymphocytes using a standard technique (McFeely, 1993). In cases where XX/XY
chimerismhas occurred with relatively lowfrequency of XY, it is important to examine many
metaphase spreads to be certain of the diagnosis of freemartinism. One study calculated
using a statistical model the number of metaphase spreads that must be examined in order
to be 95 and 99% condent that the animal is a chimera; and, based on 12,885 spreads
examined, they concluded that sample sizes of at least 26 and 168 are required respectively
(Dunn et al., 1981). In a detailed study of 17 heifers, there was no relationship between the
percentage of XY cells present in the freemartin and the severity of the masculinsation that
had occurred (Marcum et al., 1972).
Other chromosomal abnormalities occasionally coincident with XX/XY chimeras have
been reported such as 4/21 tandemfusion (Pinheiro et al., 1995), 1/29 Robertsonian translo-
cation (Zhang et al., 1994; Guanti and Minola, 1978), 61,XXY trisomy (Zhang et al.,
A.M. Padula / Animal Reproduction Science 87 (2005) 93109 99
1994),undetermined centric fusion (Zhang et al., 1994) and mixoploid chromosome con-
stitution (Hare, 1976). Various body tissues such as spleen, bone marrow, lung, connective
tissue, gonad interstitial tissue, lymphoid tissue and liver have been cultured and chimerism
demonstrated, although the percentage of XY cells was lower and more variable than re-
ported for lymphocytes (Marcum, 1974).
4.3. Blood grouping
The probability of fraternal (dizygotic) twins having identical blood groups is extremely
low, however when vascular anastomoses occur as in the freemartin, and red blood cells are
interchanged, antigenic tolerance develops and this has been used for freemartin diagnosis
(Long, 1990; Stormont, 1982; Mayr and Hager, 1978; Marcum, 1974). The blood group
of co-twin dizygotic calves will be derived from two populations. When a blood group
reagent is added that would normally lyse red blood cells of one blood group, only partial
haemolysis occurs. In practice, this test often yields inconclusive results because non-
specic haemolysis occurs due to the continued manipulation of erythrocytes (Kastli and
Hall, 1978).
4.4. Detection of Y-chromosome DNA
The development of the polymerase chain reaction (PCR) for amplication of DNA
sequences marked by oligonueclotide probes for Y-chromosome specic segments, has
revolutionised the approach to gender determination. Sexing of bovine embryo biopsies
prior to transfer has stimulated research into nding primer sequences that are useful and
reliable for sex determination (Lopes et al., 2001; Shea, 1999; Kobayashi et al., 1998). The
absence of Y chromosome sequences in a sample is regarded as exclusion from freemartin
diagnosis (Ennis et al., 1999). Internal control samples usually need to be run to ensure the
PCR reaction has occurred, although in the case of the AMX/Y primer it has the advantage
of generating its own internal control fragment (Ennis et al., 1999).
PCR assay for gender determination from entire animals has typically been reported fol-
lowing DNAextraction fromfresh whole blood; although many tissue types are suitable for
PCR following appropriate extraction procedures. Culture of skin tissue was unsuccessful
for karyotyping freemartins in one study (Short et al., 1969), although other workers have
cultured broblasts from skin biopsies and demonstrated a very low level of chimerism
(Plante et al., 1992; Wilkes et al., 1981). PCR is undoubtedly a far more sensitive assay and
would likely detect chimerism at a level where karyoytping could not.
4.5. Fluorescent in situ hybridisation (FISH)
Hybridisation without prior amplication of template DNAand conjugation of the probe
to a uorescent marker dye has been used for gender determination in animals (Zeleny
et al., 2002). Hybridisation efciency for X- and Y-chromosomes was 77 and 89% respec-
tively in one study (Meinecke et al., 2003). Chimerism was detected in cell cultures of
tissues obtained from a freemartin calf born co-twin to a hydatid mole (Meinecke et al.,
2003).
100 A.M. Padula / Animal Reproduction Science 87 (2005) 93109
4.6. Hormonal methods
Various hormones have been measured in an attempt to either establish diagnosis of
freemartinism, conrm endocrinological functionality of gonadal tissue or as predictors of
potential future fertility. Considering the varying degrees of masculinsation that is reported
to occur in the freemartin, it is not surprising that these tests lack specicity.
