Use of primers derived from a new sequence of the bovine Y chromosome for sexing Bos taurus and Bos indicus embryos

Alves, Beatriz da Costa Aguiar; Lima, Vera Fernanda Martins Hossepian de; Teixeira, Christine; Moreira-Filho, Carlos Alberto

doi:10.1016/s0093-691x(02)01191-3

Cited by 21 publications

(22 citation statements)

References 7 publications

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“…When embryo sexing is performed with male-specific target sequences, one cannot distinguish the false-negative amplification caused by sampling errors or from lack of amplification in female samples. To solve this problem, various combination of two different PCR primer sets, one for a male-specific sequence and another for a sequence common to both sexes, have been examined as a multiplex PCR to obtain internal bovine control for embryo sexing [12][13][14][15][16][17].…”

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Multiplex Polymerase Chain Reaction Used for Bovine Embryo Sex Determination

Rattanasuk

Parnpai

Ketudat-Cairns

2011

Journal of Reproduction and Development

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Abstract. Widely used bovine sexing primers were compared in terms of suitability in determining the sex of bovine embryos. Under optimized multiplex PCR conditions, the ConBV/ConEY couple primers did not show accurate results when combined together in multiplex PCR, but worked well when the couple primers were used separately. The S4BF/ S4BR primers showed accurate results; however, some unexpected bands were detected. When the BY/BSP couple primers were used to determine one-cell, two-cell, four-cell and eight-cell stage embryos of known sexed SCNT-derived embryos, the results showed 100% accuracy. The BY/BSP couple primers were also able to identify the sex of one-cell and two-cell IVF-derived embryos. Key words: Bovine sex determination, Embryo sexing, Multiplex PCR (J. Reprod. Dev. 57: [539][540][541][542] 2011) ex determination of preimplantation embryos is important for manipulating the sex ratios of domestic animals at an early stage of embryo development [1,2]. A number of approaches have been used to determine the sex of bovine embryos but have failed to gain much popularity due to the lack of either accuracy or speed [3]. Simple and precise methods for embryo sexing are important for management and improvement of bovine species. Polymerase chain reaction (PCR) has been used for sexing of bovine embryos [4,5] with various target sequences, including male-specific repetitive sequences [6,7] and single-copy genes on the Y chromosome, such as amelogenin [8,9] and ZFY gene [10][11][12]. When embryo sexing is performed with male-specific target sequences, one cannot distinguish the false-negative amplification caused by sampling errors or from lack of amplification in female samples. To solve this problem, various combination of two different PCR primer sets, one for a male-specific sequence and another for a sequence common to both sexes, have been examined as a multiplex PCR to obtain internal bovine control for embryo sexing [12][13][14][15][16][17].In the present study, three widely used primer sets were compared using genomic DNA and embryo at various developmental stages. The embryos used in this study were somatic cell nuclear transfer (SCNT)-derived embryos of known sex and in vitro fertilization (IVF)-derived embryos of unknown sex. They were used as template in the multiplex PCR reactions.To compare the suitable couple primers used for bovine sex determination, 0.42 ng of high molecular weight DNA from male and female bovine fibroblast was amplified using three different sets of bovine sexing primers (Table 1) and subjected to gel electrophoresis. The PCR products were detected after ethidium bromide staining. The results in Fig. 1 suggested that the BY/BSP couple primers were able to amplify male bovine genomic DNA, which gave two fragments of 300 bp from Y-specific primers (BY) and 538 bp from bovine-specific primers (BSP) (lane 1) and a clear single band of 538 bp (lane 2) from BSP amplified female bovine genomic DNA. The S4BF/S4BR primers showed clear accurate results with bovine genomic DN...

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Multiplex Polymerase Chain Reaction Used for Bovine Embryo Sex Determination

Rattanasuk

Parnpai

Ketudat-Cairns

2011

Journal of Reproduction and Development

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“…PCR is a rapid, easy procedure for large scale sexing, and primers derived from many Y-specific sequences have been used to screen blood, meat and blastomere samples (Alves et al, 2003;Zeleny et al, 2002). Manz et al (1998) demonstrated the potential usefulness of TSPY for sex diagnosis in equine preimplantation, and Pierce et al (2000) described a highly accurate method for determining the sex of human embryos by real-time PCR of this same gene.…”

