The freemartin syndrome: an update.

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 biology, 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 reproductive 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 common 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 efficiency 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 indeed 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 introduction of high fecundity genes into flocks. 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 Mullerian 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 to produce multiple reproductively normal female offspring from a single dam without using sex predetermination.

Effects of twinning on dystocia, calf survival, calf growth, carcass traits, and cow productivity.

This paper reports results from a long-term experiment with a primary objective to increase twinning rate in cattle at the Roman L. Hruska U.S. Meat Animal Research Center. Survival of singles was 13% higher (P < .01) than that of twins at birth, and the difference in survival in favor of singles was of similar magnitude at 72 h (12.9%, P < .01), 150 d (14.8%, P < .01), and 200 d (15.2%, P < .01). Survival of calves with no dystocia was higher than survival of calves with dystocia: 8.6% (P < .01) at birth, 10.8% (P < .01) at 72 h, 12% (P < .01) at 150 d, and 12.2% (P < .01) at 200 d. The effect of dystocia on survival was greater (P < .01) in twins than in singles at birth and at 72 h. Least squares means for dystocia were 20.4% in singles compared with 42.2% in twins. Most of the dystocia in singles resulted from a traction requirement (84.7%) of normal presentations, whereas most of the dystocia in twins (77.8%) resulted from malpresentations, with 59.2% of the malpresentations accompanied with a requirement for traction. Survival in singles ranged from 10.7% to 15.3% greater than in twins at different ages when there was no requirement for assistance in either singles or twins. Calves born as singles were 8.8 kg heavier (P < .01) at birth and 28 kg heavier (P < .01) at 200 d than calves born and reared as twins. Calf weight produced per cow calving was 53.1%, 54.7%, and 58.4% greater (P < .01) at birth, 150 d, and 200 d, respectively, in cows producing twins than in cows producing singles. Cows producing twins had 65.2% more (P < .01) live calves at 200 d than cows producing singles. Single male calves gained 74 g more per day than twin males from birth to 200 d, 45 g more (P < .01) per day from 200 d to slaughter and 57 g more (P < .01) per day from birth to slaughter. Differences between twin and single males in carcass traits were small. A sample of steers from the Twinning Project gained significantly faster and produced significantly more desirable carcasses than a sample of steers from a high performance reference population. Freemartins did not differ (P < .05) from normal females in growth traits, but freemartins had higher (P < .05) scores for marbling with a higher percentage (P < .05) of USDA Choice or better quality grade carcasses and lower estimated percentage retail product.

The possibility of early estimation for fertility in bovine heterosexual twin females.

To diagnose the possibility of early estimation for fertility in bovine heterosexual twin females, we designed a new diagnostic program. The 9 freemartins (FM) and 5 normal females (Normal) were used in this study. All 14 cases, at 4 months of age, were given Pregnant Mare Serum Gonadotrophin (PMSG) and human Chorionic Gonadotrophin (hCG) 1.5-2 days later. Thereafter, the concentration of estradiol-17 beta (E2) was determined by RIA, and that of progesterone (P) was done by RIA and EIA (Ovcheck EIA Kit). The concentration of E2 in the Group of Normal rapidly increased after administration of PMSG, but in the Group of FM, the concentration of E2 changed in very low levels over 14 days. The concentration of P in the Group of Normal rapidly increased after administration of PMSG, but in the Group of FM, the concentration of P changed in very low levels over 14 days.

Cattle twins and freemartin diagnosis.

Some cattle breeding improvement programmes particularly in continental Europe aim to reduce the costs of rearing calves by increasing the number suckled by each cow. This encourages the rearing of twins and an increase in the number of heifers born twin-to-bull. Many of these are offered in the market and bought on the assumption that they are single-born. A large proportion will be freemartins and therefore irremediably sterile. Theories of the aetiology of the freemartin condition are summarised. The anatomical, haematological and chromosomal characteristics are described. Diagnostic methods based on these characteristics are described and compared. The clinical method is recommended as reliable in all but a small proportion of cases. The clinical signs of the freemartin at age three to six weeks are the absence of the external os, and a vagina length of 5 inches (12 cm) or less. The veterinary surgeon is encouraged to make the diagnosis on these clinical signs alone.

Novel gonadal characteristics in an aged bovine freemartin.

