本文選題：地中海水牛　切入點(diǎn)：精液冷凍　出處：《廣西大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：意大利地中海水牛因其高產(chǎn)奶量和優(yōu)良的奶質(zhì)被公認(rèn)為是目前世界上河流型水牛中乳用性能最好的品種之一。廣西于2014年引進(jìn)該品種水牛,受冷凍保存配方、冷凍程序和個(gè)體差異等因素影響,目前的種公牛精液冷凍保存效果不甚理想,這極大地影響了地中海水牛的繁殖效率。因此,探討抗氧化劑等對精液冷凍保存效果的影響,研發(fā)針對意大利地中海水牛的精液稀釋液配方,對提高種公牛利用率有著重大意義。為此,本研究在傳統(tǒng)精液稀釋液基礎(chǔ)上,首先重新比較有效成分、調(diào)整其比例、同時(shí)引入新的保護(hù)劑、擬出新配方,隨后應(yīng)用改進(jìn)的配方探討添加褪黑素、谷胱甘肽和維生素E等不同抗氧化劑對地中海水牛精液冷凍保存的效果影響。研究結(jié)果如下。1.地中海水牛精液冷凍稀釋液配方的改進(jìn)研究在水牛精液常用冷凍稀釋液的基礎(chǔ)上,首先對配方中的能量物質(zhì)、抗凍物質(zhì)和抗菌物質(zhì)分別進(jìn)行效果比較。結(jié)果發(fā)現(xiàn),稀釋液成分中卵黃的冷凍保存效果優(yōu)于鮮全奶;3%甘油和2%乙二醇聯(lián)合作用效果優(yōu)于5%的甘油、5%的乙二醇和5%的DMSO三者單獨(dú)作用時(shí)的效果;青霉素和慶大霉素的抑菌效果比較明顯;其次,對于新引入的保護(hù)劑牛血清白蛋白(BSA),和未添加組相比,0.9 mg/mL的BSA顯著提高了水牛精子的活率,降低其畸形率(P0.05);改進(jìn)后的地中海水牛精液稀釋液配方為:0.10 g/mL葡萄糖、0.0006 g/mL檸檬酸鈉、0.0011 g/mL青霉素、0.0006 g/mL慶大霉素、0.9 mg/mLBSA、3%甘油、2%乙二醇、20%卵黃、75%蒸餾水。2.褪黑素、谷胱甘肽和維生素E對地中海水牛精液冷凍保存效果的影響在冷凍稀釋液中添加褪黑素(MLT)時(shí)發(fā)現(xiàn),添加0.Img/mLMLT顯著提高了受精卵分裂率和GSH-Px酶活性(P0.05),對精子活率、畸形率、運(yùn)動(dòng)性能等變化無顯著差異(P0.05);添加0.2mg/mL、0.4mg/mL MLT明顯提高了凍后精子活率、頂體完整率、GSH-Px酶活性、受精卵分裂率和囊胚率(P0.05),添加0.2mg/mLMLT還顯著降低了精子畸形率(P0.05)。添加0.8mg/mLMLT明顯降低了精子活率(P0.05),對精子運(yùn)動(dòng)性能、受精卵分裂率、囊胚率等均沒有顯著影響(P0.05)。添加谷胱甘肽(GSH)處理時(shí)發(fā)現(xiàn),添加0.1mMGSH顯著提高精子前向性和質(zhì)膜完整率,降低了精子畸形率(P0.05),對精子活率和頂體完整率無明顯影響(P0.05)。添加0.2 mMGSH明顯了提高精子活率、運(yùn)動(dòng)性能、質(zhì)膜完整率、GSH-Px酶活性和囊胚率(P0.05),顯著降低精子畸形率和MDA水平(P0.05)。0.4mMGSH增加了精子畸形率,顯著提高GSH-Px酶活性、受精率分裂率、囊胚孵化率(P0.05),對頂體完整率、SOD酶活性等無明顯影響(P0.05)。添加0.8 mMGSH增加了精子畸形率(P0.05),對精子抗氧化物酶活性和囊胚率等無顯著性影響(P0.05)。添加維生素E(VE)處理時(shí)發(fā)現(xiàn),與未添加組相比,0.1 mg/mL和0.2 mg/mLVE對精子活率無明顯影響(P0.05)。0.2mg/mL、0.4mg/mLVE明顯提高精子的GSH-Px酶活性、頂體完整率、質(zhì)膜完整率、受精卵分裂率、囊胚率和囊胚孵化率,顯著降低了精子MDA水平(P0.05)。0.4mg/mL、0.8mg/mLVE顯著提高了精子活率(P0.05),其中0.4mg/mL時(shí)精子活率最大,0.4mg/mL VE顯著降低了精子畸形率,0.8mg/mLVE導(dǎo)致畸形率增加(P0.05),0.8mg/mLVE明顯提高受精卵分裂率(P0.05),對囊胚率和囊胚孵化率無顯著影響(P0.05)。3.三種抗氧化劑配伍作用對地中海水牛精液冷凍保存效果的影響為了解兩種或多種抗氧化劑配伍添加的協(xié)同效果,根據(jù)單獨(dú)添加MLT、GSH和VE時(shí)的作用效果確定各抗氧化劑的配伍濃度,分別為MLT(0.2 mg/mL、0.4 mg/mL)、GSH(0.1 mM、0.2 mM)、VE(0.2 mg/mL、0.4 mg/mL),將其兩兩組合,共形成12個(gè)配伍組合。結(jié)果發(fā)現(xiàn),與未添加組相比,0.2 mg/mL VE+O.1 mM GSH、0.4 mg/mL VE+0.2 mg/mL MLT、0.2 mg/mL MLT+0.1 mM GSH組合均能在一定程度上提高了精子活率,降低精子畸形率,其中 0.2 mg/mL MLT+0.1 mM GSH 和 0.4 mg/mL VE+0.2 mg/mL MLT 表現(xiàn)較突出。三個(gè)配伍組合均顯著提高了精子運(yùn)動(dòng)性能、頂體完整率和質(zhì)膜完整率(P0.05),其中尤以0.2mg/mLMLT+0.ImMGSH組合提升效果最好。三個(gè)配伍組合均顯著提高了受精卵分裂率(P0.05),0.4 mg/mL VE+0.2 mg/mL MLT和 0.2 mg/mL MLT+0.1 mM GSH組合對囊胚率的提高有顯著差異(P0.05),0.4 mg/mL VE+0.2 mg/mL MLT和0.2 mg/mL VE+0.1 mM GSH顯著提高了囊胚孵化率(P0.05)。綜合考慮,0.2 mg/mL MLT+0.1 mM GSH 和 0.4 mg/mL VE+0.2 mg/mL MLT 組合優(yōu)于 0.2 mg/mL VE+0.1 mM GSH。以上結(jié)果表明,添加 0.2 mg/mL MLT、0.2 mM GSH 或 0.04 mg/mL VE可提高水牛精液的冷凍保存效果較好,采用0.4mg/mLVE+0.2mg/mL MLT或0.2 mg/mL MLT+0.1 mM GSH的組合添加劑對地中海水牛精液冷凍保存可取得較好效果。
