Anti-Müllerian hormone: A novel biomarker for detecting bovine freemartinism.

The anti-Müllerian hormone (AMH) indicates ovarian reserve in cattle, maintaining a consistent trajectory post-puberty. In heterosexual pregnancies, the development of the Müllerian duct in female foetuses is inhibited, resulting in an anticipated minimal or absent ovarian reserve capacity. This investigation aimed to compare AMH levels in healthy Holstein heifers that had reached puberty with those of freemartin animals of the same breed and age. The study incorporated Holstein heifers reaching puberty between 11 and 15 months of age in Group 1 (G1, n = 20) and freemartin animals in Group 2 (G2, n = 19, 16). AMH measurements (AMH-1/AMH-2) were recorded at 12-day intervals for the study participants. Notably, AMH levels in three freemartin animals could not be detected, prompting statistical analysis based on measurements from the remaining 16 freemartin animals in G2. A statistically significant correlation was observed between two separate measurements in G1 and G2 (p < .001). Furthermore, AMH-1 and AMH-2 levels were statistically higher in G1 than in G2 (p < .001). In G1, AMH-1 levels ranged from 227 to 677 pg/mL, with an average of 367.3 ± 25.5 pg/mL, and AMH-2 levels ranged from 234 to 645 pg/mL, with an average of 380.8 ± 24.4 pg/mL. Conversely, in G2, AMH-1 levels ranged from 10 to 72 pg/mL, with an average of 26.8 ± 4.44 pg/mL, and AMH-2 levels ranged from 12 to 68 pg/mL, with an average of 28.75 ± 4.18 pg/mL. The mean AMH levels in G1 were approximately 14 times higher than in G2 (p < .001). Consequently, ROC analysis utilizing AMH-1 and AMH-2 data established cut-off values of ≤72 and ≤ 68 pg/mL respectively for distinguishing freemartin animals. In conclusion, AMH could be used as a reliable biomarker for identifying Holstein freemartin animals.

From Enrico Sertoli to freemartinism: the many phases of the master testis-determining cell†.

Sertoli cells, first identified in the adult testis by Enrico Sertoli in the mid-nineteenth century, are known for their role in fostering male germ cell differentiation and production of mature sperm. It was not until the late twentieth century with the discovery of the testis-determining gene SRY that Sertoli cells' new function as the master regulator of testis formation and maleness was unveiled. Fetal Sertoli cells facilitate the establishment of seminiferous cords, induce appearance of androgen-producing Leydig cells, and cause regression of the female reproductive tracts. Originally thought be a terminally differentiated cell type, adult Sertoli cells, at least in the mouse, retain their plasticity and ability to transdifferentiate into the ovarian counterpart, granulosa cells. In this review, we capture the many phases of Sertoli cell differentiation from their fate specification in fetal life to fate maintenance in adulthood. We also introduce the discovery of a new phase of fetal Sertoli cell differentiation via autocrine/paracrine factors with the freemartin characteristics. There remains much to learn about this intriguing cell type that lay the foundation for the maleness.

Degree of leukochimerism with calf age in Angus-crossbred twin sets.

Twinning in cattle is infrequent and usually undesired. It can result in an increased occurrence of abortion and dystocia, reduced calf survival and a high likelihood of freemartinism in mixed-sex twins. Twin gestations are also commonly associated with the formation of placental vascular anastomoses (PVA) between twins. Through PVA they share blood, hormones (leading to freemartinism in mixed sex twins) and hematopoietic stem cells, which are the progenitors of white blood cells. The sharing of stem cells between twins can result in leukochimeric twin sets. These are twins that have white blood cells derived from both self and the co-twin owing to the fetal migration of hematopoietic stem cells from the extraembryonic mesoderm of the yolk sac to final sites like bone marrow and thymus. This study examined the degree to which this leukochimerism changes with age. DNA was extracted from hair bulbs containing mesenchymal dermal papilla to determine the individual's true genotype and blood samples were obtained at six time points from 1 week to 8 months of age to assess leukochimerism. Samples were genotyped using a medium density SNP chip, and quantitative estimates of allele frequency were determined using SNPs for which members of a twin set had alternative homozygous genotypes. The results indicate statistically significant changes in the proportion of self and co-twin with age and suggest that by 2-4 months of age the genotypic mix in white blood cells represents the hematopoetic stem cell population resident in the individual (i.e. permanently found in thymus and bone marrow).

Managing embryonic and calves losses after twin pregnancies induced by transfer of in vitro-produced Nellore embryos.

