Heterosexual Twins Research Articles

Rapid sex chromosomal chimerism analysis in heterosexual twin female calves by Loop-mediated Isothermal Amplification

We attempted to apply an embryo sexing kit with Loop-mediated Isothermal Amplification (LAMP) to sex chromosomal chimerism analysis in heterosexual twin female calves. Peripheral blood was used for the amplification of male-specific DNA, derived from XY leukocytes. When blood samples were diluted 1:1000 in LAMP reaction mixture, hemoglobin or blood coagulation did not influence the turbidity measurement of the reaction mixture for detection of amplified DNA. This procedure detected the existence of XY leukocytes of 0.01% in female blood. Furthermore, all heterosexual twin female calves, bearing sex chromosomal chimerism based on karyotyping and PCR, showed male-specific DNA from peripheral blood by LAMP. These results indicated that the embryo sexing kit with LAMP was available for sensitive detection of sex chromosomal chimerism. This procedure made it possible to detect easily Y-chromosome specific DNA in a short interval compared with PCR, and was convenient for field application of freemartin diagnosis.

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 Müllerian 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.

The ambiguity of freemartinism diagnosis in cattle revealed by cytogenetic and molecular techniques

Nineteen heifers and three male co-twins, originating from heterosexual twin pregnancies, were analysed with the use of cytogenetic and molecular techniques. A large number (50–400) of Giemsa stained metaphase spreads were studied and the proportion of XX and XY cell lines was calculated. The presence of two genes – SRY and AMEL (AMELX and AMELY) – was also analysed. Both approaches facilitated the identification of the XX/XY chimerism in 17 females and three males. The proportion of the co-twin cell line ranged from 1 to 99%. In two females no chimerism was detected: (1) 60,XY male chromosome complement in all 400 metaphase spreads; SRY-positive, AMELY-positive, AMELX-positive and (2) 60,XX female chromosome complement in all 200 metaphase spreads; SRY-negative, AMELY-negative, AMELX-positive. The usefulness of different techniques for the diagnosis of freemartinism is discussed.  

Evidence on hereditary occurrence of placental anastomoses in heterosexual twins in sheep.

Pedigree analysis of 22 heterosexual litters demonstrating the leukocyte chimerism (XX/XY) was carried out. The analysis revealed the familial occurrence of the chimerism in 15 litters. Eight litters were obtained from four ewes and the other seven ones were sired by three rams. Moreover, it was shown that ten chimeric litters had a common male ancestor. The coefficients of inbreeding ranged from 0.0 to 0.0625, with a mean of 0.019. These results suggest that the development of placental anastomoses in sheep is genetically controlled. A possible mode of the genetic control is discussed.

Freemartinism: Three Cases in Goats

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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.

Factors affecting pregnancy rate following nonsurgical embryo transfer in buffalo ( Bubalus bubalis) : A retrospective study

The objectives of this study were to determine the pregnancy rate and factors affecting it following nonsurgical embryo transfer in buffalo. Donor buffalo were superovulated with FSH, and embryos collected nonsurgically were evaluated for stage of development and quality. They were transferred nonsurgically to 91 recipients on Days 5 to 7 of the natural (n=52) or induced (n=39) estrus (estrus = Day 0). The overall pregnancy rate of 24/91(26.4%) was higher than in earlier reports for buffalo but was much lower than in cattle. Pregnancy rates were not affected by season (autumn vs winter), side of transfer (right vs left uterine horn), or type of estrus (spontaneous vs induced). The pregnancy rate was high 11/27(40.7%) when donors and recipients were closely synchronized, while it was compromised when recipients were in estrus at +12 h (1/7, 14.3%) and at ȡ12 h (5/27, 18.5%). Asynchrony beyond 12 h on either side resulted into conception failure. The pregnancy rate tended to increase with the increase in CL Size of recipients, while stage of embryonic development had no effect. The transfer of an 8-cell embryo with a 16-cell embryo led to the birth of heterosexual twins, indicating that the uterine milieu of Day 5 to 6 recipients may be tolerated by the out-of-phase 8-cell embryo, at least in the presence of a more mature embryo. Embryo quality had the greatest effect on pregnancy rate as it was higher (P<.005) after the transfer of Grade I than Grade III embryos (6/10, 60.0% vs 3/36, 13.9%). Assessment of returns to estrus indicated that among nonpregnant recipients, 17/67 (25.4%) embryos never matured sufficiently to prevent luteolysis through maternal recognition of pregnancy (MRP), while 14/67 (20.8%) embryos probably died following MRP. These results indicate that efforts to increase pregnancy rate following embryo transfer in buffalo should include prevention of luteolysis during the first week of transfer and a reduction in the incidence of embryonic mortality.