4.6.1. Steroid hormones
Overall, bovine freemartins appear to exhibit very low steroidogenic activity despite the
presence of steroid producing cells (both interstitial and granulosa) (Shore and Shemesh,
1981). Oestradiol production from freemartin ovarian tissue has been reported to be below
normal (Cavalieri and Farin, 1999; Satoh et al., 1997; Shore and Shemesh, 1981). Plasma
oestradiol concentrations followingintravenous injectionof 6000 iuof hCGintoa freemartin
showed no change over time when compared with three normal heifers (Cavalieri and Farin,
1999). Similarly, injection of eCG followed by hCG into mature freemartins demonstrated
no increase in oestradiol or progesterone (Satoh et al., 1997). Progesterone concentrations
in plasma of freemartins have been reported to uctuate around baseline levels (<0.4 ng/ml)
with occasional surges up to 1.6 ng/ml (Saba et al., 1975). The levels obviously depend
upon the presence of functional luteal tissue, and in freemartins where no gonadal tissue is
present, lowlevels would be expected. Plasma testosterone concentration of freemartins was
found to be indistinguishable from normal heifers (<10 pg/ml) and not useful for diagnosis
(Saba et al., 1975).
4.6.2. Gonadotrophins
There are very few published reports of plasma gonadotrophin levels in freemartins. In
one study, freemartin heifers showed a diminished or absent LH surge response to injection
0.25 mg or 0.5 mg of oestradiol (Saba et al., 1976). In their study group of freemartins, there
was no correlation between the degree of chimerism and the LH response to oestrogen. In
a separate study, only 7 of 12 freemartins showed a dened LH surge following injection
with oestradiol and these also released greater amounts of LHfollowing a subsequent GnRH
injection (Cunningham et al., 1977).
4.6.3. H-Y antigen detection
The male specic histocompatability antigen (H-Y) has been used to sex bovine embryos
(Utsumi and Iritani, 1993; Avery and Schmidt, 1989). Since the development of highly
sensitive and repeatable PCR-based sexing methods, detection of H-Y antigen for gender
determination is uncommon (Zeleny et al., 2002). Freemartinism has been detected using
this technique (Carlon, 1982a; Wachtel et al., 1980; Ohno et al., 1976).
4.7. M ullerian inhibiting substance
A factor produced by the foetal Sertoli cells during mammalian male sexual differen-
tiation is the glycoprotein M ullerian inhibiting substance, also known as anti-M ullerian
hormone. The development of a sensitive and specic MIS radioimmunoassay allowed for
the detection of 600 times lower concentration of MIS in plasma than in an older bioassay
A.M. Padula / Animal Reproduction Science 87 (2005) 93109 101
(Vigier et al., 1982). It was found that newborn males and freemartins have very high levels
(>700 ng/ml) of MIS in plasma while females have much lower levels (<120 ng/ml) (Rota
et al., 2002). These levels remain elevated for the rst 5 months of life for normal males
and then decline, whilst in freemartins, elevations of MIS are only observed for around 2
weeks after birth before declining to normal female levels (Rota et al., 2002).
5. Uses for freemartins in a cattle herd
In a dairy production system, freemartins are of little economic value because their ability
to conceive and subsequently lactate is impaired, unless a market for dairy beef meat exists.
In contrast, freemartin beef cattle do have carcass value but cannot contribute to the genetic
gain of a herd.
Freemartins have been successfully used for the detection of oestrus. The chronic treat-
ment of 18 freemartin intersexes with testosterone, oestradiol, and oestrone resulted in
display of oestrous behaviour (Greene et al., 1978). Standing oestrus was observed in
freemartins treated with testosterone or oestradiol. Flehmen lip curl was only noted in
freemartins treated with testosterone (Greene and Foote, 1978).
Carcass characteristics of freemartins have been studied in detail. In a study conducted
over 5 years where 463 freemartins were assessed against isosexual twins and steers, birth
weight of freemartins was signicantly heavier than normal twins (37.6 kg versus 35.2 kg),
but did not differ fromnormal twins in subsequent growth rates (0.855, 0.838, 0.843 kg/day;
single, normal twin, freemartin) (Gregory et al., 1996). In that study, freemartins had sig-
nicantly higher marbling scores and a higher proportion of carcasses that were of higher
grade than either single or normal twin calves.
