碩士 / 國立宜蘭大學 / 生物技術研究所碩士班 / 97 / 英文摘要
Pre-selection of animal sex is expected in livestock industry. They hope to select the sex of offspring by reproductive science and technology to obtain the high benefits. In mammals, the equal numbers of X- and Y-bearing spermatozoa are produced during the spermatogenesis. The theoretical X:Y ratio in ejaculates is 1:1. Numerous environmental and physiological factors were associated with variation in the sex ratio of mammals. Three experiments were carried out in present study. Experiment 1 and 2 was to establish an efficient technology for determination the proportion of X- and Y-bearing spermatozoa in boar semen samples by real-time PCR and fluorescence in situ hybridization. Experiment 3 was conducted to evaluate the effect of different pH of extender and storage period on sperm motility, viability and ratio of Y spermatozoa. We evaluated semen quality and sperm motility by phase contrast microscopy, and used the flow cytometer and FISH to evaluate the viability and Y sperm proportion of these semen samples. In experiment 1, we used two sets of primers designed according to the specific sequence of SMCX and SMCY genes in X- and Y-chromosome. The real-tim PCR method was validated by a series of accuracy and repeatability assays and by testing two sets of different X% of genomic DNA and different boar semen samples. The results showed that real-time PCR could quantity the ratio of X chromosome with 10% differences in the samples. We also found that the percentage of X -chromosome in semens was different among five boars. In experiments 2, the chromosome 1 and Y specific primers were designed and used to amplifty their specific fragments respectively by PCR. The direct probes specific for small regions of chromosome 1 and Y were produced by nick translation-labelle kit. The results showed that the two-colors direct FISH with 1 and Y chromosome-specific DNA probes prepared by nick translation provided a useful tool for determining X and Y spermatozoa. In experiments 3, the sperm motility of semen diluted with pH 7.2 and 7.8 extender was lower ( P < 0.05 ) than with pH 6.6 in the day 0. However, the sperm motility was no differences ( P > 0.05 ) for 1-3 days preservation. The results showed that the pH of extenders affected the sperm motility in day 0. The means of sperm motility of the day 4 preservation in pH 6.0 and 6.6 groups were higher ( P < 0.05 ) than that of pH 7.2. But there were no differences of sperm viability ( P > 0.05 ) between pH extenders and storage periods. Finally, there were no differences of Y sperm proportion of centrifugal supernatant semen between pH extender by FISH analysis. However, the semen sample were stored for 4 days, the Y sperm proportion were decreased significantly ( P < 0.05 ). In conclusion, we established the real-time PCR and FISH technology provided a valid support to the sperm sexing technologies. The finding suggested that the different pH extenders and storage period didn’t affect the Y spermatozoa ratio. However the motility and viability of sperm were affected. Whether this phenomenon therefored affected the fertilization ability of X and Y sperm or developmental ability of XX and XY embryo, to influence the sex ratio of offspring, needs further proof.
| Identifer | oai:union.ndltd.org:TW/097NIU07108013 |
| Date | January 2009 |
| Creators | Yen-Shi Kuo, 郭妍希 |
| Contributors | Ming-Cheng Chen, 陳銘正 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 106 |
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