[Abstract]:Italy Mediterranean buffalo because of its high milk production and excellent milk quality is recognized as the world river buffalo milk with one of the best varieties performance. Guangxi in 2014 to introduce the varieties of buffalo, the cryopreservation formula, factors affecting the freezing procedure and individual differences, the bull semen cryopreservation effect ideal, which greatly affects the reproductive efficiency of the Mediterranean buffalo. Therefore, to investigate the effect of antioxidants on the preservation effect of frozen semen in Italy, Mediterranean semen dilution formula Buffalo Research, is of great significance to improve the utilization rate of male cattle. Therefore, this study based on traditional semen dilution, first re comparison of effective components, adjust the proportion, while the introduction of new protective agents, a new formula, then using the improved formula to investigate the effects of melatonin, glutathione and vitamin E etc. Effect of antioxidants on the preservation of Mediterranean buffalo semen cryopreservation effect. The results are as follows.1. Mediterranean buffalo frozen semen dilution formula improvement based on buffalo common semen freezing diluent on the first energy formula, and antibacterial substances of antifreeze material respectively compared the effects. The results showed that the preservation effect is better than that of fresh whole milk yolk diluent ingredient in freezing; 3% glycerol and 2% ethylene glycol combination is better than 5% glycerol, 5% ethylene glycol and 5% DMSO three respectively. The effect of anti bacteria effect; penicillin and gentamicin obviously; secondly, the protective agent of bovine serum albumin (BSA), the newly introduced compared and without group, 0.9 mg/mL BSA significantly increased the survival rate of buffalo sperm, reduce the deformity rate (P0.05); the buffalo semen dilution formula of the improved: 0.10 g/mL glucose, 0. 0006 g/mL sodium citrate, 0.0011 g/mL penicillin, gentamicin 0.0006 g/mL, 0.9 mg/mLBSA, 3% glycerol, 2% ethylene glycol, 20% egg yolk, 75%.2. distilled water preservation effect of melatonin, glutathione and vitamin E on buffalo in the Mediterranean frozen semen freezing dilution liquid added melatonin (MLT) found that the addition of 0.Img/mLMLT significantly increased cleavage rate and enzyme activity of GSH-Px (P0.05), survival rate, the rate of sperm deformity, no significant difference in movement performance changes (P0.05); adding 0.2mg/mL, 0.4mg/mL MLT increased obviously after freezing sperm motility, acrosome integrity rate, GSH-Px activity, cleavage rate and blastocyst rate (P0.05) add 0.2mg/mLMLT, also significantly reduced the rate of sperm deformity (P0.05). Adding 0.8mg/mLMLT significantly reduced sperm motility, sperm motility (P0.05) on performance, cleavage rate, blastocyst rate did not significantly affect (P0.05) added Gu Guanggan. Peptide (GSH) treatment was added prior to 0.1mMGSH significantly increased and sperm membrane integrity, reduced sperm deformity rate (P0.05), the sperm motility and acrosome integrity rate had no significant effect (P0.05). Adding 0.2 mMGSH significantly improve sperm motility, exercise performance, membrane integrity rate the activity of GSH-Px and blastocyst rate (P0.05), significantly reduced the rate of sperm deformity and the level of MDA (P0.05).0.4mMGSH increased the rate of sperm deformity, increased the GSH-Px activity, the fertilization rate cleavage rate, blastocyst hatching rate (P0.05), the rate of intact acrosome, had no significant effect on the activity of SOD (P0.05) added. 