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“…A more practical and efficient approach for determining the sex of cattle is to amplify sequences of the Y chromosome by the polymerase chain reaction (PCR) using sequence-specific primers (Alves et al, 2003). The advent of PCR has increased the interest in sexing cattle, and the potential usefulness of this technique has been demonstrated (Bredbacka et al, 1995).…”

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Use of the TSPY gene for sexing cattle

et al. 2005

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The Y-encoded, testis-specific protein (TSPY) is a Y-specific gene. The copy numbers of TSPY range from 20 to 60 in men and up to 200 in bulls. In this study, we examined the possibility of using the TSPY gene to sex cattle. DNA from blood samples of 100 Nelore cattle (50 males and 50 females) from the Nelore Cattle Breeding Program (PMGRN) was screened for TSPY by PCR using TSPY-specific primers. The assay was highly specific since all male samples were TSPY-positive and all female samples were negative. Positive results were also obtained at low DNA concentrations (less than 1 ρg/µL). These results showed that TSPY was a good male-specific marker, the usefulness of which was enhanced by the high copy number of the gene. This is the first report to demonstrate the applicability of TSPY for sexing cattle.

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“…PCR assays for molecular sexing are often more useful because they provide sensitive, precise, rapid, and reliable results. To date, PCR-based sex typing has been used for embryonic sexing in cattle, mice, and pigs [7][8][9][10][11][12]. To establish a rapid and precise molecular sexing method using PCR amplification for horse breeding, we analyzed the sequence characteristics and sexual dimorphism of the ZFX and ZFY genes and compared the results to those obtained from Y chromosome-specific SRY gene amplification and phenotypic investigation.…”

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Length difference between equine ZFX and ZFY genes and its application for molecular sex determination

Han

Yang

et al. 2010

J Assist Reprod Genet

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Purpose We analyzed the sex chromosome-encoding ZFX-ZFY genes and tested molecular sexing using the amplification patterns of intron 9 of ZFX-ZFY in the horse. Methods and resultsThe amplification of the ZFX-ZFY produced two distinct patterns, reflecting sexual dimorphism based on a length difference between the X and Y chromosomes. The amplification products from foals showed two distinct bands: one was common to all foals and mares, indicating that this band was amplified from ZFX, while the other was specific to some foals, indicating that it was from ZFY. The result based on the PCR assay was identical to the results of amplification of the Y chromosome-specific SRY gene and those of investigations of the phenotypic gender in three different horse populations. Conclusion We suggest that this PCR strategy for determining sexes by comparing the amplification patterns of ZFX-ZFY genes is a convenient and precise method for discriminating sexes in horses.Most mammals have an X-Y chromosome sex determination system. Sexual differentiation is determined primarily by the presence or absence of the Y chromosome, and the sex determining region Y (SRY) gene plays a key role in inducing male development, while inhibiting female differentiation [1][2][3].Control of the sex ratio of animals is desirable in the livestock industry. In mammals, the X-Y homologous sequences for the X-and Y-linked genes zinc finger, amelogenin-X and -Y (AMELX-AMELY), and SMCX/SMCY have been compared [4][5][6]. PCR assays for molecular sexing are often more useful because they provide sensitive, precise, rapid, and reliable results. To date, PCR-based sex typing has been used for embryonic sexing in cattle, mice, and pigs [7][8][9][10][11][12]. To establish a rapid and precise molecular sexing method using PCR amplification for horse breeding, we analyzed the sequence characteristics and sexual dimorphism of the ZFX and ZFY genes and compared the results to those obtained from Y chromosome-specific SRY gene amplification and phenotypic investigation.Total of 128 genomic DNA samples were collected from three horse populations (61 Thoroughbreds, 20 Tsushima native horses from Japan, and 47 Jeju native horses from South Korea). To amplify the intron 9 of ZFX and ZFY genes, the primer pair was designed from exon 8 and 10 of the sequences of human and cattle [13]. The primer Capsule Length difference was found in PCR amplification products between equine ZFX and ZFY genes. This sexual dimorphism may be a diagnostic molecular marker for determining the sexes in the horse.

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Use of primers derived from a new sequence of the bovine Y chromosome for sexing Bos taurus and Bos indicus embryos

Cited by 21 publications

References 7 publications

Multiplex Polymerase Chain Reaction Used for Bovine Embryo Sex Determination

Multiplex Polymerase Chain Reaction Used for Bovine Embryo Sex Determination

Use of the TSPY gene for sexing cattle

Length difference between equine ZFX and ZFY genes and its application for molecular sex determination

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