The gonads from a five-year-old freemartin Holstein animal were subjected to morphological analysis and to immunohistochemistry using antibodies against developmental and functional markers. We demonstrate, for the first time, the retention of anti-mullerian hormone (AMH) producing intratubular cells (Sertoli cells) in the context of abundant steroidogenic interstitial cells, and structures consistent with clusters of luteal cells. This novel report describes the clinical, gross and histological findings accompanying this newly described gonadal immunophenotype, and its implication in the understanding of freemartin development.

Anti-Müllerian hormone: a local or long-distance morphogenetic factor?

In an attempt to determine whether anti-Müllerian hormone could exert long-distance effects, we studied the anti-Müllerian activity of gonads from bovine Freemartin fetuses. Anti-Müllerian activity was detected in 3 out of the 7 animals studied: one was 62 days old, and its gonad contained undifferentiated tissue only; the 2 others were 110 and 130 days old respectively, and their gonads contained seminiferous tubules. The gonads devoid of anti-Müllerian activity contained only rete tubules or fibrous tissue. Anti-Müllerian activity was absent in fetal male and Freemartin serum, except in 2 cases, in which low activity was present after 37-fold purification by lectin affinity chromatography. The presence of anti-Müllerian activity in Freemartin gonads with seminiferous tubules is an indication that gonadal virilization in these fetuses is functional as well as morphological. Further experiments are needed to determine whether regression of the Müllerian ducts in the Freemartin is due to anti-Müllerian hormone produced by the Freemartin gonads in situ.

Anti-müllerian hormone and freemartinism: inhibition of germ cell development and induction of seminiferous cord-like structures in rat fetal ovaries exposed in vitro to purified bovine AMH.

In 13 and 14-day old fetal rat ovaries maintained 3 to 10 days in organ culture, purified bovine anti-Müllerian hormone (AMH) (1.5 to 3 micrograms/ml) induced a characteristic freemartin effect. Gonadal volume and germ cell number were significantly reduced, compared to control ovaries cultured in anhormonal medium, and epithelial cells with large clear cytoplasm linked by interdigitations differentiated in the gonadal blastema. These cells resembling rat fetal Sertoli cells became polarized and formed seminiferous cord-like structures delineated by a basal membrane containing laminin and fibronectin as is the case of testicular seminiferous cords at the first step of their differentiation. These data indicate that AMH is probably the testicular factor responsible for the morphological modifications of bovine freemartin gonads and suggest that this hormone could also be involved in normal morphological differentiation of the testis. In contrast, in fetal rat ovaries, AMH did not trigger the testosterone production which occurs in freemartin gonads at an early stage of the gestation.

Development of and fluid accumulation in mammary glands of freemartins administered estradiol, estrone, or testosterone..

Eighteen Holstein freemartins acquired at 1 wk of age were assigned to a control and to four treatment groups, but only two of the groups received hormone the first 50 wk. Testosterone administered for the first 50 wk appeared to inhibit teat growth whereas estrone stimulated teat and udder length. At 50 wk of age, implants releasing daily approximately 12.9 microgram of testosterone, 2.9 microgram of estrone, or 2.6 microgram of estradiol-17beta per kg of body weight were implanted for 6 wk. One of three testosterone-treated and 9 of 11 estrogen-treated animals exhibited increased udder development associated with fluid accumulation. The 10 animals with fluid accumulation in the udders were milked once 39 days after implanting. The fluid contained 15.0% total solids, 4.1% fat, and 7.0% protein, values which are between those for normal milk and colostrum. These studies support the concept that administration of low concentrations of estrogens alone, released continuously from implants, were capable of stimulating fluid secretion in mammary glands of animals that had no detectable gonadal luteal tissue.

Growth and reproductive development in freemartins hormonally treated from 1 to 79 weeks of age.

