猪精液5ml细管冷冻保存技术研究
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摘要
对上海白猪精液的冷冻分装方法、冷冻方法、稀释液及添加剂、甘油浓度、精子密度、解冻温度及时间等技术环节进行了研究，并对冻融前后的精子进行了电镜观察，记录了试验过程中的温度，描绘了不同的冷冻温度曲线。旨在探索猪精液5 ml细管冷冻保存技术体系，为猪精液冷冻保存及商业化生产提供技术支撑。试验结果如下：
猪精液颗粒、0.25 ml细管、5 ml细管之间，冷冻后精子活力和活率的差异不显著(P＞0.05)。0.25 ml细管冻后精子弯尾率和顶体完整率显著高于颗粒(P＜0.05)，但与5 ml细管之间差异不显著(P>＞0.05)。按不同的始冻温度、平衡温度、入氮温度绘制了三种精液冷冻方式的冷冻温度曲线。
在使用的四种稀释液中，4号(LEY)稀释液冷冻解冻后的精子活力、弯尾率和顶体完整率显著高于其他三种稀释液(P＜0.05)，活率极显著高于其他三种稀释液(P＜0.01)。
比较5 ml细管冷冻甘油终浓度为1％、2％、3％、4％、5％的稀释精液，2％、3％的甘油浓度能有效保护冻后精子活力、活率和顶体，1％～3％的甘油浓度能有效保护精子质膜。
5 ml细管冷冻时距离液氮面不同高度冷冻，距离液氮面3 cm高冷冻，熏蒸时温度回升幅度较小，解冻后的精子质量高于距离液氮面5 cm高冷冻，但二者差异不显著(P＞0.05)。并分别绘制两者的冷冻温度曲线。
稀释液添加剂试验表明，0.05％、0.1％的N-乙酰-D-氨基葡萄糖能有效保护精子顶体，咖啡因提高了精子的冻后活力(P＞0.05)，2～8 mmol／L咖啡因显著降低了冻后精子的弯尾率(P＜0.05)，2％、5％安钠咖可显著提高精子的冻后活力(P＜0.05)。
5 ml细管冷冻密度分别为2.0×10~9个／ml、1.5×10~9个／ml、1.0×10~9个／ml的猪精子，解冻后精子质量差异不显著(P＞0.05)。
干解法和湿解法解冻颗粒冻精的效果差异不显著(P＞0.05)。5 ml细管冻精在不同温度、不同时间解冻，随着解冻温度的升高，解冻后精子活力和活率也升高，弯尾率先升高后降低，且较高的解冻温度对精子顶体有害。在所有解冻组中52℃35s和45℃70s是解冻效果最好的组合。
本试验推荐的猪精液5 ml细管冷冻保存程序为：用LEY稀释液稀释精液，甘油终浓度为2％、精子密度为2.0×10~9个／ml，5 ml细管分装后距离液氮面3 cm处冷冻，52℃35s解冻。稀释液中可添加0.05％或0.1％N-乙酰-D-氨基葡萄糖及2％或5％安钠咖。
In order to develop cryopreservation techniques of boar semen to be applied to commercial production, experiments were carried out to determine optimum package, freezing method, diluents and additives, glycerol concentration, sperm concentration, thawing temperature and time in freezing boar semen by evaluating post-thaw sperm quality. The ultrastructural changes of sperm before and after cryopreservation were evaluated under electron microscope. During freezing procedure different temperature were wrote down to make freezing temperature curve. The results were as follows:
There was no significant difference among the post-thaw motility and viability of boar semen frozen in pellets, 0.25 ml mini-straws and 5 ml maxi-straws(P > 0.05). 0.25 ml mini-straws had significantly higher post-thaw tail coiling rate and normal acrosome rate (NAR) than pellets(P < 0.05), but had no significant difference with 5 ml maxi-straws. Three freezing temperature curve were set down respectively with different beginning frozen temperature, balance temperature and enter nitrogen temperature.
Among the four diluents used, diluents 4(LEY) has significantly higher post-thaw sperm motility, tail coiling rate and NAR than the other three diluents(P < 0.05), and had extremly significant higher post-thaw sperm viability than the other three diluents(P < 0.01).
