366 SEX IDENTIFICATION OF PORCINE EMBRYOS BY PCR BASED ON THE AMELOGENIN GENE

The amelogenin (AMEL) gene exists on both X and Y chromosomes in various mammalian species. The non-coding region of this gene is different between X and Y chromosomes. The use of this gene has made sex determination much less complicated, since only one pair of primers is required to amplify the different size fragments of the AMEL gene. Therefore, AMEL had been successfully used to determine the sex in cattle, sheep, and humans. The difference of AMEL genomic sequences between X and Y chromosomes has also been found in pig. In this study, we designed primers that identified AMEL of both chromosomes. The amplicons were isolated and sequenced, and showed a length polymorphism characteristic for the X and Y chromosome in pigs. Furthermore we examined whether a single oocyte or embryo could be sexed. Genomic DNA samples were collected from various breeds of pigs (European breeds: Landrace, Large White, Duroc, Berkshire; Chinese breeds: Meishan, Jinhua). DNA was extracted from ears, tails, or leukocytes using the salting-out method and then dissolved in TE buffer. We used one set of primers for amplifying the pig AMEL gene. The polymerase chain reaction (PCR) procedure was performed with initial denaturation at 94�C for 2 min, followed by 40 cycles of one denaturation step at 98�C for 10 s, primer annealing at 60�C for 30 s, and primer extension at 72�C for 30 s in 20 �L of reaction mixture containing 50 ng genomic DNA. The PCR products were electrophoresed and documented. Some amplicons were isolated and sequenced, and showed a length polymorphism characteristic for the X and Y chromosome in every breed. Next, we tried sexing of pig oocytes and embryos. Cumulus–oocyte complexes (COCs) were aspirated from ovaries recovered from prepubertal gilts. COCs were matured in modified NCSU-37 medium for 44 h, fertilized in vitro, and then cultured in PZM5. The pre-implantation embryos were sampled at 1, 2, 3, 4, and 5–6 days after insemination. Day 1–4 embryos were treated in 5 �L of lysis solution; whole solution were used for subsequent PCR. After Day 5–6 of insemination, only blastocysts were treated in 20 �L of lysis solution, and 5 �L were used for PCR. GV oocytes and electro-activated embryos were sampled as controls. PCR amplification yielded the expected 480-bp and 301-bp products. Male pigs in all breeds are expected to show 2 bands (480 bp and 301 bp), whereas all females, one band only (480 bp). The comparison of AMEL gene DNA sequences among pig breeds showed over 99% homology for the PCR products in both the AMEL-X and the AMEL-Y gene, except for several single-base substitutions. Within GV oocytes and electro-activated embryos, 98% and 96–99% of those examined displayed one band of 480 bp. In IVF groups, 49–55% of those embryos had 2 bands, with no difference between the number of embryos displaying one band and two bands. In conclusion, our findings show that the PCR assay based on the AMEL gene is reliable for sex identification in every pig breed. The advantage of this assay is its capability of identifying sex using a genomic DNA sequence as small as that contained within a single cell such as an oocyte.