Download research paper: Bovine freemartinism - Ariel Taxdal`s E

Bovine freemartinism
Ariel Rasmussen-Taxdal
431W
Final Draft
Due: 11/18/13
Submitted: 11/21/13
Introduction:
The occurrence of the reproductive condition titled freemartinism has baffled
endocrinologists, embryologists, anatomists, and reproductive biologists around the globe.
Although this condition has been recognized by farmers and scientists for hundreds of years and
many postulates have been created, the exact cause of freemartins is still unknown (Short, R.V.
et al. 1970). The word freemartin can be more easily defined by breaking the word into two
parts: “free” meaning sterile and “martin” meaning bovine. This condition arises when a cow is
pregnant with heterozygous twins: one male and one female. As the definition suggests heifers
born twin to a bull have a 92% chance (Fujishiro et al. 1995) of being either sterile or not
reproductively intact (Kadokawa, H. et al 1995). Sterility in females is economically important to
both beef and dairy producers alike because females are not only used for meat and milk
production, but also to produce the next generation of offspring who will replace older herd
mates (Kadokawa, H. et al 1995). The first documented study of freemartins occurred in 1779,
when scientist John Hunter dissected two different heifers with phenotypic reproductive tract
abnormalities (Short, R.V. 1969). Since then, there have been numerous studies conducted
producing many different theories as to how these reproductive abnormalities arise in heifers
born twin to a bull. This review will encompass vascular anastomosis, chimerism, the biology
and diagnosis, possible effects on the male, incidence rates in multiple embryo transfer, and
connections to human medicine.
Vascular anastomosis in twins:
To further understand how this condition arises the interactions between embryos in utero
must first be understood. Bovine twin pregnancies have over a 90% chance of sharing a common
blood supply through the fusion of the chorio- allantoic membranes of each twin and subsequent
anastomosis of the blood vessels (Dominguez, M.M. et al. 1989). This fusion of the blood supply
is the fundamental connection between the two developing fetuses allows the transfer of
hormones and other substances from the male to the female and vice versa. As previously stated
92% of the time a freemartin heifer (Fujishiro et al. 1995) with masculinization of the
reproductive tract to some degree results (Padula, A.M. 2004). This fusion of the fetal
membranes occurs before sexual differential allowing the male fetus to affect the female’s gonad
development.
Chimerism:
In freemartins a condition known as chimerism, which is defined as “an individual,
organ, or part consisting of tissues of diverse genetic constitution” (chimera) exists where the
twins have both XX and XY genotypes present within their cells. Chimerism was originally
thought to only be present in leukocytes and erythrocytes, however, more recent research shows
their presence in germ cells as well. A study of heterosexual quadruplets (3 female, one male)
reported that the concentrations of XX/XY in leukocyte cells were similar among all four calves.
In the erythrocyte cells there were four distinct populations present suggesting that a quadruple
chimerism had occurred and cells from all four calves were present in a single calf’s cell
population. This new finding suggests that an exchange of blood cell precursors occurred
between all embryos leading to the quadruple chimerism, the first of its kind to be documented.
In all four of the calves there was a significantly high proportion of XX cells present, which lead
researchers to believe that it is the XY cell concentration that determines the degree of
masculinization as opposed to the XX concentration. It was then further concluded that the Y
chromosome cells interfere with female gonad production in some way. Although we know that
the presence of Y cells negatively affects the female’s reproductive tract, no Y cells had been
found in the female gonads up to this point. This phenomenon of no Y cells (in germ cells) could
possibly be explained by a case seen in the males with chimersism. Additionally, the XX cells
were observed at the beginning of meiosis and could possibly be identified and removed during
the subsequent meiotic divisions. In a study where bull calves germ cells were evaluated at 3
months old XX cells were present, but at 18 months old all the XX cells were gone; suggesting
that the individual recognized the XX cells as foreign and therefore they were either targeted for
removal or they were eliminated over time through meiotic divisions. Another theory is that
phagocytic cells in the female gonad can be triggered by the male twin’s secretions and lead to
the breakdown of the female’s own germ cells. Once the germs cells are destroyed they are not
restored. This lack of regeneration is typical in females because they are born with a
predetermined number of germ cells and are not capable making more postnatally (Basrur, P.K).