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

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

A fast, convenient diagnosis of the bovine freemartin syndrome using polymerase chain reaction

To establish the polymerase chain reaction (PCR) method for detecting the XY cells in cases suspected to have the bovine freemartin syndrome, a PCR reaction test was conducted on blood from a normal bull diluted in blood from a normal cow. From the results obtained, it was shown that the Y-specific sequence was detectable down to a concentration of 0.1%. Various types of the bovine freemartin syndrome, which occurs in heterosexual twins, single-born sterile heifers, and heifers born with Acardius amorphus, were examined by the chromosome analysis and the PCR method. The Y-specific sequence was detected in all 26 cases that showed chromosome chimerism but which was absent in the 5 cases without a chimerism. The PCR method was found to be effective and convenient for quickly diagnosing the various types of bovine freemartin syndrome.

A fast and highly sensitive method for detecting freemartinism in bovine twins using immunomagnetic beads and Y-specific PCR primers.

A method for detecting male cells in the blood of the female calf in bovine heterosexual twin pregnancies has been established. Nucleated cells were isolated from full blood by immunomagnetic separation, lysed by boiling and then subjected to polymerase chain reaction (PCR) amplification with Y chromosome specific primers. Diagnosis was achieved within one day. The method was successfully used on blood samples that had been stored at +4 degrees C for more than one month. Dilution of male blood in female blood showed that XY cells were detectable down to a concentration of 0.1%. This method should be amenable to automatization and can be adapted to any PCR-based genetic test.

Sexing of mouse and cow embryos with freemartin serum

Gonadal morphology and steroidogenic patterns of female bovine fetuses in twin pregnancies were compared with those of single male and female fetuses to determine the morphogenetic and functional correlates of ovarian alteration in freemartins. In male fetuses, seminiferous cords were detected by 40 days and Leydig cells by 45 days. The ovaries of 50-day fetuses showed clusters of steroidogenic cells forming medullary cords, which appeared to be the precursors of the follicular cells detected in the ovarian cortex of fetuses over 90 days old. Among the steroids produced by the testis of single male fetuses, testosterone was the most prominent, androstenedione was intermediate and estrone and estradiol were in trace amounts, whereas ovaries secreted low levels of testosterone and moderate amounts of androstenedione, estradiol and estrone. Ovarian estradiol secretion in the fetus peaked during 50 to 70 days of development while estrone was produced at elevated levels during 75 to 95 days. During the peak period for estradiol, steroidogenesis in the ovary appeared to follow the androstenedione-to-estrone-to-estradiol pathway. From the relative concentrations of steroids detected, it would appear that steroid synthesis declines in fetal ovaries, probably due to the inhibition of 17β-hydroxysteroid dehydrogenase, prior to that of aromatase. In contrast, in female fetuses of heterosexual twins (freemartins) an interruption of ovarian differentiation was evident as early as 55 days whereas testicular cords and cells resembling Leydig cells were apparent only in fetuses over 70 days old. The morphologic evidence of ovarian alteration was preceded by functional alterations in that the levels of estrogen were significantly reduced and androstenedione was elevated in freemartins as early as 40 days of fetal development. These initial changes were followed by elevated levels of testosterone and androstenedione after 75 days in virilized freemartin gonads coincident with the induction of Leydig cells in the ovary under the influence of the male co-twin. The higher levels of androstenedione secreted by freemartin gonads in early stages seem to further confirm that the androstenedione-to-testosterone pathway may be inherently inactive in normal bovine ovaries.

