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O Lie - One of the best experts on this subject based on the ideXlab platform.
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a fast and highly sensitive method for detecting Freemartinism in bovine twins using immunomagnetic beads and y specific pcr primers
Animal Genetics, 2009Co-Authors: Ingrid Olsaker, Claus Jørgensen, Preben D. Thomsen, A L Hellemann, O LieAbstract: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.
Páez Barón, Edwin Manuel - One of the best experts on this subject based on the ideXlab platform.
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Freemartinismo o quimerismo XX/XY en bovinos: revisión
Escuela de Ciencias Agrícolas Pecuarias y del Medio Ambiente. Universidad Nacional Abierta y a Distancia (UNAD), 2009Co-Authors: Corredor Camargo, Emma Sofía, Páez Barón, Edwin ManuelAbstract:This is a literature review in which we present, in a narrative form, the results and conclusions of original research on the pathophysiology and diagnostic methods for the identification of XX/XY chimerism or freemartin anomaly in cattle. Freemartinism affects up to 92% of the pregnancies involving different sex twins. Exchange and transfer of cells and hormones from the male to the female cause suppression of the female�s reproductive system development and lead to the development of a masculine reproductive system that produces infertile females. Since diagnosing this anomaly is important to stockbreeders, efficient detection techniques have been developed.Realizamos una revisión bibliográfica actualizada sintetizando los resultados y conclusiones de investigaciones originales sobre la fisiopatología y los métodos diagnósticos para la identificación de la anomalía conocida como quimerismo XX/XY o Freemartinismo en bovinos. El Freemartinismo afecta las hembras nacidas en aproximadamente el 92% de los partos de gemelos macho-hembra, debido a la transferencia de células y hormonas del macho a la hembra causando grados variables de supresión del desarrollo del sistema reproductivo femenino y permitiendo el desarrollo de partes reproductivas masculinas, generando hembras infértiles. El diagnóstico de esta anomalía es de gran importancia en los sistemas productivos de bovinos y por esta razón se han implementado varias técnicas efectivas para detectarla
Ingrid Olsaker - One of the best experts on this subject based on the ideXlab platform.
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A fast and highly sensitive method for detecting Freemartinism in bovine twins using immunomagnetic beads and Y-specific PCR primers.
Animal Genetics, 2009Co-Authors: Ingrid Olsaker, Claus Jørgensen, Hellemann Al, Preben D. Thomsen, Lie OAbstract:Summary 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°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.
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a fast and highly sensitive method for detecting Freemartinism in bovine twins using immunomagnetic beads and y specific pcr primers
Animal Genetics, 2009Co-Authors: Ingrid Olsaker, Claus Jørgensen, Preben D. Thomsen, A L Hellemann, O LieAbstract: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.
Marek Switonski - One of the best experts on this subject based on the ideXlab platform.
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Technical note: Droplet digital PCR as a new molecular method for a simple and reliable diagnosis of Freemartinism in cattle
Journal of Dairy Science, 2019Co-Authors: Izabela Szczerbal, Sara Albarella, Joanna Nowacka-woszuk, Marek SwitonskiAbstract:ABSTRACT 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 (
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Elevated incidence of Freemartinism in pigs detected by droplet digital PCR and cytogenetic techniques
Livestock Science, 2018Co-Authors: Izabela Szczerbal, Joanna Nowacka-woszuk, Stanisław Dzimira, A. Matuszczyk, P. Iskrzak, Marek SwitonskiAbstract:Abstract Freemartinism is a disorder of sex development (DSD) caused by the formation of placental anastomoses between fetuses of different sex. It leads to development of an abnormal reproductive tract in the female fetus, including the presence of testes or ovotestes. In pig production, such conditions can affect both reproductive performance and meat quality, through undesired boar taint. A common approach to diagnosing Freemartinism is to detect leukocyte chimerism (XX/XY). In the study described here, we performed a complex evaluation of 28 DSD pigs with ambiguous external genitalia (enlarged clitoris, presence of scrotum, etc.). Sex chromosomes were cytogenetically identified by Giemsa staining, as well as with fluorescence in situ hybridization (FISH), while Y-linked (ZFY) and X-linked (ZFX) genes were detected by PCR-RFLP. Histological analysis of the gonads was performed on eosin–hematoxylin-stained microscope slides. Chromosome studies revealed that, of 28 DSD animals, 20 were freemartins (38,XX/38,XY), 2 had a female karyotype (38,XX), and 6 had a male karyotype (38,XY). Analysis of breeding data showed that the mean litter size in which DSD animals occurred was about 17. Histology of the gonads could be performed for 8 DSD pigs, including 6 freemartins and 2 XX DSD. In all these cases, testicular tissue without signs of spermatogenesis was seen. In XX DSD pigs, a copy number variation (CNV) polymorphism downstream from SOX9, tentatively associated with this type of DSD, was detected by FISH. On the other hand, the background of the XY DSD remains unknown. Since cytogenetic analysis is labor-intensive and time-consuming, we evaluated the usefulness of the droplet digital PCR (ddPCR) approach to diagnosing XX/XY leukocyte chimerism. Estimation of X and Y chromosome copy numbers was based on the quantitative detection of AMELX and AMELY genes. Different sex chromosome sets (XX, XY and XX/XY) were unequivocally identified by ddPCR. In conclusion, we suggest that Freemartinism, beside testicular or ovotesticular XX DSD, is an emerging problem in highly prolific pigs and that detection of XX/XY leukocyte chimerism can be performed using a fast and reliable ddPCR approach.