6. Effects on male born co-twin to a freemartin
The fusion of chorionic foetal circulation of malefemale twin sets results in chimerism
in both offspring. It has been reported that the proportion of XX/XYcells in paired twins sets
is very similar and stable over many years (Marcum, 1974). Chimerism is readily detected
in the male, but the reproductive effects of this are continually debated. The literature
concerning this topic has been reviewed (Long, 1979) and only recent studies are discussed
below. There are two issues in relation to bulls that are of interest: (i) sex ratio of the offspring
produced by the male; and (ii) fertility.
The presence of XX cells in bull lymphocytes has been suggested to result in more
female offspring, though this may not be the case with spermatogonia (see Section 7).
The literature on sex ratio was reviewed by Long (1979), of which the conclusion at that
time was that there was . . . probably no shift in favour of bulls producing more female
offspring. The question as to whether or not germ cell chimerism occurs in bulls has been
contentious. Long (1979) concluded based on published literature available at the time, that
no germ cells are exchanged in utero. In support of this statement, no evidence of germ cell
chimerism was detected in 11 lymphocyte XX/XY chimeric bulls (Dunn et al., 1979). In
contrast, more recently it was shown using uorescent in situ hyribisation, spermatogonial
102 A.M. Padula / Animal Reproduction Science 87 (2005) 93109
chimerismdoes occur and was demonstrated in three bulls (Rejduch et al., 2000). The results
of their study showed a difference between the proportion of XX:XY chimeric blood cells
and spermatogonial cells within each bull.
The fertility of bulls born co-twin to a freemartin has been debated for a number of
years. In a study of 22 bulls born co-twin with freemartins, where XX/XY chimersim was
demonstrated on lymphocyte karyotype, a higher proportion (7/12) of chimeras were culled
because of poor fertility than normal controls (7/128) (Dunn et al., 1979). Severe semen
abnormalities were also noted in these chimeric bulls. When examined histologically, the
testes of the culled bulls had large and focal areas of testicular degeneration (Dunn et al.,
1979). In a survey of bulls in a large AI centre in the USA, 6 out of 606 bulls had XX/XY
lymphocyte chimerism (Seguin et al., 2000). Similarly, in a Dutch AI centre, 4 of 300
bulls showed chimerism on karyotype, although lower fertility was not recorded in those
animals (Bosma et al., 1987). Testicular hypoplasia was observed in a 4 1/2 year old bull
with XX/XY lymphocyte chimerism, although all cells cultured from the skin were only
XY (Bongso et al., 1981).
7. Other species
7.1. Ovine
Traditionally, freemartinism in sheep has been regarded as a rare, unimportant abnor-
mality (Marcum, 1974), although more recent studies suggest that it is more common than
previously reported and may be increasing in prevalence. The discovery of high fecun-
dity genes in sheep has led to introduction of genes for multiple ovulation, and the risk of
freemartinism in sheep has been shown to be greater when litter numbers are four or more
(see Smith et al., 2003; Parkinson et al., 2001). Earlier studies reported the incidence of
ovine freemartins at approximately 1% (Marcum, 1974), a similar prevalence was found in
abattoir specimens (1.2%, Long (1980)) and a eld study of male co-twin ewe lambs (see
Parkinson et al., 2001).
Diagnosis of freemartinism in ewe lambs can be made by measuring the vaginal length,
which has been reported as blind ended, less than 5 cm long and without the presence of a
recognisable cervix (Spedding and Dobson, 1989; Wilkes et al., 1978). Oestrous cycles are
absent in pubertal freemartin ewes (Smith et al., 2003) although many freemartins clearly
showmasculine patterns of behaviour (Smithet al., 2000; BosuandBasrur, 1984). Karyotyp-
ing is the denitive method of establishing the presence of 54, XX/XYchimerism(Eldridge,
1985). There are no published reports of PCR-based assays for diagnosing freemartinismin
the sheep, although suitable primer sequences for ovine embryo sexing have been described
(Gutierrez-Adan et al., 1997; Pomp et al., 1995).