Two farms applying reproductive technology for the Nellore beef cattle were selected. Both farms had the same technology programme of oestrous synchronization and embryo transfer, but management was different, especially regarding twins pregnancies. In the present study, we followed the farms from the moment of oestrous synchronization, embryo transfer (two per cow), until delivery and first care of the calves. In farm A, cows presenting twin pregnancies (5 from 13) were submitted to delivery induction, as well as calves and cows were monitored after birth. In farm B, such management was not followed with the twin pregnant cows (31 from 49). In both farms, freemartinism was detected, but this was not a problem as none of the animals would be selected for breeding. No dystocia was observed in farm A, while 48% of the twin pregnancies in farm B ended up in dystocia. Furthermore, the mortality rate of new-born calves in farm A was 10%, while in farm B it reached 32%. Although twin pregnancies remain a concern, we showed here that proper management during and after delivery minimizes animal and economic losses.

Technical note: Droplet digital PCR as a new molecular method for a simple and reliable diagnosis of freemartinism in cattle.

Freemartinism is the most common type of disorder of sex development in cattle. It leads to sterility in the female co-twin in heterosexual twin pregnancy, and is thus a serious problem in cattle production. The incidence of freemartin syndrome is directly dependent on the prevalence of twinning, which has increased in dairy cattle populations in recent years. Thus, early and rapid identification of freemartins is needed to reduce economic loss. Of the various methods used to diagnose this condition, identifying the XX and XY cell lines in blood samples using cytogenetic techniques is the gold standard; however, this technique is time consuming. Faster and more reliable techniques are thus being sought. Droplet digital PCR (ddPCR) is a third-generation PCR method and it has not previously been used to detect XX/XY leukocyte chimerism in cattle. The aim of the present study was to verify the usefulness of ddPCR to detect and quantify leukocyte chimerism in this species. The X and Y copy numbers were estimated by identifying the copy numbers of 2 genes located on the sex chromosomes: amelogenin X-linked (AMELX) on the X chromosome and amelogenin Y-linked (AMELY) on the Y chromosome. In the first step, we performed ddPCR on samples prepared from female DNA mixed with male DNA in serially diluted proportions. We determined that the sensitivity of this method was sufficient to detect a low-frequency (<5%) cell line. In the next step, ddPCR was used to analyze 22 Holstein Friesian freemartins. Cytogenetic evaluation of these cases revealed leukocyte chimerism; the proportion of XX and XY metaphase spreads varied over a wide range, from XX (98%)/XY (2%) to XX (4%)/XY (96%). The use of ddPCR facilitated the precise estimation of the ratio of the copy number of X to Y sex chromosomes. In all cases, the XX/XY chimerism detected by cytogenetic analysis was confirmed using ddPCR. The method turned out to be very simple, accurate, and sensitive. In conclusion, we recommend the ddPCR method for fast and reliable detection of XX/XY leukocyte chimerism in cattle.

Dizygotic monochorionic canine fetuses with blood chimaerism and suspected freemartinism.

Two full-term canine fetuses were found to share a placenta during Caesarean section. The fetuses were of discordant gender, with apparently normal male and female external genitalia. Genetic analysis of whole-blood samples obtained from each fetus revealed identical DNA profiles, with more than two alleles detected at six loci. Subsequent genetic analysis of myocardial tissue samples revealed dissimilar DNA profiles, with at most two alleles detected per locus. Superimposition of the tissue-derived profiles matched that derived from the blood samples exactly, except for two loci failing to amplify, and hence demonstrated blood chimaerism. Dissection of the abdomen of the male fetus revealed delayed descent of the testes towards the inguinal canals. Macroscopically, the gonads, uterus and vagina were not identifiable on dissection of the female fetus, although vestigial ovarian tissue and a vagina were detected microscopically. The hypoplastic internal reproductive tract of the female fetus was suggestive of freemartinism and is believed to be the first report of this condition in the canine.

Age-specific analysis of anti-müllerian hormone and liver biochemical parameters in freemartin holstein calves and heifers: A pilot study.

