新西兰兔精子冷冻保存研究
摘要
精液冷冻可以有效地保存动物种质资源,尤其是保存基因工程技术和诱导突变得到的动物新品系的精子,并且可以为动物人工授精技术的大规模应用提供精液来源。
在兔精液在5℃条件下的低温保存方法的基础上,比较两种冷冻保护剂和冷冻程序对兔精子低温保存的影响;分析冷冻稀释液对精子的影响,优化冷冻程序,以期提高兔精子冷冻保存的效果和效率,为兔精子冷冻保存的发展和应用提供试验依据。
1.将采集的新鲜兔精液用3种稀释液分别进行稀释,以温水浴法使精液缓慢降温,5℃低温保存,每隔12 h取出20μL精液,在37℃的CO2培养箱中平衡5 min,进行活率检测。结果表明,低温保存72 h后,稀释液Ⅲ中的精子存活率为53.0%,显著高于其他两组(P<0.05)。用稀释液Ⅲ低温保存48 h后的精液对26只母兔进行人工授精,每只母兔输精1 mL,母兔平均产仔数6.2只,成活率85.2%,与新鲜精液人工授精母兔平均产仔数(6.8只)和成活率(83.8%)相比无显著差异(P>0.05)。证明兔精液通过慢速降温保存于5℃条件下,可以有效延长精子在离体状态下的存活时间。
2.用三步降温法(程序Ⅰ)和两步降温法(程序Ⅱ)两种冷冻程序,与分别含有终浓度为2%,3%,4%和5%的甘油与乙酰胺的冷冻保护液配合,进行精液冷冻保存,统计精子的复苏率。冷冻前精液均通过200 g(1000 r/min)离心10 min除去部分精浆。结果程序Ⅱ中,3%乙酰胺组获得了45.5%的精子复苏率,与其他组比较差异具有统计学差异(P<0.05);同种冷冻保护剂相同浓度组采用不同冷冻程序,精子复苏率差异无显著性意义(P>0.05)。用程序Ⅱ中3%甘油组和3%乙酰胺组的冷冻精子进行人工授精,3%乙酰胺组获得了4.6只的产仔数,与3%甘油组的结果相比差异有显著性意义,但与新鲜精液6.8只的产仔数相比仍较低。两组获得的妊娠率和仔兔存活率分别为60%,55%和78.7%,82.4%,数据之间的差异性没有显著性意义(P>0.05)。程序Ⅱ与3%的乙酰胺配合可以取得良好的冷冻保存效果;用二步降温法进行兔精液冷冻保存与三步降温法相比可以将操作时间缩短70%。
Semen cryopreservation is an efficiently way to protect germplasm resource, especially for conserve the new stock which made by genetic engineering and induced mutation, and it can provide enough semen for animal artificial fecundation.
Studies probed into the cryopreservation approach of rabbit semen at 5℃, and compared different cryoprotectants and cryopreservation procedures of rabbit spermatozoa; determined and analysed the effects of cryoprotectant, optimized the cryopreservation procedures. To improve the effectiveness and efficiency of rabbit semen freezing, this can supply experiment foundation for development and use of rabbit semen cryopreservation.
1. The collected semen diluted with medium-Ⅰ, medium-Ⅱand medium-Ⅲrespectively, 37℃water bathed and hoarded at 5℃, pipettor 20μL the intermixture and mensurate the survive rate every 12 h interval. The results show that the survive rate of medium-Ⅲgroup was 53.0% after 72 h, distinct higher than others. Artificial insemination carried on for 26 does with the semen of medium-Ⅲgroup which refrigerated 48 h, and the inseminationdose is 1 mL. The litter size and livability are 6.2 and 85.2%. Compared with the fresh sperm group, the results of the two groups don’t have significantly different. The survivable time in vitro of rabbit semen can be extended effectively through cooling to 5℃slowly.
2. Different concentration (2%, 3%, 4% and 5%) of glycerol and acetamide were used as cryoprotectants with two different cryopreservation procedures (ProtocolⅠand ProtocolⅡ) to cryopreserved the rabbit spermatozoa, and the freeze-thawed fertility was examined. Seminal plasma is removed through centrifugal (200 g, 10 min) before the sperm cryopreserved. The motility in protocolⅡwith 3% acetamide is 45.5%, significantly higher than in other groups(P<0.05). Artificial insemination is tested with the thawed semen of 3% acetamide group and 3% glycerol group. The litter size of 3% acetamide group is 4.6, significantly different with the result of 3% glycerol group(3.9) (P<0.05),but lower than fresh semen’s (P<0.05). The results indicate that the protocolⅡwith 3% acetamide have optimal cryoprotective effects and the protocolⅡcan save about 70% manipulation time.