Freemartin biology on the cellular level:
During sexual differentiation gonads develop from sertoli cells in the testes and granulosa
cells in the ovaries. The presence of the XY genotype in the male sertoli cells leads to activation
of the SRY gene, triggering the release of anti-Mullerian hormone, Sry-box containing gene 9
(SOX9), and glial line-derived neuroptrophic factor. Since female XX cells do not have the Y
chromosome to activate the SRY gene they develop into ovaries. SOX9 is especially important
because without it the male is not able to differentiate testes or maintain their function.
Furthermore, the SOX9 genes presence or absence can lead to sexual reversal. Anti-mullerian
hormone is also suspected to have a significant impact on cellular differentiation in freemartins.
(Harikae, K 2012)
Subsequently, it has been theorized that the female’s gonadal tissues are altered and
actually begin to produce more androgens which act back on their own tissues. This theory was
tested by Dominguez et al to try to affirm that the androstenedione-to-testosterone pathway is
altered in freemartins. When looking at androstenedione production in the freemartin fetuses, as
compared to singleton female fetuses, levels were extremely high down to 40 days which
supports the theory that hormone pathways are altered early on in fetal development.
Interestingly, the freemartin’s ovaries were noticeably smaller than the singleton calf as early as
50 days of gestation. (Dominguez, M.M. et al 1990)
Diagnosis:
However, there is also a small chance, 8%, that a heifer born twin to a bull will not be a
freemartin (Shore,L., Shemesh, M. 1981). This 8% of heterozygous twins do not have a fused
blood supply, therefore, stopping the flow of cells and hormones from calf to calf. In order to
determine if a heifer calf is in fact a freemartin, both clinical observations and blood tests can be
used to determine the calf’s status. The most tried and true clinical determination is to measure
the vaginal length by inserting a rounded probe into the vagina (Padula, A.M. 2004). This probe
testing can be performed from one month old until maturity, however, the sooner you can
identify a suspected freemartin the more beneficial it will be in terms of making management
decisions. A one month old calf should have a length of 13-15 cm, where as a freemartin would
only measure about half that length of 5-8cm (Padula, A.M. 2004). In an adult bovine, the
normal vaginal length would be 30cm, and in freemartins significantly smaller at 8-10cm. In
addition to the probe test, there are other secondary characteristics that can be observed in some
freemartins including long coarse hair on the vulva, increased distance between the vulva and
anus, an enlarged clitoris, an enlarged neck, curly hair, and even the appearance of a scrotal
pouch. All of the afore mentioned secondary signs are not always present and therefore diagnosis
should not be made based solely on these secondary signs. (Padula, A.M. 2004)
Today Polymerase Chain Reaction (PCR) testing is the gold standard as compared to
yesteryears probe test. This test uses the chimeric trait of freemartins to determine if a heifer is
in fact a freemartin. The goal of the test is to determine the presence of the Y chromosome in the
freemartin’s blood serum. Freemartin calves range from a 20-80% ratio of XX/XY and have
even been found to be as low as 1% or less (Fujishiro, A. et al 1995). In a study by Fujishiro(et
al) it was determined that the presence of the Y chromosome can be detected down to a .0008
ratio, meaning that the test is very effective (Fujishiro, A. et al 1995). These tests can be
especially helpful if the calf is of significant genetic value to the producer or if the heifer was
born a singleton and suspected to be a freemartin. Unlike the probe test, a blood sample can be
taken just days after birth and therefore have results more quickly. The test is also more accurate
because it is not based on interpretation of one sole factor, but rather the animal’s entire genetic
makeup. Genetic Visions will perform the PCR test for a $25 fee all the producer needs to do is
collect at least 2ml of blood into a collection tube with anticoagulant(Genetic Visions Inc).
Multiple Embryo Transfer freemartin incidence rate:
As genetic selection increases in both beef and dairy cattle, producers continue to seek
ways to advance their herds genetics more quickly. One popular way is through embryo transfer,
in which one female is superovulated in order to create many embryos which can then be
implanted into other genetically inferior cows. Sometimes more than one embryo is implanted in
the surrogate to increase the chances of a pregnancy occurring. If both embryos attach then twins
would result and possibly freemartins however recently researchers have looked at single born
calves that were the result of multiple embryo transfer to look for potential freemartins whose
twin died early in pregnancy. In the study, 22 heifers were chromosome typed and PCR analysis
was performed. Of the 22 females, 21 were determined to be normal by both tests and one was
found to have both XX and XY by PCR analysis (Kadokawa, H. et al 1995). Based on these
results the chance of a freemartin developing from multiple embryo transfer are very low.
