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.

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.

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.

Low levels of X-inactive specific transcript in somatic cell nuclear transfer embryos derived from female bovine freemartin donor cells.

The present study compared developmental potential, telomerase activity and transcript levels of X-linked genes (HPRT, MECP2, RPS4X, SLC25A6, XIAP, XIST and ZFX) in bovine somatic cell nuclear transfer (SCNT) embryos reconstructed with cells derived from a freemartin (female with a male co-twin) or from normal female cattle (control). The rates of cleavage, development to blastocyst and hatched blastocyst stage, and the mean numbers of total and inner cell mass cells in the freemartin SCNT embryos were not significantly different from those of control SCNT embryos (p > 0.05). The levels of telomerase activity analyzed by RQ-TRAP in the freemartin SCNT embryos were also similar to those of the normal SCNT embryos. Transcript levels of HPRT, MECP2, RPS4X and XIAP, measured by quantitative real-time RT-PCR, were not significantly different between the control and freemartin SCNT embryos (p > 0.05). However, the transcript levels of SLC25A6, XIST and ZFX were significantly decreased in the freemartin SCNT embryos compared to control SCNT embryos (p < 0.05). Transfer of 71 freemartin SCNT embryos to 22 recipient cows resulted in 4 (18%) pregnancies, which were lost between days 28 and 90 of gestation. Taken together, the present study demonstrates that the transcript levels of several X-linked genes, especially XIST, showed an aberrant pattern in the freemartin SCNT embryos, suggesting aberrant X inactivation in freemartin clones which may affect embryo survival.

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.

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.

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.

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.

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.

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.

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.

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.

A case of freemartin with atresia recti and ani in Japanese Black calf.

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.

Limited contribution of circulating cells to the development and maintenance of nonhematopoietic bovine tissues.

Bone marrow-derived stem cells appear surprisingly multipotent in experimental settings, but the physiological significance of such plasticity is unclear. We have used sex-mismatched cattle twins with stably chimeric hematopoietic systems to investigate the general extent of integration of circulating cells to the nonhematopoietic cell lineages in an unmanipulated large mammal. The donor-derived (Y+) nonhematopoietic cells in female recipient tissues were visualized by Y-chromosome specific in situ hybridization combined with pan-leukocyte labeling. Y+ leukocytes were frequent in all tissues, but in 11 of 12 animals, average contribution to nonhematopoietic lineages was in any tissue below 1% (in brain <0.001%). Significantly higher integration rate was detected in regenerating granulation tissue. Also, one animal showed a high frequency of nonhematopoietic Y+ cells in several tissues, including intestinal epithelium and mammary gland stroma. In conclusion, circulating cells do not appear significant in the development and maintenance of nonhematopoietic bovine tissues, but may be important in regeneration and other special conditions.

Persistent differences in the level of chimerism in B versus T cells of Freemartin cattle.

Parabiosis during pregnancy regularly results in an exchange of hematopoietic stem cells between cattle twins. We have exploited this phenomenon and show differences in the levels of chimerism between the descendant cell types. Female recipients were screened for the levels of male donor contribution in surface IgM-bearing B lymphocytes versus CD3(+) T lymphocytes using immunomagnetic fractionation and Y-chromosome specific in situ hybridization. Two calves of 15 were discovered to have less than 10% of B cells but over 70% of T cells and other blood leukocytes of male origin. The donor cell ratios remained stable for 9 months. Analysis of lymphoid tissues revealed a similar cell type specific pattern of male cell ratios in both female calves and one twin brother. These findings are in agreement with the existence of an essentially self-sufficient population of developing B cells that gives rise to the peripheral pool of B lymphocytes in young cattle.

A mola hydatidosa coexistent with a foetus in a bovine freemartin pregnancy.

Molar transformations of the bovine placenta are extremly rare phenomenona and the aetiology of this genuine placental disease is still unknown. In the present study, an uncommon case of a German Holstein Friesian foetus co-twinned with a hydatidiform mole is described. Cytogenetic and fluorescence in situ hybridization analysis of cell cultures as well as prove of the presence of the SRY gene sequence revealed a heterosexual twin pregnancy. A chimeric condition of the mole was also established. In addition, an XO cell population was detected in the co-twin as well as in the mole. Upon examination of microsatellites of the parents, the mole and the co-twin an androgenetic origin of the mole is suggested, supporting the hypothesis that molar transformation of the bovine placenta may be caused by an androgenetic origin. Furthermore, the present observation demonstrates that the freemartin condition in cattle can be induced even in cases where severe placental transformations had subsequently occurred and no foetus proper could be detected at delivery.

Births of freemartins derived from embryos reconstructed with ear fibroblasts.

Although the combination of artificial insemination (AI) and embryo transfer (ET) is effective for preventing large offspring syndrome in clone cattle production, it may cause freemartinism. In this study, 51 reconstructed embryos were transferred to artificially inseminated recipients. Of those 9 twin pregnancies, three delivered male and female offsprings. The females had tufts of long coarse hair and short blind pouch at the vaginal end. At necropsy, hypoplastic testicles and epididymis, which connected to the uterus through the spermatic cord, were found and seminal vesicles were also noted. All females had mixed sex chromosome configuration (60, XX and 60, XY). These results suggest that the combined ET program can cause freemartinism, which reduces the efficiency of clone cattle production.

Anatomical, cytogenetic and behavioural studies of freemartin ewes.

The external phenotype and reproductive behaviour of 21 freemartin sheep and two male pseudohermaphrodite sheep were recorded with the aim of identifying any characteristics which might be used to predict a failure to breed. The size and shape of the vulva and clitoris, the length of the vagina, the size of the teats, the presence or absence of inguinal gonads, and the ultrasonographic characteristics of the inguinal gonads were recorded. A subjective assessment of the masculinity of each animal's body form was also made, and its behavioural responses to a virile ram and to an oestrus ewe were recorded. A number of physical and behavioural abnormalities were detected but the only consistent finding in all 23 animals was a short vagina which varied in length from 3.1 to 7.0 cm, compared with 10 to 14 cm in normal animals.

XX/XY chimerism and freemartinism in a female llama co-twin to a male.

A 20-month-old female llama was examined because at the time of mating, the male llama was apparently unable to achieve intromission. The female llama had been born co-twin to a male. On physical examination, the vaginal vestibule appeared to end in a blind pouch, and the uterus, cervix, and ovaries could not be identified during transrectal palpation or ultrasonography. Karyotyping was done, and 43% of blood lymphocytes had 2 X chromosomes, and 57% had 1 X and 1 Y chromosome. All skin fibroblasts had 2 X chromosomes. A diagnosis of freemartinism and XX/XY chimerism was made. Because conception of twins may be more common in llamas than birth of twins, it is possible that freemartinism could develop in singleton females, if, for instance, a male twin was conceived and died after the placentas had anastomosed. More widespread use of karyotyping in llamas with congenital defects of the reproductive tract will help to define the incidence of freemartinism.