Heterosexual Twins Research Articles

Sexing of Embryos at the Time of Twin Reduction: A Clinical Approach.

This study provides new unique information on bovine twin pairs during the late embryonic period (28-34 days of pregnancy) in relation to (1) a predictive ultrasound measurement that was differential for sexing heterosexual twins; (2) intrauterine embryonic growth patterns in twin pairs; and (3) a higher vulnerability of female embryos compared to males following an induced embryo reduction in heterosexual twins. The study population comprised 92 dairy cows carrying bilateral twins. A length difference between co-twins equal to or greater than 25% in around 50% of pregnancies served to determine the sex of embryos with 100% accuracy in heterosexual twins, which was assessed four weeks later on the remaining fetus after twin reduction. The apparent rates of growth of twin pairs and of individual male and female embryos from day 28 to 34 of gestation were similar to established growth pattern standards for singletons. Mean embryo sizes in relation to gestational age were smaller by some 5 days' growth equivalent in twins compared to singletons. After the reduction in the female embryo in heterosexual twins, the risk of male embryo loss was null. This new information allowed for sex selection at the time of twin reduction.

Twin reduction in the late embryonic period prevents the condition of freemartin in dairy cattle.

The condition of freemartinism occurs in the bovine heterosexual twin foetuses and refers to the resulting infertile female. Vascular anastomoses of the foetal membranes are the major reason of this anomaly. This study examines whether single born heifers following induced twin reduction at 28-34 days of pregnancy could develop normal reproductive functions. The study population derived from 367 lactating dairy cows carrying: unilateral twins (n=178), bilateral twins (n=174) or triplets (n=15), in which manual embryo reduction was performed. The final study population was constituted of 95 single born twin females that reached 12months of life and entered into the AI period. Of these heifers, 40 have had one unilateral co-twin, 49 one bilateral co-twin, and 6 two contralateral co-twins. A total of 1688 heifers inseminated during the same period were used as controls to compare the rates of pregnancy at 15months of age, culling due to infertility and birth before the age of 24months. No differences were detected between groups. With an accepted incidence rate of 50% heterosexuality for all twin sets, we should assume that half of our study population had a male co-twin. Our results indicate lack of effect of the male co-twin on normal development of the gonads and genital tract of his female partner during the late embryonic period.

Early Diagnosis of 60, XX/XY Chimerism in Cattle using RT-PCR

Background: Freemartinism is a special kind of cell chimerism occuring in female calf born co-twin with male calf. It is one of the most common forms of intersexuality found in cattle, arises as a consequence of vascular anastamosis between fetuses of different genders. Freemartinism causes alterations in the development of female reproductive organs and sterility. However, there were reports of fertile heterosexual twins also. Hence, the present study aimed at early diagnosis of 60, XX/XY chimerism in heterosexual pregnancies to prevent the unnecessary economic losses and preserve valuable genetic material. Methods: Chromosomal profile of female and male calves were analyzed through short term lymphocyte culture technique. The gene expression levels of ZFX and SRY genes were determined through SYBR green real-time PCR. Result: Both female and male co-twins were found to possess 60, XX/60, XY chimerism, yielding the PCR products of both the male (SRY) and female (ZFX) specific genes. A real-time PCR revealed higher levels of ZFX gene expression in female (81.66 per cent) and male (88.35 per cent) calves than SRY gene. Combination of karyotyping and real-time PCR gave a confirmative diagnosis of freemartinism, suggesting the early diagnosis soon after birth.

Do psychosocial factors mediate sexual minorities' risky sexual behaviour? A twin study.

