本文關(guān)鍵詞： 遼寧絨山羊精液 低溫液態(tài)保存 冷凍保存　出處：《沈陽(yáng)農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：精液保存技術(shù)的發(fā)展使精液的利用不受種公畜年齡及地域的限制,顯著擴(kuò)大了優(yōu)良種公畜的利用空間,有利于家畜品種的改良和育成。精液保存方法分為液態(tài)保存和冷凍保存。目前除了牛精液的冷凍技術(shù)已經(jīng)完善,豬、羊等其他家畜的精液冷凍保存效果仍不理想。冷凍精液雖然能夠?qū)崿F(xiàn)長(zhǎng)期保存,但山羊冷凍精液的受胎率較低(50%)。與之相比,液態(tài)精液的受胎率較高些,但保存時(shí)間較短。液態(tài)精液保存過(guò)程中,精子活力下降快,阻礙人工授精技術(shù)的發(fā)展和推廣。因此,提高精液保存質(zhì)量成為加速絨山羊良種擴(kuò)繁的關(guān)鍵。本試驗(yàn)利用假陰道法采集遼寧絨山羊精液,進(jìn)行了精液低溫和冷凍保存研究,篩選了遼寧絨山羊低溫保存及冷凍保存稀釋液的最佳配方,并探討了精液稀釋、降溫、平衡及冷凍方法對(duì)精液品質(zhì)的影響。其試驗(yàn)結(jié)果如下:1.遼寧絨山羊精液低溫保存稀釋液篩選將精液隨機(jī)分為四組,分別用Ⅰ、Ⅱ、Ⅲ、Ⅳ號(hào)稀釋液對(duì)精液進(jìn)行低溫保存。其結(jié)果,Ⅰ號(hào)稀釋液、Ⅱ號(hào)稀釋液對(duì)遼寧絨山羊精液低溫保存的效果在精子活力、頂體完整率、質(zhì)膜完整率皆顯著高于Ⅲ號(hào)和Ⅳ號(hào)稀釋液(P0.05)。用Ⅰ號(hào)和Ⅱ號(hào)稀釋液低溫保存精液第6d后,精子的活力分別為35.83%和30.0%,頂體完整率為75.07%和75.60%,質(zhì)膜完整率為30.54%和30.87%,有效保存時(shí)間能達(dá)到6d。2.EDTA-2Na對(duì)遼寧絨山羊精液低溫保存的影響在Ⅱ號(hào)稀釋液中添加一定濃度的EDTA-2Na,經(jīng)低溫保存可以提高精子活力、精子頂體完整率和質(zhì)膜完整率。在添加濃度分別為Ommol·L-1、0.4mmol·L-1、0.8mmol·L-1、1.2mmol·L-1,隨添加濃度提高,精液保存效果有更好的趨勢(shì)。當(dāng)EDTA-2Na的添加量為1.2mmol·L-1時(shí),精液的保存效果最好。精液低溫保存第6d后,精子活力、頂體完整率、質(zhì)膜完整率分別為39.03%、82.35%、30.09%。3.GSH對(duì)遼寧絨山羊精液低溫保存的影響在 Ⅱ 號(hào)稀釋液中分別添加 Ommol·L-1、2.5mmol·L-1、5mmol·L-1、7.5mmol·L-1、1Ommol·L-1濃度的GSH時(shí),精液低溫保存后其品質(zhì)沒(méi)有提高。當(dāng)GSH添加量為5mmol·L-1時(shí),在精液低溫保存的第5d、第6d的精子頂體完整率分別為77.76%、77.27%,顯著高于其他處理組(P0.05)。4.遼寧絨山羊精液冷凍保存液篩選分別用蔗-檸配方冷凍保存液與乳-檸配方冷凍保存液對(duì)精液冷凍保存,解凍后精子活力分別為40.0%和40.83%,兩個(gè)組之間差異不顯著(P0.05)。5.冷凍保存液中甘油添加量的篩選當(dāng)甘油添加量為4%時(shí),乳-檸配方冷凍保存液對(duì)精液冷凍-解凍后的效果最好,精子活力為23.33%。添加量為5%甘油組的蔗-檸配方冷凍保存液和5%、6%甘油組的乳-檸配方冷凍保存液對(duì)精液冷凍-解凍后的精子活力分別為40.00%、40.83%和40.51%,顯著高于添加4%甘油組(P0.05)。6.冷凍程序的篩選冷凍程序,當(dāng)細(xì)管豎直放置進(jìn)入冷凍系統(tǒng)時(shí),用5%甘油蔗-檸配方冷凍保存液與5%、6%甘油乳-檸配方冷凍保存液對(duì)精子冷凍-解凍后的效果較好,精子活力分別為41.0%、42.83%和41.53%,組間差異不顯著(P0.05)。7.精液稀釋方法的篩選采用一次稀釋法用含5%、6%甘油的乳-檸配方保存液冷凍-解凍后的效果最好,精子活力分別為45.67%、45.50%,頂體完整率分別為76.19%、80.55%,質(zhì)膜完整率為24.43%、26.62%。8.精液降溫方法的篩選含有5%、6%甘油的乳-檸配方冷凍保存液采用隔水降溫法處理,冷凍-解凍后的效果最佳。精子活力、頂體完整率與質(zhì)膜完整率依次為,45.67%、45.50%,76.19%、80.55%,28.77、26.62%。9.人工輸精試驗(yàn)本試驗(yàn)中用含1.2mmol·L-1 EDTA-2Na的Ⅱ號(hào)配方低溫保存精液,將保存3d的和4d的精液分別用于人工輸精,受胎率為66.70%和46.90%。綜上所述,Ⅰ和Ⅱ號(hào)稀釋液對(duì)精液低溫保存的效果較好,在Ⅱ號(hào)稀釋液中添加1.2mmol·L-1的EDTA-2Na時(shí),能提高精液的保存效果。在Ⅱ號(hào)稀釋液中添加GSH不能提高精液的保存效果。在乳-檸冷凍保存配方中添加5%或6%甘油;使用一次稀釋液法稀釋;降溫時(shí)采用隔水降溫法;并且在冷凍時(shí)細(xì)管豎直放置進(jìn)入冷凍程序時(shí);冷凍-解凍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.

【學(xué)位授予單位】：沈陽(yáng)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S827

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