發(fā)布時間：2018-03-07 08:44

  本文選題：奶山羊　切入點：同期發(fā)情　出處：《西北農(nóng)林科技大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：隨著我國集約化與規(guī)�；哪躺窖虍a(chǎn)業(yè)發(fā)展,高效擴(kuò)繁技術(shù)應(yīng)用與推廣越來越受到廣大養(yǎng)殖戶的重視,如同期發(fā)情技術(shù)、精液保存技術(shù)可快速提高種群質(zhì)量并便于統(tǒng)一管理。優(yōu)秀種公羊具有顯著的遺傳改良作用,充分發(fā)揮種公羊的繁殖潛力有利于擴(kuò)大良種覆蓋率及提高奶山羊整體生產(chǎn)水平。本研究以西農(nóng)薩能奶山羊為試驗對象,通過同期發(fā)情試驗方案篩選,種公羊精液常溫及冷凍保存方法,奶山羊X、Y精子分選技術(shù)參數(shù),性控精液低劑量輸精試驗以及Y染色體ZNF280BY基因拷貝數(shù)變異等奶山羊關(guān)鍵繁殖技術(shù)的系統(tǒng)研究,為建立高效奶山羊繁育技術(shù)提供了重要的理論與試驗依據(jù)。獲得了以下主要結(jié)果:1.非繁殖季節(jié)奶山羊同期發(fā)情試驗研究發(fā)現(xiàn),處理2組(CIDR+300 IU PMSG+5μg LHRH-A3)與處理3組(CIDR+450 IU PMSG+5μg LHRH-A3)中奶山羊發(fā)情率高于處理1組(CIDR+150 IU PMSG+5μg LHRH-A3)(P0.05),但各組處理母羊受胎率及產(chǎn)羔率均無差異(P0.05)。繁殖季節(jié)奶山羊同期發(fā)情試驗研究發(fā)現(xiàn),羊表現(xiàn)發(fā)情后10h促黃體激素(LH)達(dá)到峰值;處理?組(CIDR+300 IU PMSG)與處理П組(CIDR+300 IU PMSG+5μg LHRH-A3)母羊受胎率、產(chǎn)羔率均高于處理Ш組(CIDR+300 IU PMSG+40 IU FSH+5μg LHRH-A3)(P0.05),同時發(fā)現(xiàn)1胎次和2胎次母羊的受胎率均高于3胎次母羊(P0.05)。2.研究季節(jié)對西農(nóng)薩能羊種公羊精液品質(zhì)的影響,結(jié)果表明在春季、夏季及秋季種公羊精液的活率、體積、密度、單次射精總精子數(shù)以及質(zhì)膜完整率均高于冬季(P0.05),夏季與秋季種公羊精子的頂體完整率高于春季與冬季(P0.05)。將各季節(jié)采集的鮮精進(jìn)行冷凍精液制作,解凍后檢測精液品質(zhì)發(fā)現(xiàn),夏季與秋季種公羊精液活率、質(zhì)膜完整率與頂體完整率均高于春季和冬季(P0.01)。3.通過兩種精液冷凍稀釋液(Tris Citric acid Glucose buffer,TCG與Glucose Milk buffer,GM)對種公羊精液冷凍保存的研究發(fā)現(xiàn),西農(nóng)薩能奶山羊分離精漿不利于精液的冷凍保存(P0.05)。利用TCG制作的冷凍精液解凍后精子頂體完整率高于GM組(P0.05)。利用TCG與GM制備的冷凍精液用于人工授精,結(jié)果表明TCG處理組的母羊受胎率(45.9±2.06%)及產(chǎn)羔率(159.6±3.25)均高于GM組(33.3±3.15%與150±4.34%)(P0.05)。4.利用常溫保存稀釋液(TRIS_M與TRIS_A)保存種公羊鮮精12h后發(fā)現(xiàn),兩種稀釋液在19℃的保存效果均優(yōu)于4℃和15℃(P0.05)。TRIS_M與TRIS_A分別保存種公羊精液,在19℃條件下經(jīng)過長途運輸(10h)送到分選中心進(jìn)行X、Y精子分選。通過分選系統(tǒng)關(guān)鍵步驟(染色液濃度與激光強(qiáng)度)的篩選獲得精液染色液Hoechst33342的最佳濃度為28μM/m L,精子分選的紫外激光強(qiáng)度為6 W。TRIS_M與TRIS_A保存的精液分別進(jìn)行X、Y精子分選,結(jié)果表明在Hoechst33342染色前及染色后精子活率均無差異(P0.05),而分選后、降溫后TRIS_M組精子活率高于TRIS_A組(P0.01);分選后的性控精液用TCG進(jìn)行冷凍保存,解凍后TRIS_A組的精子活率為40.3±2.3%,低于TRIS_M組的精子活率(58.7±3.3%)(P0.01)。檢測TRIS_M制備的性控精液,結(jié)果表明Y精子純度為95.3±2.2%,X精子純度為95.2±1.8%,差異不顯著(P0.05),X精子與Y精子質(zhì)膜完整率及頂體完整率均無差異(P0.05)。5.性控精液腹腔鏡低劑量輸精試驗發(fā)現(xiàn),輸精劑量分別為2.2×106與4.4×106個有效精子分別獲得25%與50%受胎率、100%與187.5%的產(chǎn)羔率,100%與93.3%的后代性別符合率。性控精液常規(guī)人工輸精(4.4×106個有效精子)獲得8.3%受胎率、100%產(chǎn)羔率與100%后代性別符合率。性控后代的出生重、斷奶重與非性控后代無差異(P0.05)。6.種公羊Y染色體ZNF280BY基因拷貝數(shù)分析,結(jié)果發(fā)現(xiàn)ZNF280BY特異存在于Y染色為多拷貝基因,且在6個山羊品種中均表現(xiàn)為拷貝數(shù)變異且非正態(tài)分布。其中美姑山羊的拷貝數(shù)中值最低(19個),關(guān)中奶山羊的拷貝數(shù)中值最高151個,西農(nóng)薩能奶山羊的拷貝數(shù)中值為113個。西農(nóng)薩能奶山羊、關(guān)中奶山羊、嶗山奶山羊、文登奶山羊與云南圭山羊品種間拷貝數(shù)無差異(P0.05),美姑山羊與其他山羊品種都存在差異(P0.05)。ZNF280BY基因可作為一種分子標(biāo)記用于種公羊相關(guān)繁殖力的篩選。本研究表明,試驗所采用的同期發(fā)情方案可提高母羊的發(fā)情率、受胎率及產(chǎn)羔率;春季、夏季及秋季種公羊精液品質(zhì)良好,TCG制備的冷凍精液可提高母羊的受胎率及產(chǎn)羔率;獲得奶山羊X、Y精子流式分選技術(shù)參數(shù),TRIS_M常溫保存種公羊精液效果較好,能高效分離X、Y精子,性控精液低劑量輸精可獲得預(yù)期的性別后代;同時發(fā)現(xiàn)ZNF280BY在山羊個體與品種間存在拷貝數(shù)變異。
[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.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：S827

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