摘要
性别控制技术可以推动家畜育种进程和加快繁殖人们所期望的性别后代。尽管分离水牛X和Y精子性别控制技术体系已经建立,但分离精子用于体外受精后的囊胚发育率和人工授精后的受胎率仍低于未分离精子。精子分离及冷冻过程伴随着精液的高倍稀释、精子核染色、紫外激光照射和冷冻保存等处理程序,这些程序可能对精子的活力、质膜完整性、顶体完整性、线粒体膜电位、DNA完整性和凋亡等产生影响。本研究探讨了水牛精子分离及冷冻程序不同处理对(新鲜、染色、分离和冷冻)精子细胞器的损伤,旨在优化水牛精子分离及冷冻程序,以期提高分离精子的质量,为水牛品种改良提供有力的技术保障。具体研究内容包括以下几个部分：
1.探讨水牛精子分离过程中不同处理(新鲜、染色、分离和冷冻)精子的活力、质膜完整性、凋亡和DNA完整性的变化。结果表明,分离组和冷冻组的精子活力比新鲜组和染色组低(54.72%,52.38%vs.98.07%,96.41%,P<0.05)。荧光显微镜法和流式细胞仪法检测精子质膜完整性的试验结果均表明,新鲜组、染色组和分离组的精子质膜完整率差异不显著(P>0.05),但均显著高于冷冻组(72.12％,69.39％,77.26％vs.58.45％；64.74％,63.69％,66.25％vs.36.98％,P<0.05).精子凋亡方面,荧光显微镜法和流式细胞仪法检测的试验结果均表明,新鲜组的凋亡率最低,显著低于分离组和冷冻组(8.11％vs.13.23％,18.74％；7.56％vs.12.86％,22.4％,P<0.05)。精子染色质扩散试验(SCD)和精子染色质结构分析法(SCSA)检测精子DNA损伤率的试验结果均表明,分离组精子的DNA损伤率最低,显著低于新鲜组、染色组和冷冻组(0.47％vs.2.23％,2.25％,2.26％;0.51％vs.2.55％,2.64％,2.15％,P<0.05).
2.研究冷冻保存的不同公牛个体分离精子与未分离精子的活力、质膜完整性、凋亡和DNA完整性的变化。精子图像分析仪检测结果表明,3头尼里拉菲水牛分离精子的活力均显著低于未分离精子,分别为38.9％vs.59.6％；35.8％vs.58.1％；34％vs.59.1％(P<0.05).染色质结构分析法检测结果表明,3头尼里拉菲水牛分离精子的DNA损伤率均比未分离精子低(1.86％vs.2.47％,2.05％vs.3.0％,3.45vs.4.38％,P<0.05),其中3号公牛分离精子和未分离精子的DNA损伤率均高于其它两头公牛。荧光显微镜法检测结果表明,3头尼里拉菲水牛分离精子的质膜完整率均比未分离精子高(55.3％vs.42.4％,52.7％vs.41.7％,51.6％vs.43.3％,P<0.05).荧光显微镜法检测结果表明,3头尼里拉菲水牛分离精子的凋亡率均比未分离精子高(28.3％vs.21.1％,31.5％vs.23.6％,32％vs.23％,P<0.05),其中3号公牛分离精子的凋亡率高于其它两头公牛。
3.研究水牛精子分离及冷冻程序各稀释液(染色液、收集液和冷冻液)中添加叶酸或谷胱甘肽对精子的活力、质膜完整性、凋亡和DNA完整性的影响。试验结果表明,在水牛精子分离及冷冻程序各稀释液(染色液、收集液和冷冻液)中添加叶酸或谷胱甘肽后精子的活力、质膜完整性和DNA损伤率与对照组相比差异不显著(P>0.05)。在冷冻液中添加叶酸或谷胱甘肽后均显著降低了精子凋亡率(P<0.05)。
4.本研究旨在建立激光光镊拉曼光谱技术检测水牛分离精子的试验平台。水牛精子分离及冷冻程序不同处理的精子(新鲜、染色、分离和冷冻)拉曼光谱检测结果表明,与新鲜组相比,代表磷酸骨架(O-P-O)的788cm-1在分离组和冷冻组分别位移到783cm-1和785cm-1,代表脱氧核糖C-O伸缩振动的1010cm-1在染色组、分离组和冷冻组分别位移到1004cm-1、1005cm-1和1005cm-1。水牛精子分离及冷冻程序不同处理的精子(新鲜、染色、分离和冷冻)原始拉曼光谱经标准化处理后,主成分分析的试验结果表明,可以有效的将不同处理组的精子分开。
5.研究在水牛分离精子的染色液、收集液和冷冻液中分别添加叶酸或谷胱甘肽后对精子拉曼光谱的影响。结果表明：(1)代表脱氧核糖振动的谱线从新鲜组的1010cm-1位移到染色组的1004cm-1,在染色液中分别添加叶酸或谷胱甘肽后从对照组的1004cm-1分别位移到1008cm-1(叶酸)和1005cm-1(谷胱甘肽),冷冻液中分别添加叶酸或谷胱甘肽后从对照组的1004cm-1分别位移到1008cm-1(叶酸)和1010cm-1(谷胱甘肽)。代表碱基谱线信息的682cm-1、1341cm-1、1379cm-1和1557cm-1等也存在不同程度的位移。而分别在染色液、收集液和冷冻液中添加叶酸或谷胱甘肽后,代表磷酸骨架(O-P-O)的788cm-1及代表O=P=O对称伸缩振动的1094cm-1仅出现了特征峰强度的不同。(2)在水牛精子分离及冷冻程序的染色液、收集液和冷冻液中添加叶酸或谷胱甘肽后的拉曼光谱与对照组的差异谱线中发现了代表过氧化氢的880cm-1的存在。(3)对分离精子的拉曼光谱进行标注化处理后,主成分分析法可以对不同精子处理组进行识别和判断。
6.本研究探讨(1)不同公牛个体分离精子和未分离精子体外受精后胚胎的发育能力；(2)X精子和Y精子体外受精后的胚胎发育能力(3)精子分离及冷冻程序各稀释液中添加抗氧化剂(叶酸或谷胱甘肽)对胚胎发育的影响。结果表明：(1)3头尼里拉菲水牛分离精子体外受精后的囊胚发育率均显著低于未分离精子,分别为11.6%vs.21.8%,11.9%vs.22.6%,9.2%vs.16.4%(P<0.05)。(2)X精子和Y精子体外受精后的分裂率、囊胚率及囊胚发育速度的差异均不显著(P>0.05)。(3)在分离及冷冻程序各稀释液中添加叶酸后分离精子体外受精后的分裂率和囊胚率与对照组相比差异不显著(P>0.05),而在精子分离及冷冻程序各稀释液中添加谷胱甘肽后分离精子体外受精后的囊胚率显著高于对照组(P<0.05)。