Risky sexual behavior among sexual minorities (lesbian, gay, and bisexual individuals) are partly attributed to mental health and other social disparities; but this may be confounded by correlated genetic and environmental influences. As preregistered, the present study investigated indirect associations between sexual minority status and increased lifetime sexual partners through increased psychosocial adversity (depressive and anxiety symptoms, intimate partner violence, and early life adversities) and substance use (alcohol, cannabis, and other illicit drugs), confounding by correlated genetic and environmental influences, and sex differences in these relationships. The sample comprised sexual minority and heterosexual twins who participated in the first and second phases of the latest wave of data collection in the U.K. population-based Twins Early Development study cohort (June 2017 through February 2019; n = 9,697 and 8,718, respectively, Mage = 22.3 ± .92 years). Structural equation modeling was used to specify psychosocial adversity and substance use as mediators while genetic and environmental confounding was further determined by biometrical genetic analyses in which similarities in identical and nonidentical twins were compared. Increased psychosocial adversity and substance use fully mediated increased lifetime sexual partners in sexual minority women while this effect was partial (31.1%) in men. The best-fitting genetic models indicated that these relationships were not confounded by correlated genetic and environmental influences. The relationships between sexual minority status, psychosocial adversity, substance use, and sexual health disparities appeared independent of genetic and environmental influences. Individual and systemic interventions to reduce psychosocial disadvantage and substance use can also decrease sexual health disparities among sexual minorities. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Potential case of pseudo-hermaphroditism in Elk (<i>Cervus canadensis</i>) in Alberta, Canada

Cases of true and pseudo-hermaphroditism, in which animals possess both ovaries and testes or have a single chromosomal and gonadal sex but secondary features of the other sex, have been documented in several cervids, including Odocoileus (deer) and Capreolus (roe deer) species. Another form of intersexuality that has been well documented in Domestic Cattle (Bos taurus) and induced in Red Deer (Cervus elaphus) is freemartinism, where blood is shared between heterosexual twins leading to XX/XY chimeras. We report the first case of pseudo-hermaphroditism in wild Elk (Cervus canadensis), observed in the central east slopes of the Rocky Mountains of Alberta, Canada, from September through December 2019. The Elk had no antlers, exhibited female external genitalia, and displayed male secondary sexual characteristics, including colouring and breeding behaviour. To determine whether this is a case of true hermaphroditism, pseudo-hermaphroditism, or freemartinism would require blood analysis and inspection of internal sex organs by necropsy.

The birth of heterosexual twins after removal of the left ovary

The author, in confirmation of the fact that with the existence of only one ovary, two fetuses of different sex can develop, gives the case of the birth of twins, a boy and a girl in a woman in whom Dr. who became pregnant two months after the operation.

Comparison of the biological characteristics of umbilical cord mesenchymal stem cells derived from the human heterosexual twins

Mesenchymal stromal/stem cells (MSCs) are attracting more and more attention due to their tissue regenerative properties and immunomodulatory functions. MSCs may be the most acceptable, safe, and effective source for allogeneic cell therapy, and have been used in medical treatment. However, the similarities and differences between umbilical cord-derived MSCs (UC-MSCs) of heterosexual twins remain poorly understood. In this study, we compared the biological characteristics of UC-MSCs of heterosexual twins in vitro. We found that male fetal UC-MSCs and female fetal UC-MSCs share a similar phenotype and multi-lineage differentiation potential, and male fetal UC-MSCs show a significantly higher proliferation and adipogenic ability than female fetal UC-MSCs. UC-MSCs from heterosexual twins showed significant differences in the expression levels of NANOG, OCT4, TERT, and SOX2. In addition, male MSCs are more potent in the expression of inflammatory cytokines to lipopolysaccharide (LPS)-induced inflammation. In future clinical applications using MSCs for inflammation-related diseases, these biological characteristics differences with different genders will guide our clinical methods.

Early Diagnostics of Freemartinism in Polish Holstein-Friesian Female Calves.

