优化辽宁绒山羊精液低温与冷冻保存的研究

发布时间：2018-01-23 11:33

本文关键词： 辽宁绒山羊精液低温液态保存冷冻保存　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：精液保存技术的发展使精液的利用不受种公畜年龄及地域的限制,显著扩大了优良种公畜的利用空间,有利于家畜品种的改良和育成。精液保存方法分为液态保存和冷冻保存。目前除了牛精液的冷冻技术已经完善,猪、羊等其他家畜的精液冷冻保存效果仍不理想。冷冻精液虽然能够实现长期保存,但山羊冷冻精液的受胎率较低(50%)。与之相比,液态精液的受胎率较高些,但保存时间较短。液态精液保存过程中,精子活力下降快,阻碍人工授精技术的发展和推广。因此,提高精液保存质量成为加速绒山羊良种扩繁的关键。本试验利用假阴道法采集辽宁绒山羊精液,进行了精液低温和冷冻保存研究,筛选了辽宁绒山羊低温保存及冷冻保存稀释液的最佳配方,并探讨了精液稀释、降温、平衡及冷冻方法对精液品质的影响。其试验结果如下:1.辽宁绒山羊精液低温保存稀释液筛选将精液随机分为四组,分别用Ⅰ、Ⅱ、Ⅲ、Ⅳ号稀释液对精液进行低温保存。其结果,Ⅰ号稀释液、Ⅱ号稀释液对辽宁绒山羊精液低温保存的效果在精子活力、顶体完整率、质膜完整率皆显著高于Ⅲ号和Ⅳ号稀释液(P0.05)。用Ⅰ号和Ⅱ号稀释液低温保存精液第6d后,精子的活力分别为35.83%和30.0%,顶体完整率为75.07%和75.60%,质膜完整率为30.54%和30.87%,有效保存时间能达到6d。2.EDTA-2Na对辽宁绒山羊精液低温保存的影响在Ⅱ号稀释液中添加一定浓度的EDTA-2Na,经低温保存可以提高精子活力、精子顶体完整率和质膜完整率。在添加浓度分别为Ommol·L-1、0.4mmol·L-1、0.8mmol·L-1、1.2mmol·L-1,随添加浓度提高,精液保存效果有更好的趋势。当EDTA-2Na的添加量为1.2mmol·L-1时,精液的保存效果最好。精液低温保存第6d后,精子活力、顶体完整率、质膜完整率分别为39.03%、82.35%、30.09%。3.GSH对辽宁绒山羊精液低温保存的影响在 Ⅱ 号稀释液中分别添加 Ommol·L-1、2.5mmol·L-1、5mmol·L-1、7.5mmol·L-1、1Ommol·L-1浓度的GSH时,精液低温保存后其品质没有提高。当GSH添加量为5mmol·L-1时,在精液低温保存的第5d、第6d的精子顶体完整率分别为77.76%、77.27%,显著高于其他处理组(P0.05)。4.辽宁绒山羊精液冷冻保存液筛选分别用蔗-柠配方冷冻保存液与乳-柠配方冷冻保存液对精液冷冻保存,解冻后精子活力分别为40.0%和40.83%,两个组之间差异不显著(P0.05)。5.冷冻保存液中甘油添加量的筛选当甘油添加量为4%时,乳-柠配方冷冻保存液对精液冷冻-解冻后的效果最好,精子活力为23.33%。添加量为5%甘油组的蔗-柠配方冷冻保存液和5%、6%甘油组的乳-柠配方冷冻保存液对精液冷冻-解冻后的精子活力分别为40.00%、40.83%和40.51%,显著高于添加4%甘油组(P0.05)。6.冷冻程序的筛选冷冻程序,当细管竖直放置进入冷冻系统时,用5%甘油蔗-柠配方冷冻保存液与5%、6%甘油乳-柠配方冷冻保存液对精子冷冻-解冻后的效果较好,精子活力分别为41.0%、42.83%和41.53%,组间差异不显著(P0.05)。7.精液稀释方法的筛选采用一次稀释法用含5%、6%甘油的乳-柠配方保存液冷冻-解冻后的效果最好,精子活力分别为45.67%、45.50%,顶体完整率分别为76.19%、80.55%,质膜完整率为24.43%、26.62%。8.精液降温方法的筛选含有5%、6%甘油的乳-柠配方冷冻保存液采用隔水降温法处理,冷冻-解冻后的效果最佳。精子活力、顶体完整率与质膜完整率依次为,45.67%、45.50%,76.19%、80.55%,28.77、26.62%。9.人工输精试验本试验中用含1.2mmol·L-1 EDTA-2Na的Ⅱ号配方低温保存精液,将保存3d的和4d的精液分别用于人工输精,受胎率为66.70%和46.90%。综上所述,Ⅰ和Ⅱ号稀释液对精液低温保存的效果较好,在Ⅱ号稀释液中添加1.2mmol·L-1的EDTA-2Na时,能提高精液的保存效果。在Ⅱ号稀释液中添加GSH不能提高精液的保存效果。在乳-柠冷冻保存配方中添加5%或6%甘油;使用一次稀释液法稀释;降温时采用隔水降温法;并且在冷冻时细管竖直放置进入冷冻程序时;冷冻-解冻后精液的保存效果最佳。
[Abstract]:The development of sperm cryopreservation of semen from the use of sire age and geographical restrictions, greatly expand the use of space excellent sire, improvement of livestock breeds and breeding. Semen preservation method consists of liquid storage and cryopreservation. In addition to the current bull semen freezing technology has been improved, pig semen frozen sheep and other livestock preservation effect is still not ideal. Although it can achieve long-term preservation of frozen semen, but the pregnancy rate of goat frozen semen was low (50%). Compared with liquid semen fecundation rate higher, but the preservation time is short. The liquid semen preservation process, decreased sperm motility and fast development hindered the artificial insemination technology. Therefore, to improve the quality of semen preservation has become the key to accelerate seed multiplication of cashmere goat. The experiment using artificial vagina semen collection of Liaoning cashmere goat, the low temperature and cryopreservation of semen Study on optimum recipe of Liaoning cashmere goats during cold storage and cryopreservation dilution, and discusses the effect of semen dilution, cooling, balance and freezing methods on sperm quality. The results are as follows: 1. Liaoning cashmere goat semen cryopreservation dilution screening semen random divided into four groups, respectively, with 1, II, III, IV dilution low-temperature preservation of semen. As a result, 1 dilution, No. 2 dilution effect on cryopreservation of semen in Liaoning cashmere goat sperm motility, acrosome integrity rate, membrane integrity rate is obviously higher than that of III and IV dilution (P0.05). Semen preservation No. 6D I and II dilute