奶山羊扩繁技术、XY精子分选及Y染色体ZNF280BY基因拷贝数变异研究

发布时间：2018-03-07 08:44

本文选题：奶山羊　切入点：同期发情　出处：《西北农林科技大学》2015年博士论文　论文类型：学位论文

【摘要】：随着我国集约化与规模化的奶山羊产业发展,高效扩繁技术应用与推广越来越受到广大养殖户的重视,如同期发情技术、精液保存技术可快速提高种群质量并便于统一管理。优秀种公羊具有显著的遗传改良作用,充分发挥种公羊的繁殖潜力有利于扩大良种覆盖率及提高奶山羊整体生产水平。本研究以西农萨能奶山羊为试验对象,通过同期发情试验方案筛选,种公羊精液常温及冷冻保存方法,奶山羊X、Y精子分选技术参数,性控精液低剂量输精试验以及Y染色体ZNF280BY基因拷贝数变异等奶山羊关键繁殖技术的系统研究,为建立高效奶山羊繁育技术提供了重要的理论与试验依据。获得了以下主要结果:1.非繁殖季节奶山羊同期发情试验研究发现,处理2组(CIDR+300 IU PMSG+5μg LHRH-A3)与处理3组(CIDR+450 IU PMSG+5μg LHRH-A3)中奶山羊发情率高于处理1组(CIDR+150 IU PMSG+5μg LHRH-A3)(P0.05),但各组处理母羊受胎率及产羔率均无差异(P0.05)。繁殖季节奶山羊同期发情试验研究发现,羊表现发情后10h促黄体激素(LH)达到峰值;处理?组(CIDR+300 IU PMSG)与处理П组(CIDR+300 IU PMSG+5μg LHRH-A3)母羊受胎率、产羔率均高于处理Ш组(CIDR+300 IU PMSG+40 IU FSH+5μg LHRH-A3)(P0.05),同时发现1胎次和2胎次母羊的受胎率均高于3胎次母羊(P0.05)。2.研究季节对西农萨能羊种公羊精液品质的影响,结果表明在春季、夏季及秋季种公羊精液的活率、体积、密度、单次射精总精子数以及质膜完整率均高于冬季(P0.05),夏季与秋季种公羊精子的顶体完整率高于春季与冬季(P0.05)。将各季节采集的鲜精进行冷冻精液制作,解冻后检测精液品质发现,夏季与秋季种公羊精液活率、质膜完整率与顶体完整率均高于春季和冬季(P0.01)。3.通过两种精液冷冻稀释液(Tris Citric acid Glucose buffer,TCG与Glucose Milk buffer,GM)对种公羊精液冷冻保存的研究发现,西农萨能奶山羊分离精浆不利于精液的冷冻保存(P0.05)。利用TCG制作的冷冻精液解冻后精子顶体完整率高于GM组(P0.05)。利用TCG与GM制备的冷冻精液用于人工授精,结果表明TCG处理组的母羊受胎率(45.9±2.06%)及产羔率(159.6±3.25)均高于GM组(33.3±3.15%与150±4.34%)(P0.05)。4.利用常温保存稀释液(TRIS_M与TRIS_A)保存种公羊鲜精12h后发现,两种稀释液在19℃的保存效果均优于4℃和15℃(P0.05)。TRIS_M与TRIS_A分别保存种公羊精液,在19℃条件下经过长途运输(10h)送到分选中心进行X、Y精子分选。通过分选系统关键步骤(染色液浓度与激光强度)的筛选获得精液染色液Hoechst33342的最佳浓度为28μM/m L,精子分选的紫外激光强度为6 W。TRIS_M与TRIS_A保存的精液分别进行X、Y精子分选,结果表明在Hoechst33342染色前及染色后精子活率均无差异(P0.05),而分选后、降温后TRIS_M组精子活率高于TRIS_A组(P0.01);分选后的性控精液用TCG进行冷冻保存,解冻后TRIS_A组的精子活率为40.3±2.3%,低于TRIS_M组的精子活率(58.7±3.3%)(P0.01)。检测TRIS_M制备的性控精液,结果表明Y精子纯度为95.3±2.2%,X精子纯度为95.2±1.8%,差异不显著(P0.05),X精子与Y精子质膜完整率及顶体完整率均无差异(P0.05)。5.性控精液腹腔镜低剂量输精试验发现,输精剂量分别为2.2×106与4.4×106个有效精子分别获得25%与50%受胎率、100%与187.5%的产羔率,100%与93.3%的后代性别符合率。性控精液常规人工输精(4.4×106个有效精子)获得8.3%受胎率、100%产羔率与100%后代性别符合率。性控后代的出生重、断奶重与非性控后代无差异(P0.05)。6.种公羊Y染色体ZNF280BY基因拷贝数分析,结果发现ZNF280BY特异存在于Y染色为多拷贝基因,且在6个山羊品种中均表现为拷贝数变异且非正态分布。其中美姑山羊的拷贝数中值最低(19个),关中奶山羊的拷贝数中值最高151个,西农萨能奶山羊的拷贝数中值为113个。西农萨能奶山羊、关中奶山羊、崂山奶山羊、文登奶山羊与云南圭山羊品种间拷贝数无差异(P0.05),美姑山羊与其他山羊品种都存在差异(P0.05)。ZNF280BY基因可作为一种分子标记用于种公羊相关繁殖力的筛选。本研究表明,试验所采用的同期发情方案可提高母羊的发情率、受胎率及产羔率;春季、夏季及秋季种公羊精液品质良好,TCG制备的冷冻精液可提高母羊的受胎率及产羔率;获得奶山羊X、Y精子流式分选技术参数,TRIS_M常温保存种公羊精液效果较好,能高效分离X、Y精子,性控精液低剂量输精可获得预期的性别后代;同时发现ZNF280BY在山羊个体与品种间存在拷贝数变异。
[Abstract]:With the intensive and scale of China's dairy goat industry, efficient propagation technology application and promotion has attracted more and more attention of the majority of farmers, as the period of estrus, semen preservation technology can quickly improve the population quality and facilitate unified management. Ram has excellent genetic improvement effect, give full play to the male reproductive potential is conducive to the expansion of seed coverage and improve the overall level of production of dairy goats. In this research, nongsa goat as the test object, the test scheme of estrus