Freemartins; vaginal measurements

Keywords:  bovine, vagina, hymen, freemartin, straw, speculum

Freemartins are common in bovine twin pregnancies. Freemartins are also seen in buffalo, occasionally in small ruminants and rarely in other species. When the sexes differ between individuals in twin or triplet pregnancies, female calves can be expected to be freemartins in 90-97 percent of calvings.  In rare cases, a male twin may die after its chorionic blood vessels have anastomosed with those of its female co-twin and the surviving twin may be born a freemartin.

Interesting historical aspects of freemartinism:  The discovery of freemartinism was attributed to the Scottish Physician and surgeon J. Hunter in his 1779 account of the Free Martin (click to enlarge): However, the basic descriptions of the cause of freemartinism has been attributed to Tandler and Keller in 1911. (Deutsche Tierärzt Wochenschr 19:148-149) and later, apparently independently, by Lillie in 1916 (Science 43:611-613).  Incidentally, the etymology of the word Free Martin, (later hyphenated as Free-Martin and finally written as freemartin) is open to much speculation but remains unknown.

In the case of a potentially valuable heifer calf being born co-twin to a male, one may choose to perform a PCR test* for the presence of Y chromosomes in leukocytes. This service is becoming common in North America.

A side note on chimeras: Less commonly used tests to diagnose freemartinism include karyotyping.  Freemartins are not only gonadal chimeras but hemopoetic chimeras as well (other organs may also be chimeric but this has not been well studied). This explains why freemartins continue to produce a mixed population of XX and XY leukocytes into adulthood. The author relied on this test for many years until it was shown that karyotyping is not an accurate method of diagnosing freemartinism unless a very large number of metaphase spreads are examined.

When the potential value of a heifer calf born co-twin to a male is not great, the simplest test for freemartinism is to measure the length of its vagina.

Figure 1: The length of the vagina in this mature heifer was 10.5 cm. One reported range of vaginal lengths in adult freemartins was 8 to 10 cm. Normal mature heifers have vaginas that are approximately 30 cm long. This heifer was born co-twin to a male, therefore it was highly likely to be a freemartin.  Size available: 843 x 1004px

An explanation: In early bovine embryos, anastomosis occurs between the chorionic blood vessels of twins lying adjacent to one another in the uterus. See this entry and another in LORI for additional details. In all female mammals, the Müllerian (paramesonephric) system develops from the endoderm. The interface between the endoderm and the ectoderm becomes the hymen. A normal vagina is comprised of a vestibule and cranial vagina, separated by the hymen. In embryos, only the section of the tract that is cranial to the hymen i.e. the Mullerian system is affected by Müllerian inhibiting hormone (MIH). Freemartins have ovotestes that contain Sertoli cells. Those Sertoli cells produce MIH, suppressing the development of Müllerian system. This leaves the vestibule of the vagina unaffected. For that reason, the majority of freemartins have normal vestibules but the cranial vagina is not patent. Therefore the length of the vagina (essentially just the vestibule) is short. In a very small percentage of all freemartins, the Mullerian system is only slightly suppressed, leaving cervixes and vaginas of normal length. Therefore the measurement of vaginal length is not infallible. However it remains a valuable rudimentary tool for the diagnosis of freemartinism.

The vaginal length of a normal heifer calf less than 30 days old is 13 to 15 cm. In freemartin calves the same age, it is only 5 to 8 cm long. Because a vaginal speculum cannot be used on such young calves, the author uses Cassou artificial insemination sheaths (regular sheaths for bovine AI guns) to measure vaginal length. Using a calf of comparable age to the suspected freemartin, a sheath is inserted into its vagina to its cranial extent then kinked at the level of the vulva lips. The same process is performed in the suspect freemartin and the two sheaths are compared, explaining the significance of the test to the farmer. See figure 2.

