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KMID : 0894520010050010047
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Development & Reproduction
2001 Volume.5 No. 1 p.47 ~ p.52
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Discontinuous Percoll Gradients Enrich X-Bearing Porcine Sperms and Female Embryos
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심대용:Dae-Yong Shim
유성진:Seong-Jin Yoo/강한승:Han-Seung Kang/유정민:Jeong-Min Yu/이채관:Lee Chae-Kwan/강성구:Kang Sung-Goo
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Abstract
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Predetermination of sex in livestock of offpring is in great demand and is of critical importance to providing for the most efficient production of the animal ariculture. Such a sexing techlology would also enhance the economy of conventional artificial insemination(AI) and aid the porcine industry. The purpose of this study was to evaluate the efficiency of enriching X-bearing porcine sperm using discontinuous percoll gradients and PCR mefhod. Semen was collected from mature boars of proven fertility center (AI center KimHae). Sperm was leaded on the isotonic discontinuous percoll gradient and then it was centrifuged at 120 × g for 20 minutes. After centrifugation, sperm included in each fraction were recovered (7×106 sperms/ml) and then sperm genomic DNA was extractedfor the PCR. SRY gene was used to evaluate the ratio between X and Y sperm in the separated fractions. Ju viro ffrtilization wascarried out by adding the unseparated sperm (control) or separated (experimental poop) to the matured oocytes in TCM-199. Embryos for sex determination were obtained at 2 cell stage and then was used for SRY gene amplification. After centrifugation of discontinuous percoll gradient, the most motile sperm was obtained at 95% fiaction (94.4% ± 5.1%, p < 0.01). The PCR analysis evaluated that 30%, 50% and 65% fractions were Y sperm rich, whereas 80% and 95% fractions were X sperm rich. PCR analysis with each porcineembryo showed that 33.3% of control and 66.7% of experimental group were determined as female embryos. In conclusion, in vitro matured oocytes inseminated with sperms (95% fraction) prepared by percoll gradient centrifugation showed high fertilization rates and female embryos than control sperms.
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