Sex Determination by PCR in Single Bovine Blastomeres Biopsied by Extrusion Method at the 8-cell Stage.

Abstract

Abstract:We attempted to determine the sex by polymerase chain reaction (PCR) in single bovine blastomeres at the 8-cell stage. PCR was performed on male-specific primers attached to a bovine embryonic sex determination kit, an XY Selector. Embryos at the 8-cell stage were isolated by the EDTA method, and one (1/8 embryo), two (2/8 embryo), and four (4/8 embryo) blastomeres were subjected to PCR. The detection rates for male-specific PCR product were 25.8, 25.8 and 45.2% for 1/8, 2/8 and 4/8 embryos, respectively. In some embryos, despite detection of the male-specific product in 4/8 embryos, the male-specific product was not detected in 1/8, 2/8 or both 1/8 and 2/8 embryos derived from the same embryo (3.2, 3.2 and 16.1%, respectively). Collecting single blastomeres by the extrusion method affected neither the rate of development to blastocysts nor the number of cells in blastocysts. PCR was performed in 1/8 embryos collected by the extrusion method, and the male-specific PCR product was detected. Nevertheless, in 27.5% of embryos, despite detection of male-specific PCR product in 7/8 embryos, the male-specific product was not detected in the 1/8 embryo from the same embryo. These findings indicated that collecting single blastomeres at the 8-cell stage allows the selection of the male embryo by PCR, and also the extrusion method is useful for biopsy of embryo at the 8-cell stage. Key words: Sexing, PCR, Bovine, Embryo, Biopsy As sex determination methods for early mammalian embryos, a method using male-specific antigen, a method using sex difference in the rate of development, a method which quantifies X chromosome-linked enzymes, a method for detecting the Y chromosome, and a method that amplifies Y chromosome-specific nucleotide sequences and analyzes the product have been investigated [1,2]. Among these methods, the method using a male-specific antigen and the method using sex difference in the rate of development are not always accurate, and the overall sexing rates of the method for measuring enzymes linked to the X chromosome and the method for detecting the Y chromosome are low. Therefore, the method for detecting Y chromosome-specific nucleotide sequences by amplifying the sequence by polymerase chain reaction (PCR method) is considered to be promising, and has been developed for cattle [3][4][5][6][7], pigs [8] and mice [9,10]. As an advantage of the PCR method, tests can be rapidly and accurately performed on only a few cells, and commercial use in cattle has begun, but with the current biopsy method, blastomeres are collected by excising mural trophectoderm cells with the zona pellucida at the blastocyst stage, and there is a risk of affecting the normal development of the fetus. In mice, the extrusion method has been established, in which blastomeres at the 4-or 8-cell stage are biopsied by extruding from a small slit made in the zona pellucida [11,12]. Collecting samples for PCR by this method may reduce injury to the embryo.Therefore, in this study, we collected single blastomeres at the 8-cell s...