solution at low temperature, the sperm motility were 35.83% and 30%, acrosome integrity rate was 75.07% and 75.60%, membrane integrity rate was 30.54% and 30.87%, the effective storage time can reach 6d.2.EDTA-2Na in Liaoning cashmere goat semen cryopreservation. Adding a certain concentration in the ring II dilution in EDTA-2Na after cryopreservation can improve sperm motility, sperm acrosome and plasma membrane integrity rate. In addition concentration was Ommol - L-1,0.4mmol - L-1,0.8mmol - L-1,1.2mmol - L-1, with the concentration increased, the effect of semen preservation is better when adding EDTA-2Na trend. For 1.2mmol L-1, the best preserved effect. The preservation of semen 6D semen low-temperature, sperm motility, acrosome integrity rate, membrane integrity rate were 39.03%, 82.35%, 30.09%.3.GSH of Liaoning cashmere goat semen cryopreservation L-1,2.5mmol L-1,5mmol add Ommol - L-1,7.5mmol - L-1,1Ommol - L-1 in concentrations of GSH. II dilution, semen cryopreservation after its quality is not improved. When the amount of GSH is 5mmol, L-1, 5D in the semen cryopreservation, 6D sperm acrosome integrity rate were 77.76%, 77.27%, significantly higher than other groups (P0.05).4. in Liaoning cashmere goat semen cryopreservation liquid screening were used to cane - lime formula cryopreservation liquid and milk with formula of freezing solution to sperm cryopreservation and thawed sperm motility were 40% and 40.83%, there was no significant difference between the two groups (P0.05).5. cryopreservation screening of glycerol content in liquid when the glycerol content was 4%, milk formula on semen cryopreservation with liquid frozen thawed sperm for the best effect, 23.33%. dosage of 5% glycerol group cane - lime cryopreservation solution and formula 5%, 6% glycerol group milk formula of freezing solution. Were 40% of semen frozen thawed sperm motility, 40.83% and 40.51%, significantly higher than that of adding 4% glycerol group (P0.05) screening program.6. freezing freezing procedure, when the tube is vertically placed into the freezing preservation system, with 5% glycerol cane with frozen -. The liquid with 5%, 6% glycerol milk formula lime cryopreservation solution has good effect of frozen sperm, sperm motility were 41%, 42.83% and 41.53%, no significant difference between the groups (P0.05) screening.7. semen dilution method using a dilution method with 5%, the best 6% glycerol milk formula. Preservation of frozen thawed sperm motility effect were 45.67%, 45.50%, acrosome integrity rate were 76.19%, 80.55%, plasma membrane integrity rate was 24.43%, the screening of 26.62%.8. semen cooling method containing 5%, 6% glycerol - lime milk formula of freezing solution method of water cooling process after freezing thawing best. Sperm motility, acrosome integrity rate and membrane integrity rate were 45.67%, 45.50%, 76.19%, 80.55%, and 2 formula of cryopreservation of semen 28.77,26.62%.9. artificial insemination experiment in the experiment with 1.2mmol L-1 EDTA-2Na, will save 3D and 4D semen respectively For artificial insemination, pregnancy rate was 66.70% and 46.90%. to sum up, I and II dilution better on semen cryopreservation effect, add 1.2mmol L-1 in No. 2 in the dilution of EDTA-2Na, can improve the preservation effect of semen. Addition of GSH can improve the preservation effect of semen dilution in No. 2. Add 5% or 6% glycerin in milk - lime cryopreservation formula; using a dilution dilution method; cooling by water cooling method; and in the freezing tube vertically into the freezing process; frozen thawed semen preservation effect best.

【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S827

【相似文献】