Freemartinism is the most common congenital anomaly among sexual disorders in dairy cows. This syndrome typically occurs in different-sex twin pregnancies and causes vascular anastomoses to form with the placenta in the early stages of fetal development. The study aims to determine the effectiveness of Anti-müllerian hormone (AMH) levels in calves and heifers of different age groups for diagnostic factors and to investigate the potential consequences of different hormone levels in different age groups on some liver biochemical parameters. The study involved 50 cattle from diverse age categories, divided into the freemartin group (FM Group, n=25) and the control group (C Group, n=25). Both FM and control groups were further divided into early-age (3-5 months), middle-aged (5-9 months), and older-aged groups (9-12 months). Serum AMH levels, along with total protein, albumin, and total cholesterol levels, were measured. While no statistically significant difference in AMH levels was observed in the early-age group (P:0.53), significant differences were determined in the middle (P:0.015) and older-age groups (P:0.01), where the FM group exhibited significantly decreased AMH levels compared to the control group. The evaluation of liver biochemistry revealed a statistically significant difference in total protein levels between the FM and control groups in the older age group (P:0.033). Consequently, it is reasonable to suggest that AMH levels may serve as a valid parameter for diagnosing freemartin syndrome in calves aged older than five months. Conversely, particularly in young calves, no significant differences in liver functionality were observed between freemartin-affected and healthy calves.

Evidence for almost complete sex-reversal in bovine freemartin gonads: formation of seminiferous tubule-like structures and transdifferentiation into typical testicular cell types.

During mammalian sex determination of XY fetuses, SRY induces SOX9 in Sertoli cells, resulting in formation of testes with seminiferous tubules, interstitial Leydig cells and peritubular myoid cells. Meanwhile XX fetuses without SRY develop ovaries. In cattle, most XX heifers born with a male twin, so-called freemartins, develop nonfunctioning ovaries and genitalia with an intersex phenotype. Interestingly, freemartins sometimes develop highly masculinized gonads with seminiferous tubule-like structures despite the absence of SRY. However, in these cases, the degree of masculinization in each gonadal somatic cell type is unclear. Here, we report a rare case of a freemartin Japanese black calf with almost complete XX sexreversal. Gross anatomical analysis of this calf revealed the presence of a pair of small testis-like gonads with rudimentary epididymides, in addition to highly masculinized genitalia including a pampiniform plexus, scrotum and vesicular gland. Histological and immunohistochemical analyses of these masculinized gonads revealed well-defined seminiferous tubule-like structures throughout the whole gonadal parenchyma. In epithelia of these tubules, SOX9-positive supporting cells (i.e., Sertoli cells) were found to be arranged regularly along the bases of tubules, and they were also positive for GDNF, one of the major factors for spermatogenesis. 3ß-HSD-positive cells (i.e., Leydig cells) and SMA-positive peritubular myoid cells were also identified around tubules. Therefore, for the first time, we found the transdifferentiation of ovarian somatic cells into all testicular somatic cell types in the XX freemartin gonads. These data strongly support the idea of a high sexual plasticity in the ovarian somatic cells of mammalian gonads.

[Development of immune tolerance in liver transplantation]. / Desarrollo de inmunotolerancia en el trasplante hepático.

The liver is a privileged organ and has a lower incidence of rejection than other organs. However, immunosuppressive regimens are still required to control the alloreactive T-lymphocyte response after transplantation. These treatments may lead to severe complications, such as infectious diseases, cancers, cardiovascular diseases and chronic renal insufficiency. In clinical transplantation there is increasing evidence that some liver transplant recipients who cease taking immunosuppressive (IS) drugs maintain allograft function, suggesting that tolerance is already present. This strategy is feasible in 25-33% of liver transplant recipients. A series of experimental and clinical observations indicates that liver allografts can even provide "tolerogenic" properties for other organ grafts. In the clinical setting, clinical operational tolerance (COT) is defined as the absence of acute and chronic rejection and graft survival with normal function and histology in an IS-free, fully immunocompetent host, usually as an end result of a successful attempt at IS withdrawal. The exact mechanisms involved in achieving transplant tolerance remain unknown, although animal models suggest a possible role for regulatory T cells (Treg). Recent data have demonstrated an increase in the frequency of CD4+ CD25(high) T cells and FoxP3 transcripts during IS withdrawal in operationally tolerant liver transplant recipients. The data obtained from transcriptional profiling of the peripheral blood of IS-free liver transplant recipients suggest that there is a molecular signature of tolerance that could be employed to identify tolerant liver transplant recipients and that innate immune cells are likely to play a major role in the maintenance of COT after liver transplantation.

Blood chimerism in a girl with Down syndrome and possible freemartin effect leading to aplasia of the Müllerian derivatives.