Figure 2: The sheath-kink test used to measure the vaginal length of a suspect freemartin calf. In two calves both less that 30 days of age, the sheath at left shows the likely presence of a freemartin calf. Size available: 1500 x 1055px

Naturally, the presence of a persistent hymen in a normal calf can confound the sheath-kink test but less than 1 percent  of heifers (approximately 0.8 percent in one large study) will be expected to have persistent hymens. A commercial probe is available to measure the length of vaginas but at far greater cost that a couple of disposable AI sheaths.

*Diagnostic tests for freemartins (PCR for Y chromosomes)

https://www.vdl.umn.edu/node/4396

https://www.geneticvisions.net/freemartin.aspx

Selected references:

Basile, J. R. and Megale F. 1974. Developmental abnormalities of genitalia of Zebu cows in the State of Minas Gerias. Argentina Biologica e Tecnologica 17:136-150.

Esteves, A. et al 2012  Freemartinism in Cattle in Ruminants. In: Anatomy, Behavior and Diseases. Ed. Marques, R.E. Chapter 7. pp.99-120. Nova Science Publishers, Inc. ISBN: 978-1-62081-064-4

Freeman, G. 2007 Explaining the freemartin: Tandler and Keller vs. Lillie and the question of priority. J Exp Zool B Mol Dev Evol. 15: 105-112

Hunter, J. 1779. Account of the Free Martin. Transactions of the Royal Society of London, 1779

Kästli, F. and Hall, J.G. 1978 Cattle twins and freemartin diagnosis. Vet Rec.102: 80-83.

McEntee, M. 1990. Ed. Reproductive Pathology of Domestic Mammals. Academic Press. ISBN 0323138047, 9780323138048

Padula, A.M. 2005. The freemartin syndrome:an update. Anim Reprod Sci 87:93–109

Freemartinism

Keywords: Freemartin, bovine, infertility

The image below shows the reproductive tract from a freemartin heifer. Although there is great variation in the manifestation of Freemartinism, this tract shows some characteristics that are common to many of these animals.

Image size: 1000 x 557px

Note how the development of the Müllerian tubes (precursors of the cranial vagina, cervix, uterus and uterine tubes) was suppressed by anti-müllerian hormone (AMH). AMH was presumably produced by the small gonads seen here where ovaries are usually found. Although it was not verified histologically in this case, these gonads would have contained cells from the male co-twin, including Sertoli-like cells that produce AMH. Of course, it is normal for a male fetus to produce AMH because the Müllerian system must be suppressed if the male is to develop normally. However, in female fetuses where the gene for AMH production is inactive, AMH production by the gonads is disastrous.

As alluded to above, gene coding for AMH production is active in the male co-twin. Therefore gonadal cells derived from the male produce AMH in the co-twin's (Freemartin) gonad. In addition, AMH from the male twin circulates in the female co-twin, amplifying the suppressive effect on the female tract. That has occurred here; the uterus is a mere thread, the cervix is absent and the cranial vagina is also absent. The caudal vagina develops from the ectoderm in a fetus, a system that is not affected by AMH, so it is virtually normal in Freemartins. In this image, a probe has been inserted into the vagina, demonstrating the presence of a normal vestibule i.e. cranially, up to the level of what would have been the hymen. More cranial to that, normal tract is almost absent.

As discussed below, there is a considerable range in the manifestation of Freemartinism;  the tract may be barely affected or may be extremely masculinized. The specimen shown here would probably be considered modal within that range.

Notes: 

In 2005, A.M. Padula  published an excellent review of  Freemartinism (Padula. A.M. 2005. The freemartin syndrome: an update. Anim. Reprod. Sci. 87: 93–109). For additional detail, one should consult that article and others cited within.

At approximately 28 to 30 days of gestation, the allantochorion membranes of most ruminant co-twins fuse. This causes vascular anastomoses to form between the two conceptuses, resulting in freemartinism, a condition that is especially common in cattle. Freemartins occur in other ruminants, cervids and even pigs. However, the condition is comparatively rare in those species (except perhaps for sheep). This author is not aware of any well-documented case in a horse.

