Multiplex Polymerase Chain Reaction Used for Bovine Embryo Sex Determination

Abstract

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...

Characterization of X-Chromosome Gene Expression in Bovine Blastocysts Derived by In vitro Fertilization and Somatic Cell Nuclear Transfer

“…Sexes of IVF or sham-SCNT blastocysts were determined by PCR with Y-specific primers (BY; 5′-CTCAGCAAAGCACACCAGAC-3′ and 5′-GAACTTTCAAGCAGCTGAGGC-3′) and bovine-specific primers (BSP; 5′-TTTACCTTAGAACAAACCGAGGCAC-3′ and 5′-TACGGAAAGGAAAGATGACCTGACC-3′) as previously reported (Rattanasuk et al, 2011). One-tenth volume of genomic DNA extracted from single blastocysts was amplified by PCR using AccuPower PCR PreMix (Bioneer) and PCR product was resolved on 2% agarose gel.…”

A fast and simple method for the polymerase chain reaction-based sexing of livestock embryos

“…Moreover, the procedure for sperm cell sorting can alter the semen IVF performance of certain bulls (Blondin et al, 2009), reducing the options for genetic diversity in breeding programs. In turn, embryo sexing by the PCR-based detection of X-and/or Y-specific regions in retrieved blastomeres is an alternative means of overcoming some of the limitations of the sexed semen for in vivo-or in vitro-produced embryos, allowing the producer to choose which embryo should be transferred to synchronous recipients based on sex determination (Mara et al, 2004;Carneiro et al, 2011;Rattanasuk et al, 2011). The molecular method is efficient for field applications, resulting in pregnancy rates comparable to nonbiopsied embryos, even after cryopreservation (Tominaga, 2004;Tominaga and Hamada, 2004).…”

Novel Scientific Methods and Technology in the Reproductive Medicine

“…Nearly 95% embryos may be sexed by Y-specific chromosome probe using the polymerase chain reaction (PCR) [39], or the fluorescent in situ hybridization (FISH) [47]. A considerable improvement in the PCR technique such as multiplex PCR [48] or in the loop-mediated isothermal amplification method, constituting a new generation of innovative gene amplification techniques, allows for a rapid sex determination in the embryo [49], with 100 % reported accuracy of sex prediction [50]. Therefore, the PGD constitutes a procedure with the success rates of sex predetermination in embryo being as high as 99.9%.…”