Postnatal growth, steroid hormone profiles and response to steroid hormone treatment were studied in 18 freemartins and one normal female born co-twin with a bull. They were either treated postnatally with testosterone or estrone at one week of age, or left untreated until 50 weeks of age when they received silastic implants calculated to release either 12.9 microgram of estrone or 2.6 microgram of estradiol per day per kg of body weight. Later a dihydrotestosterone-treated group was added. Reproductive development was studied by palpation per rectum and by examination when animals were slaughtered at 79 weeks of age. Treated animals grew slightly faster than untreated animals. Testosterone in untreated freemartins averaged 76 and 87 pg/ml of blood plasma during weeks 1 to 48 and 52 to 56. Corresponding values for those animals with small testosterone implants (weeks 1 to 48) and with larger implants (weeks 52 to 56) were 130 and 272 pg/ml. Estrone and estradiol values appeared to fluctuate between 10 and 50 pg/ml but values are uncertain because they were below the sensitivity of the assay then available. Thus, circulating steroid hormone concentrations were similar to those reported for castrates. Testosterone stimulated clitoral development prenatally and postnatally. None of the treatments influenced vaginal depth, which averaged 4.0, 9.0 and 10.9 cm at 1, 24 and 52 weeks of age. Vaginal depth at birth was not a reliable indicator of freemartinism. Androgen may have inhibited udder development, whereas estrogen appeared to be stimulatory. The reproductive organs of the freemartin were characterized by differences in underdevelopment and the general presence of seminal vesicles. The latter structures, plus clitoral development at birth in 3 animals and postnatal response of the clitoris to testosterone is interpreted to indicate that the presence of androgens is one factor in abnormal development. Otherwise, gross morphology of the reproductive tract was not related to hormone treatment, postnatal gonadal histology, endocrinology or lymphocyte chromosomal karyotypes.

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.

To understand the behaviour of the gonads, in terms of hormonal secretion, in a model of intersexual development naturally occurring in mammals, we determined plasma concentrations of testosterone, progesterone, and anti-Müllerian hormone (AMH) in bovine freemartins, and compared them to normal levels measured in males and females from birth to puberty. We found that newborn males and freemartins have very high concentrations of AMH (over 700ng/ml). Conversely, plasma AMH concentration is always below 120ng/ml in females. While values remain stable in males for the first five months of life, they sharply decrease in the freemartins within the first fortnight, and reach female levels, which demonstrates that AMH is essentially originated in the male twin. In young bulls the trend of plasma testosterone concentrations is opposite to that of the AMH. The rise in testosterone production at puberty corresponds to a sharp decline in AMH concentrations. Bovine plasma concentrations of AMH are surprisingly higher than those measured in other mammals, including man and mouse. The results obtained are discussed in reference to comparative aspects of endocrine functions.

Effects of testosterone on pedicle formation and its transformation to antler in castrated male, freemartin and normal female red deer (Cervus elaphus).

Pedicles and antlers are male deer secondary sexual characters. As such, development of these structures is under the control of androgen hormones. Pedicle growth is caused by increasing and elevated plasma testosterone (T) levels, whereas first antler transformation from a fully formed pedicle occurs when the T levels are decreasing. Castration prior to pedicle initiation abrogates future pedicle and antler formation. Female deer also have the potential to develop pedicles and antlers, but they do not normally express this phenotype due to lack of sufficient androgen stimulation. Previous studies have shown that female white-tailed deer could be readily induced to grow pedicles as well as antlers by singular administration of exogenous androgens (EA), but in red deer (Cervus elaphus) singular or irregular EA treatment could only stimulate castrated male, normal or ovariectomised females to grow pedicles, but not antlers. The present study was set out to test whether these EA-induced pedicles in red deer failed to give rise to antlers was because they were constitutively incapable of doing so, or because the plasma T profile naturally exhibited in intact stags was not achieved by the androgen treatment used in these previous studies. Eight castrated red deer stag calves, 3 freemartins (females which were born co-twin to males), and 3 normal female red deer were used in the present study and treated with EA, either as biweekly injections for the castrates or as implants for freemartin and females until the late stage of pedicle growth. Blood sampling was carried out biweekly for the analyses of plasma T and IGF1 concentration. The results showed that the natural plasma T profile in the experimental deer was successfully mimicked through regular EA treatment and subsequent withdrawal at late pedicle growth stage. All castrated males, 2 out of 3 freemartin, and 1 out of 3 normal female red deer formed not only pedicles, but also antlers. Based on these results, we conclude that EA-induced pedicles at least in red deer of the genus Cervus, like those in the genus Odocoileus, are constitutively capable of giving rise to antlers, if they are of sufficient height.