Diluents with 2% and 3% glycerol concentration had the highest post-thaw sperm motility, viability and NAR among the five different glycerol concentrations. 1%~3% glycerol concentration was effective for protecting post-thaw sperm membrane.
猪精液颗粒、0.25 ml细管、5 ml细管之间，冷冻后精子活力和活率的差异不显著(P＞0.05)。0.25 ml细管冻后精子弯尾率和顶体完整率显著高于颗粒(P＜0.05)，但与5 ml细管之间差异不显著(P>＞0.05)。按不同的始冻温度、平衡温度、入氮温度绘制了三种精液冷冻方式的冷冻温度曲线。
在使用的四种稀释液中，4号(LEY)稀释液冷冻解冻后的精子活力、弯尾率和顶体完整率显著高于其他三种稀释液(P＜0.05)，活率极显著高于其他三种稀释液(P＜0.01)。
比较5 ml细管冷冻甘油终浓度为1％、2％、3％、4％、5％的稀释精液，2％、3％的甘油浓度能有效保护冻后精子活力、活率和顶体，1％～3％的甘油浓度能有效保护精子质膜。
5 ml细管冷冻时距离液氮面不同高度冷冻，距离液氮面3 cm高冷冻，熏蒸时温度回升幅度较小，解冻后的精子质量高于距离液氮面5 cm高冷冻，但二者差异不显著(P＞0.05)。并分别绘制两者的冷冻温度曲线。
稀释液添加剂试验表明，0.05％、0.1％的N-乙酰-D-氨基葡萄糖能有效保护精子顶体，咖啡因提高了精子的冻后活力(P＞0.05)，2～8 mmol／L咖啡因显著降低了冻后精子的弯尾率(P＜0.05)，2％、5％安钠咖可显著提高精子的冻后活力(P＜0.05)。
5 ml细管冷冻密度分别为2.0×10~9个／ml、1.5×10~9个／ml、1.0×10~9个／ml的猪精子，解冻后精子质量差异不显著(P＞0.05)。
干解法和湿解法解冻颗粒冻精的效果差异不显著(P＞0.05)。5 ml细管冻精在不同温度、不同时间解冻，随着解冻温度的升高，解冻后精子活力和活率也升高，弯尾率先升高后降低，且较高的解冻温度对精子顶体有害。在所有解冻组中52℃35s和45℃70s是解冻效果最好的组合。
本试验推荐的猪精液5 ml细管冷冻保存程序为：用LEY稀释液稀释精液，甘油终浓度为2％、精子密度为2.0×10~9个／ml，5 ml细管分装后距离液氮面3 cm处冷冻，52℃35s解冻。稀释液中可添加0.05％或0.1％N-乙酰-D-氨基葡萄糖及2％或5％安钠咖。
In order to develop cryopreservation techniques of boar semen to be applied to commercial production, experiments were carried out to determine optimum package, freezing method, diluents and additives, glycerol concentration, sperm concentration, thawing temperature and time in freezing boar semen by evaluating post-thaw sperm quality. The ultrastructural changes of sperm before and after cryopreservation were evaluated under electron microscope. During freezing procedure different temperature were wrote down to make freezing temperature curve. The results were as follows:
There was no significant difference among the post-thaw motility and viability of boar semen frozen in pellets, 0.25 ml mini-straws and 5 ml maxi-straws(P > 0.05). 0.25 ml mini-straws had significantly higher post-thaw tail coiling rate and normal acrosome rate (NAR) than pellets(P < 0.05), but had no significant difference with 5 ml maxi-straws. Three freezing temperature curve were set down respectively with different beginning frozen temperature, balance temperature and enter nitrogen temperature.
Among the four diluents used, diluents 4(LEY) has significantly higher post-thaw sperm motility, tail coiling rate and NAR than the other three diluents(P < 0.05), and had extremly significant higher post-thaw sperm viability than the other three diluents(P < 0.01).
Diluents with 2% and 3% glycerol concentration had the highest post-thaw sperm motility, viability and NAR among the five different glycerol concentrations. 1%~3% glycerol concentration was effective for protecting post-thaw sperm membrane.
引文
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