0.8 mMGSH increased the rate of sperm deformity of sperm (P0.05), antioxidant enzyme activity and blastocyst rate had no significant effect (P0.05). The addition of vitamin E (VE) treatment was found, compared with not adding group, 0.1 mg/mL and 0.2 mg/mLVE of sperm survival rate had no significant effect (P0.05).0.2mg/mL, 0.4mg/mLVE obviously To improve the GSH-Px activity of sperm, acrosome, plasma membrane integrity rate, cleavage rate, blastocyst rate and blastocyst hatching rate, significantly reduced the level of sperm MDA (P0.05).0.4mg/mL, 0.8mg/mLVE significantly increased the survival rate of sperm (P0.05), the 0.4mg/mL 0.4mg/mL VE maximum motility significantly reduced sperm. The deformity rate, deformity rate increased due to 0.8mg/mLVE (P0.05), 0.8mg/mLVE significantly increased the cleavage rate (P0.05), had no significant effect on blastocyst rate and blastocyst hatching rate (P0.05) of.3. three compatible effect of antioxidant effect on preservation of Mediterranean buffalo frozen semen for understanding the synergistic effect of two or more antioxidants added compatibility. According to the addition of MLT, the effect of GSH and VE to determine the concentration of each combination of antioxidants, respectively MLT (0.2 mg/mL, 0.4 mg/mL), GSH (0.1 mM, 0.2 mM), VE (0.2 mg/mL, 0.4 mg/mL), the combination of 22, A total of 12 combinations. The results showed that compared with not adding group, 0.2 mg/mL VE+O.1 mM GSH mg/mL VE+0.2 mg/mL, 0.4 MLT, 0.2 mg/mL MLT+0.1 mM GSH combination can to some extent improve sperm motility, reduce the rate of sperm deformity, including 0.2 mg/mL MLT+0.1 mM GSH VE+0.2 mg/mL MLT and 0.4 mg/mL more prominent. Three combinations significantly increased sperm movement performance, acrosome and plasma membrane integrity rate (P0.05), especially 0.2mg/mLMLT+0.ImMGSH combination to enhance the effect of three. The best combination can significantly increase the fertilized egg split rate (P0.05), 0.4 mg/mL VE+0.2 mg/mL MLT and MLT+0.1 mM GSH 0.2 mg/mL the combination has significant difference to improve the blastocyst rate (P0.05), 0.4 mg/mL VE+0.2 mg/mL MLT mg/mL VE+0.1 mM and 0.2 GSH significantly increased the blastocyst hatching rate (P0.05). Considering the 0.2 mg/mL and 0.4 mg/m MLT+0.1 mM GSH L VE+0.2 mg/mL MLT mg/mL VE+0.1 mM is better than that of combination of above 0.2 GSH. results showed that the addition of 0.2 mg/mL MLT, 0.2 mM GSH or 0.04 mg/mL buffalo VE can improve the sperm cryopreservation effect is good, the sea buffalo semen cryopreservation can achieve good effect with the combination of 0.4mg/mLVE+0.2mg/mL MLT or mg/mL MLT+0.1 0.2 additive mM GSH.

【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S823.83

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