screening, ram semen at room temperature and cryopreservation method, dairy goat X, Y sperm sorting technology parameters, system control research of low dose test and copy semen insemination Y chromosome the number of ZNF280BY gene mutation in dairy goat breeding key technology, provides theoretical and experimental basis for the establishment of efficient important dairy goat breeding technology were obtained. The following main results: 1. experimental study on non estrus breeding season of dairy goats found at the same time, treatment group (2 CIDR+300 IU PMSG+5 g LHRH-A3 (CIDR+450) and treatment group 3 IU PMSG+5 g LHRH-A3) in dairy goat estrus rate higher than the treatment group (1 CIDR+150 IU PMSG+5 g LHRH-A3 (P0.05)), but each treatment of ewes the pregnancy rate and lambing rate showed no significant difference (P0.05). Experimental study on dairy goat breeding season was found at the same time, sheep showed estrus after 10h luteinizing hormone (LH) treatment group reached the peak;? (CIDR+300 IU PMSG) and treatment group (n CIDR+300 IU PMSG+5 G LHRH-A3) female pregnancy rate, lambing rate was higher than that of treatment group III (CIDR+300 IU PMSG+40 IU FSH+5 g LHRH-A3) (P0.05), also found that 1 parity and 2 parity ewes pregnancy rate was higher than 3 (P0.05).2. parity ewes of seasonal sheep ram semen quality of Xinong showed that in spring, summer and Autumn The survival rate, ram semen volume, total sperm density, single ejaculation number and membrane integrity rate were higher than those in winter, summer and autumn (P0.05) male sperm acrosome integrity rate was higher than that in spring and winter (P0.05). The fresh semen collected seasonal frozen semen production, thawed semen quality detection that summer and autumn ram sperm motility, membrane integrity and acrosome integrity rate were higher than those in spring and winter (P0.01).3. by two kinds of frozen semen dilution (Tris Citric acid Glucose buffer, TCG Glucose and Milk buffer, GM) on ram semen cryopreservation, Xinong Saanen dairy goat sperm separation is not conducive to sperm cryopreservation (P0.05). TCG produced by the use of frozen semen thawed sperm acrosome integrity rate higher than that of GM group (P0.05). Using TCG and GM preparation of frozen semen for artificial insemination, the results showed that TCG treatment group parent The sheep pregnancy rate (45.9 + 2.06%) and lambing rate (159.6 + 3.25) were higher than that of group GM (33.3 + 3.15% and 150 + 4.34%) (P0.05).4. using room temperature preservation of dilution (TRIS_M and TRIS_A) to save ram of fresh semen found after 12h two dilution in preservation were better than 19 DEG C 4 degrees and 15 degrees (P0.05) of.TRIS_M and TRIS_A were preserved ram semen, under the condition of 19 DEG C after long-distance transport (10h) of X to Y sperm sorting sorting center. Through the