Cytogenetic and molecular genetic analysis in a case of sex-discordant dizygotic twins revealed blood chimerism in the girl (46,XY in blood and 47,XX, + 21 in fibroblasts) caused by feto-fetal transfusion from her healthy brother. The girl presented with Down syndrome, aplasia of the uterus and the Fallopian tubes and normal female external genitalia. We propose that the lack of Müllerian structures is caused by the effect of the Müllerian inhibiting substance transferred from the male to the female twin in early pregnancy. This disorder of sex development is known as freemartin phenomenon in female cattle from sex-discordant twin pairs.

Multi-aspect testing and ranking inference to quantify dimorphism in the cytoarchitecture of cerebellum of male, female and intersex individuals: a model applied to bovine brains.

The dimorphism among male, female and freemartin intersex bovines, focusing on the vermal lobules VIII and IX, was analyzed using a novel data analytics approach to quantify morphometric differences in the cytoarchitecture of digitalized sections of the cerebellum. This methodology consists of multivariate and multi-aspect testing for cytoarchitecture-ranking, based on neuronal cell complexity among populations defined by factors, such as sex, age or pathology. In this context, we computed a set of shape descriptors of the neural cell morphology, categorized them into three domains named size, regularity and density, respectively. The output and results of our methodology are multivariate in nature, allowing an in-depth analysis of the cytoarchitectonic organization and morphology of cells. Interestingly, the Purkinje neurons and the underlying granule cells revealed the same morphological pattern: female possessed larger, denser and more irregular neurons than males. In the Freemartin, Purkinje neurons showed an intermediate setting between males and females, while the granule cells were the largest, most regular and dense. This methodology could be a powerful instrument to carry out morphometric analysis providing robust bases for objective tissue screening, especially in the field of neurodegenerative pathologies.

Mechanisms of gonadal differentiation.

Sex differentiation is the result of the translation of genetic sex into gonadal sex. Without recognizable masculinizing signals the embryonic gonad will undergo ovarian differentiation. The main determinant of gonadal differentiation appears to be the presence or absence of a cell surface antigen, called H-Y antigen. The regulation of H-Y antigen expression is complex and involves the interaction between regulatory sites on the Y chromosome, the X chromosome, and possibly the autosomes.

Sex chromosome chimerism and the freemartin syndrome in Rideau Arcott sheep.

In cattle, nearly all heifers born co-twin to a male are freemartins, XX/XY chimeras that exhibit a characteristic masculinized phenotype. However, in sheep, while litters containing males and females are common, freemartins are relatively rare. The primary aim of this study was to determine the frequency and features of XX/XY chimerism in female Rideau Arcott sheep. Also, breeding records were used to investigate the effect of litter size and sex ratios, as well as the genetic basis of the condition. Finally, the migration and transcriptional competence of cells of the opposite sex in the XX/XY female and male chimeras was explored. Genomic DNA (gDNA) from peripheral blood cells of ewes was screened by PCR for the male-specific SRY gene. Of 230 lambs screened, 10 were identified as chimeras. Litter size and sex ratio showed no statistically significant effect on the frequency of chimerism. PCR and FISH analysis confirmed the presence of opposite sex cells in female and male chimeras, and in the case of ewes, their migration to tissues other than blood. Transcriptional activity of SRY and AMH was detected in gonads of ewes, whereas XIST expression was detected in white blood cells of chimeric rams. It was concluded that the frequency of sex chromosome chimerism in Rideau Arcott sheep is estimated at 4.35%, with no significant effect of litter size and sex ratio. Moreover, as it was shown that opposite sex cells can migrate to tissues other than blood and be transcriptionally active in chimeric sheep, we speculate on the role they can play in these animals.

Freemartinism in replacement ewe-lambs of the Ripollesa sheep breed.

The freemartinism syndrome affects almost all female calves born as co-twins to male calves, whereas little is known about this phenomenon in female sheep. Within this context, 1,185 ewe-lambs from the Ripollesa sheep breed were genotyped for the presence of oY1 polymorphism (a non-autosomal region of the Y chromosome). Neither ewe-lambs from single births (856) nor ewe-lambs from all-female multiple births (170) were revealed as freemartins, whereas five of 159 ewe-lambs from multiple births with male co-twins were freemartins (3.15 ± 1.38%). All freemartin ewe-lambs were confirmed by physical examination of external genitalia. The results confirm a low incidence of freemartinism from heterosexual twin pregnancies in Ripollesa sheep.

The bovine anterior hypothalamus: Characterization of the vasopressin-oxytocin containing nucleus and changes in relation to sexual differentiation.

