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タイトル: 胚盤胞への発生能および受胎性の高いウシ体外受精卵の選別方法の確立に関する研究
その他のタイトル: Study on establishment of a kinetics-based selection system of in vitro fertilization embryos with high competence for development into blastocysts and conception
著者: 高山, 茉莉
発行日: 12-May-2017
抄録: In recent years, the number of in vitro fertilization (IVF) embryo transfers has increased worldwide. However, the conception rate for IVF embryos is lower than that for in vivo-derived embryos. This study was conducted to determine the efficacy of five factors to predict high developmental competence and conception rate for embryos. Effect of a kinetics-based selection system on high developmental competence of b o vin e OP U-I VF embryos. This study aimed to determine the efficacy of five predicting factors for selecting embryos with a strong ability to develop into blastocysts. Cumulus oocyte complexes (COCs) were collected by ovum pick-up (OPU). The collected COCs were cultured for 22 h. Capacitated sperm (at a final concentration of 5 x 10^6 spermatozoa/ml) were incubated with the COCs for 6 h. The presumptive zygotes were cultured for 9 days in CR1aa supplemented with 5% CS in a micro-well culture dish (Dai Nippon Printing, Tokyo, Japan). The kinetics of embryo development was observed at 27, 31, and 55 h post insemination (hpi) by stereomicroscopy. The number of blastocysts(BL) and produced embryos (PE: transferable embryos) was measured at 7, 8, and 9 days post-IVF. The five factors used to select embryos were as follows: (1) The zygotes were divided 3 groups (2cell, 3cell or more and no cleavage) at 27 hpi; (2) In case cleavage rate was less than 50% at 27 hpi and more than 50% at 31 hpi in each culture dish, divided 3 groups (2cell, 3cell or more and no cleavage) at 31 hpi; (3) The zygotes were divided 3 groups (2cell, 3cell or more and no cleavage) at 31 hpi; (4) The embryos at the 2cell stage at 31 hpi were divided into 2 groups: 2cell with normal cleavage and 2cell with abnormal cleavage (unequal cleavage, 2 cells with fragments); (5) The embryos were divided 2 groups (8cell or more and less than 8cell) at 55 hpi. The results showed that embryos with high development competence had 2 cells with normal cleavage at 27 and 31 hpi and 8 cells or more at 55 hpi. In addition, observation at 31 hpi was effective for selecting embryos with high development competence in the case of embryos with slow first cleavage. Effect of sex-sorted sperm on the timing of first cleavage in b o vin e IVF embryos This study examined the effect of the sex-sorted of sperms on the timing of the first cleavage. COCs were collected from bovine ovaries from a local slaughterhouse. We performed IVF using sex-sorted sperms from 2 Holstein bulls (female sperm: bull A, bull B) and non-sorted sperms from another Holstein bull (bull C). The presumptive zygotes were cultured for 9 days in a micro-well culture dish. The cleavage rate was analyzed at 27, 31, and 55 hpi. The blastocyst rate was analyzed at 7, 8, and 9 days post-IVF. The cleavage rate of embryos from bull A, B, and C at 31 hpi was significantly (P < 0.01) higher than that at 27 hpi. In addition, the cleavage rate at 27 hpi was less than 50%, but at 31 hpi, it was more than 50%, which indicated a delay in first cleavage for embryos from not only sex-sorted sperms, but also from non-sorted sperms. Further, the cleavage rate at 27 hpi of bull C was significantly higher than that of bull A (P < 0.01) and bull B (P < 0.05). Thus, this study confirmed that the timing of first cleavage in embryos from sex-sorted sperms was significantly later than that of embryos from non-sorted sperms. However, the cleavage rates at 31 and 55 hpi and the BL rates of bulls A, B, and C were not significantly different. These results demonstrated that late first cleavage did not affect the development of the blastocyst. Impact of a kinetics-based selection system on conception rate of fresh embryo transfer from bovine OPU-IVF This study aimed to determine the efficacy of five factors for selecting embryos with high conception rate from fresh embryo transfer. Bovine COCs were collected by OPU and cultured for 22 h. The presumptive zygotes were cultured for 9 days in a micro-well culture dish. The kinetics of embryo development was observed at 27, 31, and 55 hpi by stereomicroscopy. The factors used to select the embryos were the same as the ones used in the previous experiment. The conception rate of fresh embryo transfer selected using factors 1+3+4+5 was 61.9% (13/21), while that selected using factors 2+4+5 was 47.4% (9/19). Thus, embryos with early first cleavage should be selected when OPU-IVF is used. These results demonstrated the applicability and efficiency of the five factors for selecting embryos with high competence for conception. In conclusion, these results demonstrated the applicability and efficiency of the five factors for selecting highly competent embryos, verified using stereomicroscopy and a micro-well culture dish. In addition, this study confirmed that the timing of first cleavage of sex-sorted sperm was significantly slower than that of non-sorted sperm, Although the slow first cleavage did not affect the cleavage and BL rates, the conception rate was affected.
URI: http://hdl.handle.net/10659/4912
学位授与年月日: 2016
学位名: 酪農学
学位授与機関: 酪農学園大学
出現コレクション:学位論文(修士)

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