Simple SummaryFreemartinism is the most common type of gender developmental disorder, resulting in infertility of heifers from multiple-sex twin pregnancies. The frequency of this syndrome is related to the frequency of multiple pregnancies, the number of which has significantly increased in dairy cattle populations (HF). Therefore, rapid diagnostics is necessary to enable early elimination of heifers with freemartinism from breeding. The aim of the study was to compare and identify the best method for early identification of freemartinism. The use of cytogenetic and molecular methods (PCR, short tandem repeats (STRs), real-time PCR) allowed us to conclude that molecular methods are more effective and guarantee fast and precise diagnosis. An additional advantage of molecular methods is the easy way to collect test material, which can be frozen, unlike blood samples for cytogenetic analysis, which must be fresh and delivered within 24 h to the laboratory, which generates further costs.Freemartinism in females born from heterosexual multiple pregnancies is characterized by the presence of XX/XY cell lines due to the formation of a shared blood system by anastomoses between fetal membranes of co–twins and leads to disturbed development of the reproductive system, including infertility. The aim of this study was to estimate the most precise and effective diagnostic method, especially useful for early identification of freemartinism in young female calves. The cytomolecular evaluation results of 24 Holstein-Friesian heifers from heterosexual twins was verified by molecular techniques: PCR, short tandem repeats (STRs), and relative quantitative PCR. The molecular analyses have been found to be a more efficient testing strategy, with a higher diagnostic success rate than karyotype evaluation. In 21 heifers, leucocyte chimerism determined by the 60, XX/60, XY karyotype was revealed—the proportion of the 60, XY male cell line in individual animals was in the range of 4–66%. In three cases, a normal karyotype 60, XX was identified, which indicates that anastomoses did not occur in 12.5% of studied twins and suggests that these potentially fertile heifers can be qualified for further breeding. The precise and early identification of freemartinism can be the basis for guidelines and selection recommendations concerning the reproductive performance of heifers born from heterosexual multiple pregnancies.

Cytogenetic Analyses in Ewes with Congenital Abnormalities of the Genital Apparatus.

Simple SummaryThe disorders of sex development (DSDs) are congenital conditions characterized by inconsistency among chromosomal, gonadal, and anatomical sex development. Reproduction and prolificacy are two main parameters in the sheep industry; thus, DSDs in sheep are very detrimental. Interestingly, no DSDs-associated gene mutations have been reported in sheep so far, probably due to the fact that affected animals are not detected and studied. With the aim to deepen the knowledge about DSDs in sheep and improve diagnostic tools, screening of a sheep farm aimed to detect and study DSDs in ovine species has been started, and the actual findings relative to the first two sheep flocks analysed are reported here. In our opinion, this study demonstrates that, despite the low number of studies on ovine DSDs, if compared with other species, this problem is actually present and needs more attention.The Disorders of Sex Development (DSDs) are congenital conditions characterized by inconsistency among chromosomal, gonadal, and anatomical sex development. The aim of this research is to report the clinical and cytogenetic findings of four DSD cases and 13 couples of heterosexual twins in sheep. To this purpose, C- and R-banding techniques were used, and the analyses of the SRY (Sex Determining Region Y) and AMEL (Amelogenin) genes were carried out. Moreover, morphopathological analyses were performed in one case. The four DSD sheep cases were registered as females at birth, and for none of them it was possible to establish whether the subjects were born from heterosexual multiple births. Three of the four cases were diagnosed as XX/XY blood lymphocyte chimaeras, while the fourth case was diagnosed as a 54, XY SRY-positive DSD sheep. None of the heterosexual twins showed XX/XY blood chimaerism. This finding suggests that the blood chimaeric cases detected could also be due to a zygote/embryo fusion. Moreover, no gene variants involved in sheep DSD are known, the identification of which would be very useful for the sheep industry.

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.