Effects on the Male:
It has been theorized that bulls twin to freemartins may also be affected reproductively as
a results of chimerism. One possible affect is that chimera bulls may have a higher proportion of
female offspring as compared to singleton bulls. It is theorized that the XX cells that inhabit the
lymphocytes could possibly lead to a higher heifer to bull ratio, but this has yet to be proven
(Padula, A.M. 2004). The other effect that has been evaluated is bull fertility, however studied
effects have shown great variability.
Connection to human medicine:
The study of bovine freemartin syndrome could has the potential be used to help us
further understand a rare condition in human pregnancy known as twin transfusion syndrome.
This syndrome results when twins share one placenta and vascular anastomosis occurs leading to
an unequal sharing of the blood supply, which can be fatal to both twins. Due to the fact that
freemartins also experience this vascular anastomosis, the bovine is an excellent animal model to
further study this homo sapien syndrome (Padula, A.M. 2004).
In turn, human medicine could also help cattle by adapting a technology developed for
sexing fetuses in humans for use in livestock. It was discovered that fetal DNA can be found in
the maternal blood plasma and PCR tests can be run to look for the presence of the Y
chromosome much like the chimera test. Although this type of fetal testing has not been assessed
in livestock species, it could be beneficial to determining the presence of freemartins in early
pregnancy. (Padula, A.M. 2004).
Conclusion:
It has only been a little over 230 years since John Hunter first studied a freemartin tract.
Since then significant progress has been made in the understanding of freemartin biology and
diagnosis of freemartins through genetic testing like PCR. However, there is still no universally
agreed upon cause of freemartinism aside from the anastomosis of blood vessels in the placentas
of heterozygous twins. Furthermore, there is still much room for additional research into the
potential effects on the male. Understanding the biology of freemartins can not only help us find
potential ways of preventing the condition but also allow us to further understand reproductive
embryology in all species.
References
P.K. Basrur, S. Kosaka, and H.Kanagawa Blood Cell Chimerism and Freemartinism in
Heterosexual Bovine Quarduplets J of Heredity.
M.M. Dominguez, R.M. Liptrap, B.A. Croy, P.K. Basrur. 1990
Hormonal correlates of ovarian alterations in bovine freemartin fetuses 22:181-201
A. Fujisho, K. Kawakura, Y-I. Miyake, and Y. Kaneda. 1995. A fast, convenient
diagnosis of the bovine freemartin syndrome using polymerase chain reaction
Theriogenelogy 43:883-891
K. Harikae, N. Tsunekawa, R. Hirmatsu, S. Toda, M. Kurohmaru, Y. Kanai. 2012.
Evidence for Almost Complete Sex- Reversal in Bovine Freemartin gonads: Formation of
Seminiferous Tubule- like structures and Transdufferation into typical testicular cell
types. J. of Reproduction and Development 58:654-650
H. Kadokawa,M. Minezawa, Y. Yamaoto, M. Takahashi, K. Shimada, H. Takahashi, and
T. Karyia. 1995. Freemartinism among singleton bovine females born from multiple
embryo transfer. Theriogenelogy 44:295-306
A. M. Padula. 2004 The freemartin syndrome: an update. Animal Reproduction Science
87(2005) 93-109
Ada Rota, Cristina Ballarin, Bernard Vigier, Bruno Cozzi, and Rodolfo Rey
2002. Age dependent changes in plasma anti-Müllerian hormone concentrations in the
bovine male, female, and freemartin from birth to puberty: relationship between
testosterone production and influence on sex differentiation. General and Comparative
Endocrinology 129 (2002) 39–44
L. Shore and M.Shemesh. 1981. Altered steroidogenesis by fetal bovine freemartin ovary.
J Reprod. Fert. 63:309-314
R. V. Short, A. Jost, G. W. Harris and C. E. Ford. 1970. The Bovine Freemartin: A New
Look at an Old Problem [and Discussion]Phil. Trans. Roy. Soc. Lond. B 259:141-147
chimera. 2013. In Merriam-Webster.com.
Retrieved November 16, 2013, from http://www.merriamwebster.com/dictionary/chimera
"Genetic Visions, Inc.®." Genetic Marker Test: Freemartin (FM). N.p., n.d. Web. 14 Nov. 2013.
`
`