In an effort to systematically describe the neurochemical anatomy of the bovine anterior hypothalamus, we used a series of immunocytochemical markers such as acetylcholine esterase (AChE), arginine-vasopressin (AVP), calbindin (Calb), galanin (Gal), neuropeptide-Y (NPY), oxytocin (OXT), somatostatin (SST), and vasoactive intestinal peptide (VIP). We also investigated the potential sex difference present in the suprachiasmatic nucleus (SCN) and the vasopressin-oxytocin containing nucleus (VON) of six male and six female Bos taurus. Our study revealed that the cytochemical structure of the cattle anterior hypothalamus follows the blueprint of other mammals. The VON, which was never described before in cattle, showed a sex difference with a 33.7% smaller volume and 23.2% fewer magnocellular neurons (approximately 20-30 µm) in the male. The SCN also did show a sex difference in VIP neurons and volume with a 36.1% larger female nucleus with 28.1% more cells. Additionally, we included five heifers with freemartin syndrome as a new animal model relevant to sexual differentiation in the brain. This is, to the best of our knowledge, the first freemartin study in relation to the brain. Surprisingly, the SCN of freemartin heifers was 32.5% larger than its control male and female counterparts with 29% more VIP cells. Conversely, the freemartin VON had an intermediary size between male and female. To analyze our data, a classical statistical analysis and a novel multivariate and multi-aspect approach were applied. These findings shed new light on sexual dimorphism in the bovine brain and present this species with freemartins as a valuable animal model in neuroscience.

Chimerism and tetragametic chimerism in humans: implications in autoimmunity, allorecognition and tolerance.

The presence of cells or tissues from two individuals, chimeras, or the presence of cells and tissues that include the gonads, tetragametic chimerism can be detected by the analysis of cytogenetics and analysis of polymorphic genetic markers, using patterns of pedigree inheritance. These methodologies include determination of sex chromosomes, major histocompatibility complex (MHC) polymorphisms and panels of short tandem repeats (STRs) that include mitochondrial DNA markers. Studies routinely involve cases of temporal chimerism in blood transfusion, or following allotransplantation to measure the outcome of the organ, lymphopoietic tissues or bone marrow grafts. Demonstration of persistent chimerism is usually discovered in cases of inter-sexuality due to fusion of fraternal twins or in cases of fusion of embryos with demonstrable allogeneic monoclonality of blood which, excluded maternity or paternity when blood alone is used as the source of DNA. In single pregnancies it is possible to produce two kinds of microchimerism: feto-maternal and materno-fetal, but in cases of fraternal twin pregnancies it is possible to identify three different kinds which are related to cases of vanishing twins that can be identified during pregnancy by imaging procedures; (1) hematopoietic, (2) gonadal, and (3) freemartins when the twins have different sex and the individual born is a female with either gonadal or both gonadal and hematopoietic tissues. Fraternal twin pregnancies can also produce fusion of embryos. Such cases could be of different sex presenting with inter-sexuality or in same sex twins. One of such cases, the best studied, showed evidence of chimerism and tetragametism. In this regard, the case was studied because of disputed maternity of two of her three children. All tissues studied, except for the blood, demonstrated four genetic components but only two in her blood of four possible showed allogeneic monoclonality consistent with the interpretation that her blood originated from one hematopoietic stem cell. Also, microchimerism, due to traffic of cells via materno-fetal or feto-maternal has been prompted by reports of their potential association with the development of autoimmune disorders including systemic lupus erythematosus (SLE) and systemic sclerosis, and in allotransplantation. In addition, their relevance of chimerism in the positive and negative selection of T cells in the thymus has not been addressed. T lymphocytes play a central role in controlling the acquired immune response and furthermore serve as crucial effector cells through antigen specific cytotoxic activity and the production of soluble mediators. Central tolerance is established by the repertoire selection of immature T lymphocytes in the thymus, avoiding the generation of autoreactive T cells. Expression of chimeric antigens in the thymus could modify the generation of specific T cell clones in chimeric subjects and these mechanisms could be important in the induction of central tolerance against foreign antigens important in allo-transplantation. In this review, we discuss the genetics of chimerism and tetragametism and its potential role in thymic selection and the relevance in allotransplantation and autoimmune disorders.

Direct observation of hematopoietic progenitor chimerism in fetal freemartin cattle.

