【作者】 张立；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2005， 博士

【摘要】 本试验主要对牛羊胚胎工程技术产业化的关键环节进行了研究。针对影响牛羊同期发情、超数排卵、胚胎冷冻、胚胎移植及性别鉴定与控制等主要因素进行试验研究,以期加快我国牛羊良种化进程和胚胎工程技术产业化发展。试验研究结果表明: 1. 应用CIDR 加氯前列烯醇法比两次氯前列烯醇法对受体牛同期发情效果好,但差异不显著(p>0.05)。在牛的同期发情处理中,准备的受体牛至少应有3 个月以上的适应期;哺乳期牛同期发情率低,产犊超过3 个月以上才能得到较理想的同期发情效果;受体牛右侧黄体68.64 %,显著高于左侧黄体31.36 % (p<0.01)。2. 胚胎移植的供受体牛羊需加强饲养管理,提高供受体牛羊自身的营养水平,饲养管理和营养水平是胚胎移植成功的关键。西门塔尔、利木赞、海福特、安格斯肉牛的超排效果好于夏洛来牛;育成牛平均可用胚数和胚胎可用率比成年牛高,但差异不显著(p>0.05);在发情周期第11、12 d 起针超排效果较好,第9、10 d 效果最好,可用胚比例高;超排后输精1~4 次胚胎可用率之间差异不显著(p>0.05);用国产FSH 超排荷斯坦育成牛最佳总剂量,14~15 月龄为8.0 mg,16~17 月龄为8.8 mg,18~19 月龄为10.4 mg。3. 本研究采用两次重复超排间隔时间39 d 超排方案,既大大缩短重复超排间隔时间,又达到理想重复超排效果。第一次和第二次重复超排间隔27d,平均可用胚数为6.22±2.45枚和4.25±3.25枚,两者之间差异显著(p<0.05),发情利用率为91.42 %和75.00 %,两者之间差异极显著(p<0.01);第一次和第二次重复超排间隔39 d,平均可用胚数为5.44±2.78 枚和5.17±2.65 枚,介于两者之间差异不显著(p>0.05),发情利用率为95.03%和91.57%,两者之间差异不显著(p>0.05);第一次和第二次重复超排间隔为47 d 或67 d,平均可用胚数和发情利用率两者之间均无显著差异(p>0.05)。在内蒙古典型放牧条件下,海福特肉牛最佳超数排卵时间为5~6 月份;安格斯肉牛最佳超数排卵时间为8~9 月份。用加拿大进口与中科院国产FSH 对青年荷斯坦母牛进行超排,进口药超排平均可用胚数为4.60±3.52 枚,国产药平均为5.34±2.68 枚,两者之间差异不显著(p>0.05)。4. 在牛胚胎移植中,鲜胚移植显著高于冻胚移植妊娠率(p<0.05),分别为61.74 %和45.32 %;一步法冷冻的胚胎与常规法冷冻的胚胎,解冻移植后妊娠率差异不显著(p>0.05);6 d 的囊胚移植于6 d 的受体,妊娠率高达90.91%,显著高于6 d 的桑椹胚和7 d 的囊胚妊娠率(p<0.05),与7 d 的桑椹胚妊娠率相比差异极显著(p<0.01);我国不同地区最佳胚胎移植季节不同,北方地区以夏、秋季节为好,夏季最佳;华中地区以春、秋季节为好,秋季最佳;南方地区以春、冬季节为好,冬季最佳。5. 对不同地区绵羊超排处理,东北地区以10~12 月初效果较好,西北地区最佳超更多还原

【Abstract】 In order to quicken the well-bred step of cattle and sheep popularization and embryo engineering industrialization development, the key technical factors of embryo engineering industrialization, mainly including oestrus synchronization, superovulation, embryo freezing/thawing, embryo transfer, and sex identification and control were studied in this paper. The results were showed bellow: 1. Efficiency of oestrus synchronization for recipient cows by CIDR + PG treatment was better than that by double PG treatment, but no difference between two methods(p>0.05). Recipient cows, which were prepared more than 3 months before treatment could reach the ideal result of synchronization. Cattle during lactating period got low efficiency of oestrus synchronization, so synchronization should be performed 3 months after delivery. Among the synchronized recipient cows, corpus luteum rate in the right side (68.64 %) was remarkably higher than that (31.36 %) in the left side (p<0.01). 2. Donors and recipients of cattle and sheep should be strengthened feeding management and improved nutrition level, because feeding management and nutrition level have a key role to success of embryo transfer. Effects of superovulation were different among different breeds, and superovulation effects of Simmental, Limousion, Angus, Hereford was better than that of Charolais. The average number of available embryos and available embryo rate of heifers were higher than that of adult cows. Superovulation was more effective on the 11th and 12th day in oestrous cycle, the best result on the 9th and 10th day, and available embryos were in high proportion. Meanwhile, available embryo rate had no significant difference among 1 to 4 times insemination after superovulation (p>0.05). The best superovulation dosage of FSH made in China was 8 mg to age of 14 to 15 months heifers, 8.8 mg to age of 16 to 17 months heifers, and 10.4 mg to age of 18 to 19 months heifers, respectively. 3. The scheme of 39 d repeat superovulation interval time was implemented in this research, which could obviously shorten interval time of repeat superovulation, but also achieve ideal effect. When the interval between first and second repeat superovulation was 27 days, the average numbers of available embryos were 6.22 ±2.45 and 4.25 ±3.25, respectively, significant difference between two groups (p<0.05), and oestrus utilization rate were 91.42 % and 75.00 %, respectively, significant difference between two groups(p<0.01); When 39 days, the average numbers of available embryos were 5.44±2.78 and 5.17±2.65, and oestrus