Few endocrine studies of freemartin sheep have been reported. In one study, plasma
LH concentrations were signicantly elevated (>10 ng/ml) and plasma progesterone low
(<0.4 ng/ml) in two freemartin ewes studied, and this variable was suggested as a useful
diagnostic test of freemartinism when considered with the history of the animal (Spedding
andDobson, 1989). Ina more detailedstudyof the endocrine variables of 15freemartinewes,
plasma LH was signicantly elevated compared to normal ewes and rams, but the response
A.M. Padula / Animal Reproduction Science 87 (2005) 93109 103
to injection of 250 ng and 10 g GnRH was similar amongst those groups (Parkinson
et al., 2001). Resting concentrations of oestradiol were similar in freemartin ewes and
normal ewes, and were increased in both after administration of 1000 iu equine chorionic
gonadotrophin (Parkinson et al., 2001). Plasma testosterone concentrations were higher in
freemartin ewes than normal ewes but lower than rams and were unchanged after eCGtreat-
ment (Parkinson et al., 2001). This nding was consistent with earlier reports on smaller
numbers of sheep (Kenny et al., 1992; Bosu and Basrur, 1984; Braun et al., 1983; Saba et al.,
1977). Combined treatment with progesterone via sponges and 25 g oestradiol suppressed
LH to baseline levels in freemartins (Parkinson et al., 2001), yet oestradiol alone could not
induce a normal LH surge (Parkinson et al., 2001). Basal FSH was signicantly higher in
freemartins than normal ewes and was not suppressed by injection of semi-puried inhibin
obtained from bovine follicular uid (Parkinson et al., 2001).
Freemartin ewes generally have a higher degree of masculinisation of the reproductive
tract than their bovine counterparts (Smith et al., 2000, 2003; Parkinson et al., 2001). One
theory put forward to account for this is that the degree of masculinsation is a result of the
stage of gestation at which vascular anastomoses occurred, the earlier the more masculine
(Parkinson et al., 2001). Freemartin sheep are reported to have gonads that resembles testes,
and these commonly descend into an inguinal or lowabdominal position (Smith et al., 2003).
Histologically, they can be classied into male type or undifferentiated based on the degree
of masculinisation and lack ooctyes (Smith et al., 2003).
Freemartinsim was described in two Rocky Mountain big horn sheep from a captive
herd (Kenny et al., 1992). Both animals had XX/XY karyotype and masculinisation of the
reproductive tract, one animal demonstrated male behaviour as it matured (Kenny et al.,
1992).
7.2. Caprine
Freemartinism and XX/XY chimerism has been reported in goats (Bosu and Basrur,
1984; Smith and Dunn, 1981; BonDurant et al., 1980; Omura, 1977; Marcum, 1974). Al-
though these reports include very little information other than descriptions of the associated
anatomical changes.
7.3. Cervidae
Two cases of XX/XY chimera red deer (Cervus elaphus) have been described (Stewart-
Scott et al., 1990). These two animals were the result of seven twin pregnancies obtained
using eCG and progesterone. Testosterone treatment of three red deer freemartins resulted
in antler development, analogous to that which occurred in castrated males similarly treated
(Li et al., 2003).
7.4. Porcine
XX/XY chimerism was reported in leukcocyte cultures of intersex pigs consistent with
freemartinism (Somlev et al., 1970; Bruere et al., 1968) although the condition appears to
be very rare due to the limited number of reports.
104 A.M. Padula / Animal Reproduction Science 87 (2005) 93109
7.5. Equine
Freemartinism is rare in the horse, and questionable if it occurs at all. However, a study
of placental circulation of 51 twins showed vascular anastomoses had occurred in 50% of
cases examined (Bouters and Vandeplassche, 1972). However, in none of those cases were
gonads or genital tracts abnormal. It was suggested that the anasotomoses occurred after
the critical time for abnormalities to be induced. Five chimeric mares born co-twin to a
male in one study went on to develop normally and three subsequently produced live foals
(Marcum, 1974). A case report describes a Welsh pony that was a true hermaphrodite with
XX/XY karyotype and bilateral ovotestes (Bouters and Vandeplassche, 1972).
7.6. Camelidae
Freemartinismappears to be rare in camelidae, perhaps due to the generally lowincidence
of twins inthis family. Acase of anXX/XYchimera was describedfor a female co-twinnedto
a male (Hinrichs et al., 1999). Culture of skin brobalasts did not reveal chimerismalthough
blood culture was diagnostic. It was suspected that twin ovulation and twin conception may
occur much more commonly than twin births, and that single born freemartins due to in
utero exposure to a male, may be more common than previously suspected (Hinrichs et al.,
1999). A PCR probe was developed and shown to be reliable for sex determination in llama
embryos, and may be useful for surveying this condition in this family (Pomp et al., 1995).