key steps of sorting system (dye concentration and laser intensity) the best concentration of semen staining Hoechst33342 obtained for screening 28 M/m L, the UV laser intensity of sperm sorting is 6 W.TRIS_M and TRIS_A preserved semen were analyzed by X and Y sperm sorting, the results show that the Hoechst33342 staining before and after staining sperm showed no significant difference (P0.05), and after sorting, after cooling TRIS_M group sperm motility rate higher than that of TRIS_A group (P0.01 points); Semen control after the election with TCG cryopreserved thawed TRIS_A group of sperm motility was 40.3 + 2.3%, lower than the TRIS_M group of sperm motility (58.7 + 3.3%) (P0.01). The detection of TRIS_M prepared by sexed semen, the results showed that Y sperm purity was 95.3 + 2.2%, X sperm purity 95.2 + 1.8%, the difference was not significant (P0.05), X sperm and Y sperm membrane integrity and acrosome integrity rate were no difference (P0.05).5. Sexing Semen test found low dose laparoscopic insemination, insemination dose were 2.2 * 106 and 4.4 * 106 respectively 25% and 50% effective sperm fertility rate, 100% 187.5% and the lambing rate, 100% and 93.3%. The coincidence rate of offspring sex control semen artificial insemination (4.4 x 106 effective sperm) 8.3% pregnancy rate, lambing rate of 100% and 100%. The coincidence rate of offspring sex control offspring birth weight, weaning weight difference and non control offspring (P0.05).6. male Y chromosome ZNF280 The number of copies of the BY gene, the results showed that ZNF280BY existed in specific staining for Y gene copy, and in 6 goat breeds showed copy number variation and non normal distribution. The copy number of the lowest median Meigu goats (19), Guanzhong dairy goats the copy number of the highest median 151, Xinong the median number of dairy goats can copy 113. Xinong Saanen dairy goat, Guanzhong dairy goat, Laoshan dairy goat, Wendeng dairy goat and Yunnan goat breeds the copy number differences (P0.05), there are differences in Meigu goat and other goat breeds (P0.05).ZNF280BY gene can be used as a molecular marker for screening male fertility related. This study shows that the synchronization scheme test can improve the ewe oestrus rate, pregnancy rate and lambing rate; spring, summer and autumn of ram semen quality is good, TCG preparation of frozen semen can improve the mother The pregnancy rate and the rate of sheep lambing; obtained dairy goat X, Y sperm flow parameter separation technology, TRIS_M ram semen preservation effect is good, can effectively separate X, Y sperm, semen control low dose insemination can obtain expected offspring sex; at the same time found the presence of ZNF280BY copy number variation in goats and varieties.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S827

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