RESUMO
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.
Assuntos
Amelogenina/genética , Quimerismo/veterinária , Freemartinismo/diagnóstico , Reação em Cadeia da Polimerase/veterinária , Cromossomos Sexuais/genética , Animais , Bovinos , Feminino , Freemartinismo/genética , Leucócitos , Masculino , Reação em Cadeia da Polimerase/métodos , Gravidez , Sensibilidade e Especificidade , Cromossomo X/genética , Cromossomo Y/genéticaRESUMO
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.
Assuntos
Transtornos do Desenvolvimento Sexual/veterinária , Doenças do Cão/diagnóstico , Freemartinismo/diagnóstico , Gravidez Múltipla , Animais , Bovinos , Impressões Digitais de DNA , Transtornos do Desenvolvimento Sexual/diagnóstico , Transtornos do Desenvolvimento Sexual/genética , Doenças do Cão/genética , Cães , Feminino , Freemartinismo/genética , GravidezRESUMO
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.
Assuntos
Autoimunidade , Quimerismo , Tolerância Imunológica , Animais , Bovinos , Quimerismo/embriologia , DNA Mitocondrial/análise , Feminino , Freemartinismo/diagnóstico , Freemartinismo/genética , Marcadores Genéticos/genética , Células Germinativas/citologia , Hematopoese , Humanos , Masculino , Transplante Homólogo/imunologiaRESUMO
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.
Assuntos
Núcleo Celular/genética , Sondas de DNA/metabolismo , Freemartinismo/diagnóstico , Hibridização in Situ Fluorescente/veterinária , Interfase/genética , Linfócitos/citologia , Cromossomo Y/genética , Animais , Bovinos , Feminino , Masculino , Reprodutibilidade dos TestesRESUMO
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.
Assuntos
Proteínas do Esmalte Dentário/genética , Freemartinismo/diagnóstico , Freemartinismo/genética , Reação em Cadeia da Polimerase/veterinária , Amelogenina , Animais , Bovinos , Análise Citogenética/veterinária , DNA/química , DNA/genética , Eletroforese em Gel de Ágar/veterinária , Feminino , Masculino , Reação em Cadeia da Polimerase/métodos , Polimorfismo Genético , Sensibilidade e Especificidade , Cromossomo X/genética , Cromossomo Y/genéticaRESUMO
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.
Assuntos
Freemartinismo , Animais , Camelídeos Americanos , Bovinos , DNA/análise , Cervos , Feminino , Freemartinismo/diagnóstico , Freemartinismo/epidemiologia , Freemartinismo/genética , Freemartinismo/fisiopatologia , Cabras , Cavalos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Carne , Gravidez , Diagnóstico Pré-Natal/veterinária , Ovinos , Suínos , Cromossomo Y/genéticaRESUMO
A female Japanese Black calf was born on 25 March 2003 at Hiroshima University Farm as a co-twin to a male Japanese Black calf. The male calf showed no external urogenital abnormalities. The absence of anal opening and external features of freemartinism were observed in the female. A small opening to the vulva (about 1.5 cm in length) with fused lips and a prominent clitoris were seen. The hair around the vulva was 3.5 cm in length and was heavy and dense. The distance from the vulva to the atretic anus was 9.0 cm. There were no other detectable abnormalities on physical examination. The PCR-based DNA test showed male-specific sequences confirming the calf to be freemartin. At autopsy 1 day after the calf birth, the gonads were found to be small and hard and the left uterine horn showed segmental aplasia near its proximal end. Two seminal glands (remnants of mesonephric duct) were located on both sides of the uterine body. A cervix was absent. The vagina was underdeveloped and looked like a tubual structure. The rectal end was closed, while the distance from the end of the atretic rectum to the absent anal opening was about 4.0 cm. On histological examination, the gonads exhibited extensive morphologic alteration; there was no cortex with the absence of ovarian structures. The seminal glands consisted of hypoplastic glandular tissue surrounded by extensive fibrous connective tissue. In conclusion, this is a case report of a freemartin with atresia recti and ani.
Assuntos
Canal Anal/anormalidades , Freemartinismo/complicações , Reto/anormalidades , Animais , Bovinos , DNA/análise , Feminino , Freemartinismo/diagnóstico , Freemartinismo/genética , Genitália/anormalidades , Reação em Cadeia da Polimerase , Vulva/anormalidadesRESUMO
An unusual case of a live Holstein freemartin calf co-twinned with schistosomus reflexus fetus is presented here. Delivery of the schistosomus reflexus was achieved by fetotomy 24 h after manual delivery of a live heifer calf. The dam subsequently experienced concurrent metritis and left displacement of the abomasum; however, she conceived following insemination 85 d post partum. Cytogenetic examination of the live heifer using lymphocyte culture and cutaneous fibroblast cell culture failed to demonstrate chromosomal chimerism, whereas polymerase chain reaction (PCR) detected the presence of the bovine Y-chromosome marker BRY-1. Low concentrations of testosterone, estradiol and progesterone at 3, 6, 24 and 48 h after administration of hCG were detected in the serum of the freemartin heifer. Genetic, hormonal, histological and clinical findings established the live female co-twin calf was a freemartin, an abnormality of phenotypic sex. Failure to detect any significant peripheral concentrations of androgen supports the hypothesis that masculinization of the freemartin reproductive tract arises from diffusion of androgen and possibly other substances from the male co-twin rather than from endogenous production of androgen within the freemartin. This report documents that the freemartin condition can be induced by a male fetus with severe developmental abnormalities.