BACKGROUND: Cattle twins are well known as blood chimeras. However, chimerism in the actual hematopoietic progenitor compartment has not been directly investigated. Here, we analyzed fetal liver of chimeric freemartin cattle by combining a new anti-bovine CD34 antibody and Y-chromosome specific in situ hybridization. RESULTS: Bull-derived CD34+ cells were detected in the liver of the female sibling (freemartin) at 60 days gestation. The level of bull-derived CD34+ cells was lower in the freemartin than in its male siblings. Bull (Y+) and cow hematopoietic cells often occurred in separate clusters. Around clusters of Y+CD34+ cells, Y+CD34- cells were typically observed. The thymi were also strongly chimeric at 60 days of gestation. CONCLUSION: The fetal freemartin liver contains clusters of bull-derived hematopoietic progenitors, suggesting clonal expansion and differentiation. Even the roots of the hematopoietic system in cattle twins are thus strongly chimeric from the early stages of fetal development. However, the hematopoietic seeding of fetal liver apparently started already before the onset of functional vascular anastomosis.

Diagnosis of bovine freemartinism by fluorescence in situ hybridization on interphase nuclei using a bovine Y chromosome-specific DNA probe.

A heifer co-twin to a bull, in most cases, is a sterile freemartin which needs to be identified and culled from replacement stock. Various methods are available for the diagnosis of freemartinism, but none is ideal in terms of speed, sensitivity, or specificity. The present study was thus conducted to develop and validate a satisfactory fluorescence in situ hybridization procedure on interphase nuclei (I-FISH) for identifying the bovine XX/XY-karyotypic chimerism, the hallmark of freemartinism. A 190-bp DNA FISH probe containing the bovine male-specific BC1.2 DNA sequence was synthesized and labeled with digoxigenin by PCR. The FISH was performed on metaphase spreads and interphase nuclei of blood lymphocytes. Upon FISH, the probe expectedly bound to the nucleus of the male cell or to a region of the p12 locus of the Y chromosome. Twenty-four young heterosexual twins (Holstein-Friesian and Korean Cattle breeds; 10 pairs and 4 singletons) were analyzed in the present study; all but three exhibited the XX/XY-karyotypic chimerism to varying extents in both I-FISH and karyotyping. One heifer was identified to have 100% XX cells by both analyses, whereas two bulls were judged as 100% XY- and XX/XY-chimeric karyotypes by karyotyping and I-FISH, respectively. Nevertheless, the ratios of the XY to XX cells in these animals were very similar between the two analyses. In conclusion, the present I-FISH was a rapid and reliable procedure that can be used for early-life diagnosis of bovine freemartinism.

Comparison of cytogenetics and polymerase chain reaction based detection of the amelogenin gene polymorphism for the diagnosis of freemartinism in cattle.

A polymerase chain reaction (PCR) assay which detects a sex-based polymorphism in the bovine amelogenin locus was modified and compared to conventional cytogenetic analysis for diagnosis of freemartinism (XX/XY chimerism) in cattle. The PCR assay is more sensitive than cytogenetic analysis for detection of XY cells, with the limit of detection of the assay falling between 0.2% and 1% XY cells. Seventy-three heifer blood samples submitted for evaluation of freemartinism to the University of Minnesota Diagnostic Laboratory were tested using both cytogenetic and PCR techniques. Poor-quality samples precluded successful lymphocyte culture and recovery of mitotic nuclei for cytogenetic evaluation in 17 cases (23%). Two of these samples (2.7%) also failed to amplify with PCR. There was 100% agreement in the results from the 56 samples that were suitable for testing using both techniques. This PCR-based assay provides an alternative to the more laborious cytogenetic evaluation for diagnosis of freemartinism.

Freemartinism in river buffalo: clinical and cytogenetic observations.

During the last five years forty phenotypic female river buffalos with reproductive problems and two phenotypic male co-twins of females, raised in the provinces of Caserta and Salerno (southern Italy) underwent cytogenetic investigation. Of the 42 animals studied, 10 freemartins (8 females and 2 males) were found with variable percentages of male and female blood cells, the majority however showing similar percentages of both. Of the eight females, six showed normal body conformation, vagina and clitoris, while two showed some male traits (tight pelvis). The two males were apparently normal with only a reduced size of one testicle in one animal. Clinical observations performed in the internal reproductive organs of the female carriers by both rectal palpation (5 females) and direct observation after mating (3 females) revealed serious damages varying from complete lack of internal sex adducts (closed vagina) to hypoplasia of Mullerian ducts and absence (or atrophy) of ovaries. All freemartin females were sterile. To our knowledge, this is the first detailed description of freemartinism in river buffalo by combining clinical and cytogenetic analyses.