7.研究水牛精子分离及冷冻程序各稀释液(染色液、分离液和冷冻液)中分别添加叶酸或谷胱甘肽后对分离精子人工授精受胎率的影响。结果表明：(1)在精子分离及冷冻程序各稀释液中添加叶酸后分离精子人工授精后的受胎率与对照组相比差异不显著(P>0.05),但流产率显著低于对照组(P<0.05)。(2)在精子分离及冷冻程序各稀释液中添加谷胱甘肽后分离精子人工授精后的受胎率显著高于对照组(P<0.05),但流产率与对照组相比差异不显著(P>0.05)。
Flow-cytometric sorting of X-and Y-sperm is a promising technology for accelerating the genetic improvement and altering the sex ratio of buffalo. Though the technology of buffalo sperm sorting had been established, however, the blastocyst development rate after IVF and the conception rate after AI with sorted sperm were still lower than that with unsorted sperm. There were potential damages inflicted on sperm during the flow sorting procedure involves high levels of dilution, nuclear staining, exposure to UV lasers, cooling and cryopreservation which affected on sperm quality such as motility, plasma membrane, acrosomal integrity, mitochondrial membrane potential, DNA integrity, apoptosis. The objectives of this study were to detect the sperm damage in the different stages of flow sorting process (fresh, stained, sorted and frozen), so as to optimize the sorting procedure, improve sperm quality and finally accelerate buffalo breed improvement. A series of experiments were carried out in this study as follows:
1. Evaluation of sperm quality, such as motility, plasma membrane integrity, apoptosis and DNA integrity was studied during the flow sorting procedure including four different stages (fresh, stained, sorted and frozen). The results indicated that sperm motility of sorted and frozen semen samples was significantly lower than that of fresh and stained samples (54.72%,52.38%vs.98.07%,96.41%, P<0.05). The percentage of sperm with plasma membrane integrity was higher in fresh, stained and sorted samples compared to the frozen samples by fluorescence microscopy (72.12%,69.39%,77.26%vs.58.45%) and by flow cytometry64.74%,63.69%,66.25%vs.36.98%, P<0.05). The percentage of apoptosis was significantly increased in sorted and frozen samples compared to the fresh samples by fluorescence microscopy (13.23%,18.74%vs.8.11%) and by flow cytometry (12.86%,22.4%vs.7.56%, P<0.05). The percentage of DNA fragment was much lower in the sorted sample than those of in fresh, stained and frozen samples by sperm chromatin dispersion test (SCD,0.47%vs.2.23%,2.25%,2.26%) and by sperm chromatin structure assay (SCSA,0.51%vs.2.55%,2.64%,2.15%, P<0.05).