8. Future research opportunities
8.1. Detection of foetal DNA in maternal plasma
The recent discovery of cell-free foetal DNA circulating in human maternal plasma
has generated great interest in exploiting this phenomenon for human pre-natal diagnos-
tic testing (Bianchi, 2004; Pertl and Bianchi, 2001). This discovery has been investigated
as a non-invasive, early means of gender determination by probing for Y-chromosome
DNA segments using PCR in maternal blood samples, rather than using the more in-
vasive amniocentesis method (Bianchi, 2004). The foetal gender of 72 pregnant rhesus
monkeys was determined with 100% reliability for identifying a male foetus at 2032
days of gestation using real time PCR with primers designed to detect the SRY gene
(Jimenez and Tarantal, 2003). The problem of only being able to detect male foetuses
raises the issue of the half-life of foetal DNA in plasma. In the study using rhesus mon-
keys, no false positives were generated when a female monkey that had previously given
birth to a male foetus, but was currently carrying a female foetus, were detected with Y-
chromosome DNA (Jimenez and Tarantal, 2003). The half-life of foetal DNA in humans
has been measured to be only 16 min (Bianchi, 2004), is continuously liberated in large
copies into maternal circulation by the foeto-placental unit and undergoes rapid turnover.
Associations between the concentrations of foetal DNA in maternal plasma and chromo-
somal abnormalities (e.g. trisomy 21, Down syndrome) have been described (Bianchi,
2004).
A.M. Padula / Animal Reproduction Science 87 (2005) 93109 105
The detection of foetal DNA in maternal plasma of domestic animals has yet to be
demonstrated, but may offer an early means of detection of gender determination, detection
of freemartins and subsequent intervention. Another diagnostic application in animal repro-
duction may be in diagnosing foeto-maternal blood group incompatibilities (e.g. neonatal
isoerythrolysis in the horse) prior to parturition, which has already been investigated, and
shown to be reliable in human tests (Bianchi, 2004).
8.2. Anti-tumour properties of MIS
Measurement of serum levels of MIS have proved useful in human medicine for dif-
ferential diagnosis of patients with intersex disorders in infants (Teixeira et al., 2001) and
cryptorchidism (Lee et al., 2003). In female humans with granulosa cell tumours, MIS has
been found to be extremely elevated (Teixeira et al., 2001) and an obvious application in
animals may be to improve the diagnosis of this relatively common condition in horses.
There is great research interest in human medicine in producing MIS in vitro and using it as a
chemotherapeutic agent for tumour types that express MIS receptors such as ovarian cancers
(Teixeira et al., 2001). Similar unexplored applications may exist in domestic animals.
8.3. Model for the human twin-transfusion syndrome
The bovine freemartin syndrome may represent a useful model in which to study the
human condition called twin transfusion syndrome. In this condition, twins originating from
a single placenta form vascular anastomoses which may lead to unequal sharing of blood
supply and ultimately the impaired development or death of one or both foeti (Jain and
Fisk, 2004). The infrequent nature of the condition makes comparison of treatment options
difcult and the bovine freemartin may represent an animal system in which to study and
compare treatments.
9. Conclusions
Signicant progress has been made in the last 30 years in the understanding and diagnosis
of the freemartin syndrome. As twinning rates increase in Holstein cows, and high fecundity
genes are introduced into sheep ocks, the prevalence of freemartins will increase. Diagno-
sis of freemartinism has been rened through the use of molecular biology techniques and
is now a reliable option to establish gender in animals. Further clarication of the effects on
fertility of males co-twin to freemartins needs to be established, although the effect appears
to be small. The occurrence of freemartins in sheep ocks may be expected to increase, al-
though due to the generally lower economic value of this species, some diagnostic methods
may not be applicable. It is interesting to ponder the reasons why the freemartin syndrome
should occur at all, especially with such frequency in cattle, considering the consequences
for reproduction. In an evolutionary sense, the freemartins genes are not passed on and their
direct contribution to genetic gain and species perpetuation is not obvious. The freemartin
syndrome will always be a limiting factor in cattle and to a lesser extent sheep production
systems in which the goal is to produce a reproductively normal female offspring useful
106 A.M. Padula / Animal Reproduction Science 87 (2005) 93109
for future breeding and lactation. The problem is accentuated when the incidence of
heterosexual offspring is increased above that which would normally occur, either through
genetic selection for twinning or by multiple ovulation and embryo transfer programs.
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