在兔精液在5℃条件下的低温保存方法的基础上,比较两种冷冻保护剂和冷冻程序对兔精子低温保存的影响;分析冷冻稀释液对精子的影响,优化冷冻程序,以期提高兔精子冷冻保存的效果和效率,为兔精子冷冻保存的发展和应用提供试验依据。
1.将采集的新鲜兔精液用3种稀释液分别进行稀释,以温水浴法使精液缓慢降温,5℃低温保存,每隔12 h取出20μL精液,在37℃的CO2培养箱中平衡5 min,进行活率检测。结果表明,低温保存72 h后,稀释液Ⅲ中的精子存活率为53.0%,显著高于其他两组(P<0.05)。用稀释液Ⅲ低温保存48 h后的精液对26只母兔进行人工授精,每只母兔输精1 mL,母兔平均产仔数6.2只,成活率85.2%,与新鲜精液人工授精母兔平均产仔数(6.8只)和成活率(83.8%)相比无显著差异(P>0.05)。证明兔精液通过慢速降温保存于5℃条件下,可以有效延长精子在离体状态下的存活时间。
2.用三步降温法(程序Ⅰ)和两步降温法(程序Ⅱ)两种冷冻程序,与分别含有终浓度为2%,3%,4%和5%的甘油与乙酰胺的冷冻保护液配合,进行精液冷冻保存,统计精子的复苏率。冷冻前精液均通过200 g(1000 r/min)离心10 min除去部分精浆。结果程序Ⅱ中,3%乙酰胺组获得了45.5%的精子复苏率,与其他组比较差异具有统计学差异(P<0.05);同种冷冻保护剂相同浓度组采用不同冷冻程序,精子复苏率差异无显著性意义(P>0.05)。用程序Ⅱ中3%甘油组和3%乙酰胺组的冷冻精子进行人工授精,3%乙酰胺组获得了4.6只的产仔数,与3%甘油组的结果相比差异有显著性意义,但与新鲜精液6.8只的产仔数相比仍较低。两组获得的妊娠率和仔兔存活率分别为60%,55%和78.7%,82.4%,数据之间的差异性没有显著性意义(P>0.05)。程序Ⅱ与3%的乙酰胺配合可以取得良好的冷冻保存效果;用二步降温法进行兔精液冷冻保存与三步降温法相比可以将操作时间缩短70%。
Semen cryopreservation is an efficiently way to protect germplasm resource, especially for conserve the new stock which made by genetic engineering and induced mutation, and it can provide enough semen for animal artificial fecundation.
Studies probed into the cryopreservation approach of rabbit semen at 5℃, and compared different cryoprotectants and cryopreservation procedures of rabbit spermatozoa; determined and analysed the effects of cryoprotectant, optimized the cryopreservation procedures. To improve the effectiveness and efficiency of rabbit semen freezing, this can supply experiment foundation for development and use of rabbit semen cryopreservation.
1. The collected semen diluted with medium-Ⅰ, medium-Ⅱand medium-Ⅲrespectively, 37℃water bathed and hoarded at 5℃, pipettor 20μL the intermixture and mensurate the survive rate every 12 h interval. The results show that the survive rate of medium-Ⅲgroup was 53.0% after 72 h, distinct higher than others. Artificial insemination carried on for 26 does with the semen of medium-Ⅲgroup which refrigerated 48 h, and the inseminationdose is 1 mL. The litter size and livability are 6.2 and 85.2%. Compared with the fresh sperm group, the results of the two groups don’t have significantly different. The survivable time in vitro of rabbit semen can be extended effectively through cooling to 5℃slowly.
2. Different concentration (2%, 3%, 4% and 5%) of glycerol and acetamide were used as cryoprotectants with two different cryopreservation procedures (ProtocolⅠand ProtocolⅡ) to cryopreserved the rabbit spermatozoa, and the freeze-thawed fertility was examined. Seminal plasma is removed through centrifugal (200 g, 10 min) before the sperm cryopreserved. The motility in protocolⅡwith 3% acetamide is 45.5%, significantly higher than in other groups(P<0.05). Artificial insemination is tested with the thawed semen of 3% acetamide group and 3% glycerol group. The litter size of 3% acetamide group is 4.6, significantly different with the result of 3% glycerol group(3.9) (P<0.05),but lower than fresh semen’s (P<0.05). The results indicate that the protocolⅡwith 3% acetamide have optimal cryoprotective effects and the protocolⅡcan save about 70% manipulation time.
引文
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