2. Sperm motility, plasma membrane integrity, apoptosis and DNA integrity of sorted frozen and unsorted frozen sperm from three Nili-Ravi buffalo bulls were evaluated. The percentage of sperm motility was significantly lower in sorted frozen sperm than unsorted frozen sperm by computer assisted sperm analysis system (CASA,59.6%vs.38.9%;58.1%vs.35.8%;59.1%vs.34%, P<0.05. for bull No.1,2and3, respectively). The DNA fragment index (DFI) was much lower in the sorted group than that in the unsorted one by sperm chromatin structure assay (SCSA,1.86%vs.2.47%,2.05%vs.3.0%,3.45 vs.4.38%, P<0.05), however, bull3had higher DFI than the other two bulls regardless of sorted or unsorted sperm. The percentage of sperm with plasma membrane integrity from sorted group was higher than that from unsorted group by fluorescence microscopy (55.3%vs.42.4%,52.7%vs.41.7%,51.6%vs.43.3%, P<0.05). The percentage of apoptosis was significantly increased in the sorted sperm compared to that of unsorted sperm by fluorescence microscopy (28.3%vs.21.1%,31.5%vs.23.6%,32%vs.23%, P<0.05). The percentage of apoptosis from sorted sperm was significantly higher in bull3than the other two bulls.
3. Effects on buffalo sperm motility, plasma membrane integrity, apoptosis, DNA integrity of different antioxidant substances (folic acid and GSH) supplemented in the semen stained, sorted and frozen semen extenders were evaluated. The results of the study indicated that sperm motility, membrane integrity and DNA fragment were no significant differences (P>0.05) either with or without folic acid or GSH supplemented in the semen extender (stained, sorted and frozen). The percentage of apoptosis was much lower in frozen semen supplemented with folic acid or GSH in the extender compared to that in the control (P<0.05).
4. A detection system based on laser tweezers Raman spectroscopy of sorted buffalo sperm was established. The results of the study indicated that the band assigned to PO2diester symmetric stretching shifted from788cm-1to783,785cm-1in fresh group compared to that in sorted and frozen groups. The band assigned to C-O stretching at1010cm-1shifted to1004,1005and1005cm-1in fresh group compared to that in stained, sorted and frozen groups. The scatter plots of principal component analysis could be distinguished the buffalo sperm from fresh, stained, sorted and frozen stages after the normalization of original Raman spectrum.
5. Effects of different antioxidant substances (folic acid and GSH) supplemented to the semen stained, sorted and frozen extenders on sperm Raman spectrum were evaluated. The results showed that:(1) The band assigned to C-O stretching at1010cm-1in the fresh group shifted to1004cm-1in the stained group. When supplemented with folic acid or GSH in stained extender, the band assigned to C-O stretching at1004cm-1in control group shifted to1008cm-1(folic acid) and1005cm-1(GSH) in treatment groups, while for the frozen semen the C-O stretching from1004cm-1in the control semen shifted to1008cm-1(folic acid) and1010cm-1(GSH). The band assigned to PO2diester symmetric stretching at788cm-1and the PO2-group symmetric stretching band at1094cm-1only had different Raman peak intensity in the stained, collected and frozen semen supplemented with folic acid or GSH.(2) There was a peak of880cm-1considered as characteristic frequency of H2O2which was only existed when differential Raman spectrum was subtracted between the control and the stained, collected and frozen semen supplemented folic acid or GSH.(3) The scatter plots of principal component analysis (PCA) could be indentified from different semen treatments after the normalization of original Raman spectrum.
6. Three experiments were conducted to evaluate in vitro embryonic development after IVF with (1) sorted and unsorted sperm from three Navi-Ravi buffalo bulls;(2) X-and Y-sperm and (3) sorted sperm treated with folic acid or GSH in the stained, sorted and frozen semen extender. The results showed that (1) the rates of cleavage and blastocyst after IVF were much lower (P<0.05) for sorted sperm than that for unsorted sperm (11.6%vs.21.8%,11.9%vs.22.6%,9.2%vs.16.4%, for the three Navi-Ravi buffalo bulls, respectively).(2) there were no significant differences (P>0.05) in cleavage and blastocyst rates and the percentage of blastocyst developed on Day6, Day7, Day8after insemination with X sperm and Y sperm.(3) there were no defferences (P>0.05) in cleavage and blastocyst rates between the control and the sperm treated with folic acid in the stained, sorted and frozen semen extender after IVF. However, the blastocyst rate was increased (P<0.05) when the sperm was treated with GSH in the stained, sorted and frozen semen extender after IVF compared to the control group.
7. Pregnant rate was investigated after AI with X sperm treated with folic aicd or GSH in the stained, sorted and frozen semen extenders. The results showed that (1) There were no significant difference (P>0.05) in conception rate after AI with X sperm between the folic acid treatment and control groups, but the abortion rate for the treatment group was much lower (P<0.05) than that for the control.(2) A significant higher conception rate was found in X sperm treated with GSH than that in the control (P<0.05), but no differerence in abortion rate (P>0.05).
引文
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