Jorge H. Calvo - One of the best experts on this subject based on the ideXlab platform.
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Freemartinism and FecXR allele determination in replacement ewes of the Rasa Aragonesa sheep breed by duplex PCR
Theriogenology, 2009Co-Authors: A. Martínez-royo, Enkeleda Dervishi, J. J. Jurado, Jose Luis Alabart, Jose Folch, Jorge H. CalvoAbstract:Abstract A new naturally occurring mutation in the fecundity gene BMP15 in the Rasa Aragonesa sheep breed ( Ovis aries ) has been found to affect prolificacy. This mutation ( FecX R allele) is a deletion of 17 base pairs that leads to an altered amino acid sequence, and this alteration increases prolificacy in heterozygous ewes but causes sterility in homozygous ewes. Selection of repository lambs with the FecX R allele increases rates of twins and multiple lambing and thereby also increases the probability of lambing freemartins that will become sterile. In this sense, an accurate, reliable, and quick method was developed by duplex polymerase chain reaction (PCR) for sex, amplifying an ovine-specific Y chromosome repetitive fragment, and BMP15 genotype determination in replacement ewe lambs. The BMP15 fragment served as an internal control of the amplification and detected the FecX R allele, avoiding a false negative and then a mistake in freemartin detection. This assay uncovered 6 freemartin females among 195 replacement ewes from 7 different commercial flocks and 1 experimental flock. Furthermore, 1554 rams from 64 commercial flocks were also analyzed to identify FecX R rams. This analysis identified 103 rams hemizygous for the FecX R allele and 1 heterozygous ram. Because this gene is located on the X chromosome, this heterozygous animal is a freemartin ram that is co-amplifying the DNA from XX and XY lymphocytes. These results confirm the usefulness of this multiplex PCR assay for detecting phenotypically sexed females, freemartins, and the BMP15 genotype to detect highly prolific ewes in commercial flocks and to assist breeders in selection of repository lambs.
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Technical note Freemartinism and FecX R allele determination in replacement ewes of the Rasa Aragonesa sheep breed by duplex PCR
2009Co-Authors: A. Martínez-royo, Enkeleda Dervishi, Jose Folch, Jorge H. CalvoAbstract:A new naturallyoccurring mutationinthefecundity geneBMP15intheRasa Aragonesasheep breed(Ovisaries) hasbeen found to affect prolificacy. This mutation (FecX R allele) is a deletion of 17 base pairs that leads to an altered amino acid sequence, and this alteration increases prolificacy in heterozygous ewes but causes sterility in homozygous ewes. Selection of repository lambs with the FecX R allele increases rates of twins and multiple lambing and thereby also increases the probability of lambing freemartins that will become sterile. In this sense, an accurate, reliable, and quick method was developed by duplex polymerase chain reaction (PCR) for sex, amplifying an ovine-specific Y chromosome repetitive fragment, and BMP15 genotype determination in replacement ewe lambs. The BMP15 fragment served as an internal control of the amplification and detected the FecX R allele, avoiding a false negative and then a mistake in freemartin detection. This assay uncovered 6 freemartin females among 195 replacement ewes from 7 different commercialflocks and 1 experimental flock. Furthermore, 1554 rams from 64 commercial flocks were also analyzed to identify FecX R rams. This analysis identified 103 rams hemizygous for the FecX R allele and 1 heterozygous ram.Becausethis geneis locatedontheXchromosome, this heterozygous animalis afreemartin ram thatis co-amplifyingtheDNA from XX and XY lymphocytes. These results confirm the usefulness of this multiplexPCR assay for detecting phenotypically sexed females, freemartins, and the BMP15 genotype to detect highlyprolific ewes in commercialflocks and to assist breeders in selection of repository lambs.