Applying real-time quantitative PCR to diagnosis of freemartin in Holstein cattle by quantifying SRY gene: a comparison experiment

BackgroundFreemartinism generally occurs in female offspring of dizygotic twins in a mixed-sex pregnancy. Most bovine heterosexual twin females are freemartins. However, about 10% of bovine heterosexual twin females are fertile. Farmers mostly cull bovine fertile heterosexual twin females due to the lack of a practical diagnostic approach. Culling of such animals results in economic and genetic-material losses both for dairy and beef industry.MethodsIn this study, a comparative test, including qualitative detection of SRY gene by polymerase chain reaction (PCR), quantitative detection of relative content of SRY by real-time quantitative polymerase chain reaction (qPCR), and quantitative detection of H-Y antigen, was performed to establish the most accurate diagnosis for freemartin. Twelve Holstein heterosexual twin females were used in this study, while three normal Holstein bulls and three normal Holstein cows were used as a positive and negative control, respectively.ResultsPolymerase chain reaction results revealed that SRY gene were absent in three heterosexual twin females and only two of them were verified as fertile in later age. The qPCR results showed that relative content of SRY was more than 14.2% in freemartins and below 0.41% in fertile heterosexual twin females. The H-Y antigen test showed no significant numerical difference between freemartin and fertile heterosexual twin female.DiscussionOur results show that relative content of SRY quantified by qPCR is a better detection method for diagnosis of freemartin in Holstein cattle as compare to qualitative detection of SRY gene by PCR or quantitative detection of H-Y antigen. To the authors’ knowledge, this is the first time we applied qPCR to diagnosing freemartin by quantifying SRY gene and got relative SRY content of each freemartin and fertile heterosexual twin female. We concluded that low-level of SRY would not influence fertility of bovine heterosexual twin female.

Мировоззрение и традиции населения андроновской историко-культурной общности (по данным погребальной обрядности)

Purpose. We aimed at studying the traditions and world views of the Andronovo population of the steppe bronze. Due to the absence of direct written sources and zoo-anthropomorphic pictorial tradition on the subject, the burial practice of the Andronovo population, whose detailing presupposes the existence of extensive mythological ritual knowledge concentrated in the worldview sphere, is the foreground of research as the main informative base. Results. The earliest evidence that specifies certain aspects of the worldview of the ancient society appears at the dawn of the Andronovo era. The finds of stone and bronze maces are curious, which, obviously, marked the patrimonial military aristocracy, closely connected with the cult of the military deity. Of great interest are paired and double burials in which a man and woman were buried. It can be assumed that such a burial rite is a practical realization of the sacred marriage, the participants of which are heterosexual twins, close in content to Yama-Yami or Yima-Yimak. Regular reproduction in the funeral practice of the ritual of twin burials indicates that the heterosexual twins were given a significant place in the religious and mythological system of the ancient society. A certain place in the system of religious priorities was occupied by twins of the same sex, in particular males, such as, for example, the Vedic Ashvins. Double burials of the deceased of the same sex in specially prepared burial chambers, where skeletons of different sexes are usually located, are excluded, which excludes their marriage relations and makes us see in the ritual contemplated a twin, possibly, a ritual burial. There was another, more complex and rare rite of the triple burial, which includes a woman occupying the central place and two men located on each side. Such triple burials symbolize the triune image of the goddess and two twins, obviously the elder and younger, widespread in Indo-European mythology. Conclusion. Based on the well-known mythology of the funeral rite reproduced in the ritual, a whole series of sacred actions are observed pointing to the developed cults of various deities close to the Indo-Iranian pantheon and playing a fundamental role in the religious mythological representations of the ancient society. However, it should be borne in mind that the polytheism of antiquity is a dynamically changing system rather than a static, «petrified» structure, which visually demonstrates the successive stages of the social and economic development of the society itself.

Haematopoietic chimerism expressivity in bovine heterosexual twins

The aim of the experiment was to study the haematopoietic chimerism in bovine heterosexual twins conceived through artificial insemination and post-embryo transfer. Both animal groups were revealed to have a wide individual range of variability for the expressivity of chimerism that varied from 0 to 96% of cells with the chromosomes of an opposite sex. The study also revealed the tendency towards increased frequency of cytogenetic anomalies in immigrant cells.