Assuntos
Abdome/anormalidades , Anormalidades Congênitas/veterinária , Freemartinismo/diagnóstico , Animais , Bovinos , Doenças dos Bovinos/patologia , Células Cultivadas , Gonadotropina Coriônica , Anormalidades Congênitas/patologia , Estradiol/sangue , Feminino , Freemartinismo/sangue , Freemartinismo/patologia , Linfócitos/patologia , Masculino , Progesterona/sangue , Testosterona/sangue , Gêmeos , Cromossomo YRESUMO
The majority of heifers born co-twins to bulls are infertile freemartins. It is important that the condition be diagnosed at an early age as freemartins have no potential for use as replacement stock. A rapid, robust, reliable technique for freemartin diagnosis is described. Three Y-specific polymerase chain reaction (PCR) primer pairs: BOV97M, BRY.1 and AMX/Y were used to detect male cells in the blood of heifers born co-twins to bulls. PCR -based tests have advantages over currently used methods of freemartin diagnosis.
Assuntos
DNA/química , Freemartinismo/genética , Animais , Sequência de Bases , Bovinos , Feminino , Freemartinismo/diagnóstico , Masculino , Dados de Sequência MolecularRESUMO
A total of 727 blood samples from female calves born co-twin to male calves were examined cytogenetically for freemartinism between 1978 and 1992. Six hundred calves (82.5%) were determined to be freemartins, and 127 (17.5%) were determined not to be freemartins. The percentage of calves determined not to be freemartins was substantially higher than the 8% reported for an unselected population of female co-twins. We concluded that some obvious freemartins were eliminated prior to submission of samples for confirmatory cytogenetic diagnosis, and that only a small percentage of the estimated 93,000 female calves born co-twin to male calves annually are so examined. Therefore, probably a large number of female co-twins that are not truly freemartins are sold to slaughter every year. We propose that obvious freemartins be identified by use of the vaginal-length test and that the remaining clinically questionable calves be differentiated cytogenetically. This combination of procedures could prevent unnecessary economic losses and preserve important genetic material. Three animals with chromosomal anomalies were found during examination of samples for freemartinism. Cytogenetic evaluation for freemartinism thus offers the added value of simultaneous surveillance for cytogenetic aberrations in male and female cells of a sample.
Assuntos
Freemartinismo/diagnóstico , Animais , Animais Recém-Nascidos , Bovinos , Feminino , Freemartinismo/epidemiologia , Freemartinismo/genética , Cariotipagem/veterinária , Masculino , Gêmeos , Estados Unidos/epidemiologia , Vagina/patologiaRESUMO
The membrane of RBC is literally peppered with a great variety of antigenic determinants (blood factors). Some are fixed genetically, ie, they occur on the RBC of all members of the species under study. Others segregate genetically, ie, they occur on the RBC of some but not all members of the species under study. It is these segregrating determinants that form the blood groups proper, the classic example being blood factors A and B of the ABO system of human blood groups. The number of blood group determinants varies considerably between species (eg, greater than 80 in domestic cattle to only 1 in the new world Camelidae). Genetic analysis has shown that the blood factors belong to one or another of a limited number of blood group systems. Some of these systems, similar to the B system of cattle, are enormously complex, whereas others involve but a single blood factor. The object of the present paper is to report on the extent of genetic variation in blood groups and how information on blood groups is being used clinically.
Assuntos
Animais Domésticos/sangue , Antígenos de Grupos Sanguíneos , Animais , Artiodáctilos , Antígenos de Grupos Sanguíneos/genética , Tipagem e Reações Cruzadas Sanguíneas/veterinária , Transfusão de Sangue/veterinária , Cruzamento , Gatos , Bovinos , Galinhas , Reações Cruzadas , Cães , Feminino , Freemartinismo/diagnóstico , Cavalos , Masculino , Gravidez , Coelhos , Ovinos , Suínos , Reação TransfusionalRESUMO
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.
Assuntos
Bovinos/fisiologia , Freemartinismo/diagnóstico , Gêmeos , Animais , Bovinos/genética , Feminino , Freemartinismo/genética , Freemartinismo/fisiopatologia , Masculino , Gravidez , Cromossomos SexuaisRESUMO
The preliminary results of comparative studies designed to make it possible to establish a diagnosis of 'freemartin' are reported. Three methods were used, namely typing of the blood, chromosome analysis and determination of the length of the vagina. In a very large number of cases, the diagnosis may even be established in very young calves by measuring the length of the vagina. Laboratory studies may provide a solution in doubtful cases.
Assuntos
Freemartinismo/diagnóstico , Animais , Tipagem e Reações Cruzadas Sanguíneas , Bovinos , Cromossomos/análise , Feminino , Masculino , Vagina/anatomia & histologiaRESUMO
Methods are described for the detection of freemartins. The first step is to measure the length of the vagina, followed, if necessary, by laboratory investigations--blood group, chromosome, or DNA typing.
Assuntos
Freemartinismo/diagnóstico , Prenhez , Gravidez Múltipla , Animais , Antígenos de Grupos Sanguíneos , Bovinos , Cromossomos/ultraestrutura , Feminino , Freemartinismo/genética , Cariotipagem/veterinária , Reação em Cadeia da Polimerase/veterinária , GravidezRESUMO
The percentage of freemartins among blood samples tested by chromosome analysis amounted to 83.9%, by blood group serologie 71.4%. 126 blood samples have been tested by blood group serology and PCR. Employing blood group serology, 71.3% and using PCR with BOV97M primers 85.8% of the animals proved to be freemartins. 40 blood samples were additionally analysed using PCR with zinc-finger-gene primers. 36 animals (90%) were identified as being freemartins by means of BOV97M and 34 animals (85%) by means of the zinc-finger-gene primer. The PCR method proved to be a rapid and very sensitive method for the diagnosis of freemartins and also suitable for routine testing. The BOV97M primer showed to have a higher Y chromosome specificity than the zinc-finger-gene primer.