Non-Freemartin Rate in Holstein Heterosexual Twins

Bovine freemartinism is the sexual modification of a female twin by in utero exchange of blood from a male fetus. This condition was first described by Hunter in 17791 and was well known to result in sterility. The freemartin syndrome occurs in around 92% of bovine females born as a result of heterosexual twin pregnancies2, which indicates 8% of female heterosexual twins will be normal. The diagnosis of freemartinism can be established by one of the following methods: 1) clinical genital abnormalities; 2) presence of sex chromatin bodies in circulating leukocytes in male co-twin; 3) presence of sex chromosome chimerism (XX/XY) of hemopoietic cells; 4) blood typing; and 5) skin grafting. In order to promote the preservation of ideal genetic traits in newborn heterosexual co-twin heifer calves in the Holstein breed, the American Holstein Association authorized our laboratory to cytogenetically test calves for potential freemartinism. The purpose of this paper is to review the cytogenetical results of testing Holstein heifer heterosexual twins referred to our laboratory in the last 13 years.

Detection of leucochimaerism in bovine twins by DNA fingerprinting.

Karyotyping and hypervariable genetic markers indicate extensive leucochimaerism between pairs of dizygotic twins in cattle, a result of placental vascular anastomosis. The extent of this chimaerism includes both kind and number of cells exchanged. All heterosexual twin pairs harboured two types of leucocytes, having either XX or XY chromosome pairs, and 30 of 31 pairs of twins shared identical DNA fingerprints. Although chromosome results from skin fibroblasts indicate that some chimaerism occurs in the skin, the low level allows for differentiation of genotypes between twins. The results warrant against the common practice of using blood samples for DNA typing if twinning is not properly documented.

Hormonal correlates of ovarian alterations in bovine freemartin fetuses

Gonadal morphology and steroidogenic patterns of female bovine fetuses in twin pregnancies were compared with those of single male and female fetuses to determine the morphogenetic and functional correlates of ovarian alteration in freemartins. In male fetuses, seminiferous cords were detected by 40 days and Leydig cells by 45 days. The ovaries of 50-day fetuses showed clusters of steroidogenic cells forming medullary cords, which appeared to be the precursors of the follicular cells detected in the ovarian cortex of fetuses over 90 days old. Among the steroids produced by the testis of single male fetuses, testosterone was the most prominent, androstenedione was intermediate and estrone and estradiol were in trace amounts, whereas ovaries secreted low levels of testosterone and moderate amounts of androstenedione, estradiol and estrone. Ovarian estradiol secretion in the fetus peaked during 50 to 70 days of development while estrone was produced at elevated levels during 75 to 95 days. During the peak period for estradiol, steroidogenesis in the ovary appeared to follow the androstenedione-to-estrone-to-estradiol pathway. From the relative concentrations of steroids detected, it would appear that steroid synthesis declines in fetal ovaries, probably due to the inhibition of 17β-hydroxysteroid dehydrogenase, prior to that of aromatase. In contrast, in female fetuses of heterosexual twins (freemartins) an interruption of ovarian differentiation was evident as early as 55 days whereas testicular cords and cells resembling Leydig cells were apparent only in fetuses over 70 days old. The morphologic evidence of ovarian alteration was preceded by functional alterations in that the levels of estrogen were significantly reduced and androstenedione was elevated in freemartins as early as 40 days of fetal development. These initial changes were followed by elevated levels of testosterone and androstenedione after 75 days in virilized freemartin gonads coincident with the induction of Leydig cells in the ovary under the influence of the male co-twin. The higher levels of androstenedione secreted by freemartin gonads in early stages seem to further confirm that the androstenedione-to-testosterone pathway may be inherently inactive in normal bovine ovaries.