Cellular or humoral influences (or a combination of both) circulate between the two fetuses and affect gonadal development. The earlier the fusion, the more masculinized the female tract will be.  In a few cases, the tract is so highly masculinized that it is only slightly ambiguous. The opposite is also true i.e. females can be so mildly masculinized that they are not even recognizable as freemartins, apart from being subfertile.

Interestingly, exchange of cellular material early in gestation also makes freemartins highly tolerant of organ allografts.

Although it is often stated that male co-twins to freemartins are also affected and show deficient spermatogenesis, this is not always so. In fact, males are often completely fertile into adulthood.

On occasion, a male co-twin may die in utero after vascular anastomosis has occurred and although it is unusual (recall that their blood supplies are fused) the female co-twin may survive. In such cases a freemartin may be born as a singlet.

Typically, the Müllerian system in freemartins is severely suppressed; in most cases almost impossible to delineate clearly by transrectal palpation (see the image in this entry). This is because the gonads are a mixture of testicular and ovarian tissue, (freemartins are true hermaphrodites). Sertoli cells in the testicular tissue appear to produce müllerian inhibiting factors as they do in normal males. The gonads are also very small and very difficult or impossible to define on transrectal palpation. The vagina of a freemartin is very short (usually less that 12 cm) and it has a blind ending, the cervix being part of the müllerian system.

Statistically speaking, a female calf born co-twin to a male has approximately a 95% chance of being a freemartin. However one may need further assurance that this is the case.  In newborn calves, where a transrectal examination is impossible, this author uses the cover sheath of Cassou artificial insemination pipette as a vaginal ‘depth gauge’. The sheath is inserted into the vagina of the suspect freemartin and bent at the level of the vulva lips. A second sheath is inserted into the vagina of a normal calf and bent at the same level. One then compares the length of  sheaths to the point at which they were bent.  The length of the section of interest is far shorter in the suspect freemartin calf than the normal calf. The only chance of an error with this method is in normal heifer calves that have imperforate hymens. In a farm setting, one may have to wait for a suspect freemartin heifer to reach an age at which she can undergo transrectal palpation safely.

If a diagnosis of freemartinism is still uncertain after considering the history, palpable findings, and external and internal appearance of the heifer, numerous other tests can be performed, mostly of a genetic nature. Sex chromosome chimerism (xx and xy) is present within nucleated blood cells and contrary to older data, in lower numbers in other cells throughout the body as well.

The origin of the term freemartin (also free-martin) is a subject of great uncertainty. As discussed by J.B. Marcum in the Commonwealth Bureau of Animal breeding and Genetics abstracts in 1974 (42: 227) the term may have originated in 1593 when "Martin" was a word used a England and Scotland for a cow or ox. The etymology of the prefix "Free-" is more obscure, possibly arising as a contraction of  'farrow" meaning a barren or non-lactating cow or perhaps the German term "farre" meaning bull, denoting a cow that was like a bull.

Freemartin placentation

Keywords: freemartins, bovine, slip, membrane, diagnosis, pregnancy, chorion, infertility, twins

This image of a 38 day twin bovine pregnancy shows the outer chorio-allantoic membrane  and the two inner amnionic vesicles. Although the allantoic cavities remain separate (as shown here; one stained with iodine and the other not stained) the chorionic membranes fuse early, usually before this stage of gestation, allowing cell and hormonal exchange between the developing embryos. If one of the embryos is a female, it causes freemartinism in that embryo.

Image size: 1067 x 664 px

At approximately 28 to 30 days of gestation, the allantochorion membranes of most (not all) ruminant co-twins fuse, which causes vascular anastomoses to form between the two conceptuses. This results in freemartinism.  Freemartinism is discussed in some detail elsewhere in LORI.