utilization rate were 95.03 % and 91.57 %, no significant difference between groups (p>0.05). In 47 and 67 days, there were no significant differences of the average numbers of available embryos between first and second times (p>0.05). Under the grazing condition in Inner Mongolia, the best time of superovulation Hereford’s beef cows was between May and June, and the best time of Angus’ beef cattle was between August and September. The superovulation results showed that average number of available embryo of Holstein heifers which be superovulated by FSH made in Canada and China were 4.60±3.52 and 5.34±2.68, no significant difference between two FSHs (p>0.05). 4. To cattle embryo transfer, the pregnancy rate of fresh embryos was 61.74%, remarkable higher than that of frozen embryo 45.32 % (p<0.05). There was no significant difference between pregnancy rate of thawed embryo by one-step method or routine method (p>0.05). Pregnancy rate was up to 90.91 % when 6 d blastocysts were transfered into recipients of 6 d by oestrus synchronization treatment, remarkably higher than the pregnancy rate of 6 d morulae and 7 d blastocysts(p<0.05), and was significant difference with pregnancy rate of 7 d morulae(p<0.01). In China, the best embryo transfer season is different in different areas, summer and autumn are better to embryo transfer in the northern area, summer is the best season; Spring and autumn are better in central area, autumn is the best season; Spring and winter are better in southern area, winter is the best season. 5. Superovulating to different district sheep, the result showed: The effect is better from October to beginning of December in the Northeast; Best time of the Northwest is from the last ten days of October to December, North China from the last ten days of November to January and the central plain area from December to January. Ideal time of superovulation for Boer Goats is from January to April and from October to December, the best in November and December. The average number of available embryos of Boer Goat were significant different between 9 days and 15 days after CIDR placed (p<0.01). Oestrus rate of sheep donors which were superovulated simultaneously were 18.10% in 12 h, 75.00 % in 24 h, 6.90 % in 36 h, respectively. The average number of available embryos were the highest in 24 h, no significant difference with that in12 h (p>0.05). Both numbers in 24 h and 12 h were significant different with that in 36 h (p<0.01). 6. All 6 repeated superovulations were up to the ideal result in succession by adoptingdifferent working techniques with routine in the embryo recovery and transfer course. In sheep’s embryo transfer, the pregnancy rates of 4-cell embryos, 8-cell embryos, morulae and blastocysts had no significant difference (p>0.05). However, the pregnancy rates of them were remarkably higher than 2-cell embryos, significant difference between them (p<0.01). Pregnancy rate is influenced remarkably when embryos are stayed outside the body more than 2 h. 7. The average number of available embryos from frozen sexed sperm of Canada and China were 4.90±3.81 and 4.69±3.67, no significant difference between them (p>0.05); Average available embryo rate was 51.64 %, AI rate was 73.24 %, regression embryo rate was 21.60 %, and accurate rate of sex control was 91.07 %. High AI rate and available embryo rate were obtained with 2 million low dosage sperm by inseminating into deep uterus angle. 8. Sex of 291 fresh cattle embryos in vivo transferred were identified by LAMP method, pregnancy rate was 54.30 %, and accurate rate was 98.10 %; 47 cows were transferred with frozen embryos, pregnant rate was 36.17 % and accurate rate was 100 %. Fresh embryo transfer was higher than frozen embryo, significant difference between two groups (p<0.01). The average accurate rate of LAMP method was 98.29 % in this experiment. This method for embryo sex identification is simple, convenient, fast, accurate and economy.更多还原