The effect of CMRL-1066 and Menezo B2 media on coculture of rhesus embryos

The objectives of this study were to determine the pregnancy rate and factors affecting it following nonsurgical embryo transfer in buffalo. Donor buffalo were superovulated with FSH, and embryos collected nonsurgically were evaluated for stage of development and quality. They were transferred nonsurgically to 91 recipients on Days 5 to 7 of the natural (n=52) or induced (n=39) estrus (estrus = Day 0). The overall pregnancy rate of 24/91(26.4%) was higher than in earlier reports for buffalo but was much lower than in cattle. Pregnancy rates were not affected by season (autumn vs winter), side of transfer (right vs left uterine horn), or type of estrus (spontaneous vs induced). The pregnancy rate was high 11/27(40.7%) when donors and recipients were closely synchronized, while it was compromised when recipients were in estrus at +12 h (1/7, 14.3%) and at ȡ12 h (5/27, 18.5%). Asynchrony beyond 12 h on either side resulted into conception failure. The pregnancy rate tended to increase with the increase in CL Size of recipients, while stage of embryonic development had no effect. The transfer of an 8-cell embryo with a 16-cell embryo led to the birth of heterosexual twins, indicating that the uterine milieu of Day 5 to 6 recipients may be tolerated by the out-of-phase 8-cell embryo, at least in the presence of a more mature embryo. Embryo quality had the greatest effect on pregnancy rate as it was higher (P<.005) after the transfer of Grade I than Grade III embryos (6/10, 60.0% vs 3/36, 13.9%). Assessment of returns to estrus indicated that among nonpregnant recipients, 17/67 (25.4%) embryos never matured sufficiently to prevent luteolysis through maternal recognition of pregnancy (MRP), while 14/67 (20.8%) embryos probably died following MRP. These results indicate that efforts to increase pregnancy rate following embryo transfer in buffalo should include prevention of luteolysis during the first week of transfer and a reduction in the incidence of embryonic mortality.

Chromosome anomalies and infertility in the mare.

Nine reproductively normal mares, 25 infertile mares and one set of heterosexual twins were examined cytogenetically using conventional giemsa staining, C-banding and G-banding. It was concluded that G-banding was necessary to identify even gross anomalies. Three (12 per cent) of the infertile mares, but none of the controls, had a chromosomal anomaly. One was 63,XO, one a 63,XO/64,XX mixoploid and one a 64,XY sex reversed male. It is argued that a cytogenetic examination is a useful diagnostic technique but that routine screening of the whole population would be uneconomic.

A new type of freemartinism born co-twin with acardius amorphus.

Three females born with acardius amorphus in cattle were cytogenetically examined. Two females showed the sex chromosomal chimera (XX/XY), but the other female did not show any aberrations. The present study indicated that freemartinism can also occur in females born with acardius amorphus, in addition to the females of heterosexual twins and multiple births or single-born animals. The acardius amorphus and its twin were found to be dizygotic twins of different sex with XX/XY chimerisms.

Sex chromatin as a useful tool for detection of freemartinism in bovine twins

An attempt was made to detect freemartins among seven pairs of heterosexual twins by studying their sex chromatin patterns at birth. Examination of blood smears revealed the presence of sex chromatin (drumstick) bodies specific for females in both male and female calves. In three female calves the sex chromatin appendages, however, were absent. A common blood circulation, the basic factor responsible for freemartinism in females, was suggested due to the chimerism of neutrophils indicated by the presence of drumsticks in all the male cotwins examined. A follow-up study of seven female cotwins revealed six developed into freemartins and the seventh was culled due to its abnormal growth. The diagnostic role of sex chromatin studies in the investigation of freemartinism is briefly reviewed and possible cytological explanations for the absence/low frequency of sex chromatin bodies in freemartins discussed.

An additional case of fertile bovine heterosexual twin female with sex-chromosomal chimerism XX/XY.

A Holstein-Friesian female born as twins to a male showed sex chromosomal and blood type chimerisms; the ratio of XX/XY was 63/87 in a total of three examina-tions at different times; incomplete hemolysis was detected for antigenic factors in the B, C, L and S systems of blood type. Nevertheless, she attained sexual maturity and repeat-ed almost regular estrous cycles. By rectal palpation, no abnormality was found in the internal genitalia. The serum levels of estrone, estradiol-17 β and progesterone changed according to the stages of estrous cycle. She was artificially inseminated and successful conception was achieved.

Some rare cases of chimerism in twin cattle and their proposed use in determining germinal cell migration.

Three dizygotic, heterosexual twins with chimerisms carrying marker chromosomes are described. Phenotypic and cytogenetic methods were used to identify these animals. The occurrence of germinal cell migration causing gonad chimerism can be detected by the marker chromosome event under conditions described in this report.