A high incidence of leukocyte chimerism (60,XX/60,XY) in single born heifers culled due to underdevelopment of internal reproductive tracts

Freemartinism, a primary disorder of sex development (DSD) in cattle, is associated with leukocyte chimerism (60,XX/60,XY). The diagnosis of DSD is easy if it is known that a heifer with abnormally developed reproductive tracts originates from a heterosexual twin birth, but it is not so obvious in the case of single born calves. In the present study twelve DSD heifers which were single born (singletons) and culled due to the abnormal development of internal genitalia were studied using cytogenetic and molecular techniques. Among the heifers 7 appeared to be chimeric (60,XX/60,XY and the presence of the genes residing in the Y chromosome: SRY and AMELY) and 5 had a normal female karyotype (60,XX and a lack of the Y-linked genes). In addition, milk productivity was analyzed in relation to the incidence of twinning at a local Dairy Cattle Breeding Centre, from which 8 studied singletons (6 chimeric and 2 with a normal female karyotype) originated. It was found that in the years 2005&amp;ndash;2013 an upward trend for average milk yield (from 9700 kg in 2005 to 11 500 kg in 2013) was associated with the increase of twin births (from 1.5% in 2005 to 5.9% in 2013). Our study showed that approximately 60% of single born heifers with abnormally developed internal genitalia were freemartins (a male co-twin died during pregnancy), while DSD etiology of the other cases (60,XX and a lack of the Y-linked genes) remains unknown. It cannot be excluded that some of these heifers represent a testicular/ovotesticular DSD (60,XX and SRY-negative). In conclusion, our study suggests that the occurrence of freemartins and other DSD in single born heifers seems to be an underestimated problem in cattle breeding.

A Rapid Multiplex PCR Method for the Diagnose of Freemartin Syndrome in Domestic Cattle (Bos taurus)

The majority of heifer calves born as hetero–sexual twin births are sterile freemartins. It is important that this condition be diagnosed at an early age because freemartin heifers can’t be used as replacement stock. Different methods are available for the diagnosis of freemartinism, however molecular methods are preferred due to their accuracy and shorter duration of process. In the present study, freemartinism status of 40 female calves of heterosexual multiple births was investigated by multiplex polymerase chain reaction (multiplex PCR). The reaction was carried out using two primer sets for the Sry and K–casein genes of domestic cattle. The PCR product was analyzed by agarose gel (3%) electrophoresis, which allowed to identify the genotypes of the animals studied: normal males (453 bp and 163 bp), normal females (453 bp), and 39 (97.50%) study cases that showed chromosome chimerism (freemartin, 453 bp and 163 bp). Compared to the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) technique, this method (i.e., multiplex PCR) proved a far cheaper and quicker (approximately 27 minutes) way to diagnose freemartinism.

The relationship between help-seeking attitudes and masculine norms among monozygotic male twins discordant for sexual orientation.

In general, heterosexual men are less favorable to asking for help compared to women and gay men. This can be problematic if a man avoids professional help when he is experiencing significant psychological distress. Yet, it is unclear to what degree such attitudes among men are due to innate differences or social environments. Studying twins provides one avenue for teasing apart these relationships. We recruited 38 pairs of monozygotic male twins (Mage = 35.87 years, SD = 9.52) raised together and who were discordant for sexual orientation. They completed measures of psychological distress (Symptom Checklist-90-Revised), positive attitudes toward psychological help-seeking behavior, and emphasis with fulfilling traditional masculine norms. Contrary to predictions, the heterosexual twins expressed more symptoms of specific distress-hostility (r = .30), paranoid ideation (r = .26), and psychoticism (r = .24)-than their gay cotwins. As predicted, heterosexual men were less favorable to seeking help (r = .25) and expressed greater emphasis on masculine norms (r = .26) than their cotwins. Within each group of men, unique aspects of masculine norms were significantly related to attitudes toward psychological help-seeking behavior. The findings lend credence to the hypothesis that social environments influence attitudes and behaviors that are stereotypically masculine and potentially detrimental to men's health.

Chimaerism detection in bovine twins, triplets and quadruplets using sex chromosome-linked markers

The phenomenon of chimaerism occurs in the majority of cattle twin pregnancies. The objectives of this study were to develop a powerful diagnostic test for chimaerism in bovine male and female co-twins using X and Y chromosome-linked markers and to determine the extent of chimaerism in twins, triplets and quadruplets. We developed a multiplex PCR set of three polymorphic markers on chromosome X (DIK2865, DIK2283, AGLA257), where the presence of >1 and >2 alleles per marker is sufficient to prove chimaerism in males and females, respectively. In addition, a specific segment on chromosome Y (BOV97M) is included in the set to indicate chimaerism in females. Visualization of chimaeric alleles was best for DNA extracted from blood, fair for DNA from vaginal smears and failed for DNA extracted from hair. The power of chimaerism identification using this set of markers for DNA extracted from blood was calculated as 99% in males and virtually 100% in females. All females and males in heterosexual twins, triplets and quadruplets displayed evidence of a chimaeric allele in at least one and maximum of three of three X chromosome markers analysed. In addition, all females showed the presence of the BOV97M segment and were validated as chimaeric by the standard clinical diagnosis of impaired vaginal length. Quantitative PCR analysis of BOV97M copies in all twins vs. their sires showed a mean ratio of 45-68% in females and 39-49% in males, indicating a substantial symmetrical exchange of cells among all co-twins. The proposed analysis of X and Y chromosome-linked markers is advantageous to previous methods based on Y chromosome sequences only, because it detects chimaerism in both male and female co-twins.

Genetic factors predisposing to homosexuality may increase mating success in heterosexuals

There is considerable evidence that human sexual orientation is genetically influenced, so it is not known how homosexuality, which tends to lower reproductive success, is maintained in the population at a relatively high frequency. One hypothesis proposes that while genes predisposing to homosexuality reduce homosexuals' reproductive success, they may confer some advantage in heterosexuals who carry them. However, it is not clear what such an advantage may be. To investigate this, we examine a data set where a large community-based twin sample ( N=4904) anonymously completed a detailed questionnaire examining sexual behaviors and attitudes. We show that psychologically masculine females and feminine men are (a) more likely to be nonheterosexual but (b), when heterosexual, have more opposite-sex sexual partners. With statistical modelling of the twin data, we show that both these relationships are partly due to pleiotropic genetic influences common to each trait. We also find a trend for heterosexuals with a nonheterosexual twin to have more opposite-sex partners than do heterosexual twin pairs. Taken together, these results suggest that genes predisposing to homosexuality may confer a mating advantage in heterosexuals, which could help explain the evolution and maintenance of homosexuality in the population.

XX/XY Chimerism in Cattle: Clinical and Cytogenetic Studies

The freemartin condition represents the most frequent form of intersexuality found in cattle, and occasionally in other species. Freemartinism arises when vascular connections occur between placentae of developing heterosexual twin foetuses, XX/XY chimerism develops, and ultimately there is masculinisation of the female tubular reproductive tract to varying degrees. The aim of this work was to report the clinical and cytogenetic studies performed in 28 cattle co-twins, 24 of which were cytogenetically chimeric (2n = 60, XX/XY), raised in the region of Campania (southern Italy). Clinical findings of the 16 freemartin females examined varied greatly, from a more female phenotype (normal body conformation with the presence of a blind-ending vagina and primordial ovarian and uterus structures) to a nearly male phenotype (body conformations with male traits and presence of primordial prepuce, penis and testicles). The 8 freemartin males, in spite of the presence of XX cells, had a normal body conformation and external genitalia and some of them were fertile. In addition to cytogenetic diagnosis we also verified chromosome fragility by testing for chromosome aberration (CA: aneuploidy, gaps, chromatid breaks, chromosome breaks and fragments) and sister chromatid exchange (SCE). Freemartins showed a higher percentage of aneuploid cells and significant statistical differences in mean values of gaps, chromatid breaks and chromosome breaks when compared with control animals. To our knowledge, this is the first time that chromosome instability has been evaluated by analyses of CA and SCE in freemartin cattle.

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.