中国农业科学 ›› 2009, Vol. 42 ›› Issue (5): 1875-1880 .doi: 10.3864/j.issn.0578-1752.2009.05.048

• 研究简报 • 上一篇    

猪精液0.5 ml细管快速冷冻和解冻方法的优化

  

  1. 江苏省农业科学院畜牧研究所

  • 收稿日期:2008-08-25 修回日期:2008-11-19 出版日期:2009-05-10 发布日期:2009-05-10
  • 通讯作者: 刘铁铮

Optimization of Freezing and Thawing Protocol of Rapid Freezing Techniques of Boar Semen in 0.5 ml Straws

  1. 江苏省农业科学院畜牧研究所

  • Received:2008-08-25 Revised:2008-11-19 Online:2009-05-10 Published:2009-05-10
  • Contact: LIU Tie-zheng

摘要:

【目的】优化冷冻-解冻方法,提高0.5 ml细管快速冷冻保存猪精液的质量。【方法】冷冻方法:分别将细管水平置于液氮面上方3、5、7、9和11 cm处,每一水平位置细管分别熏蒸3、5、10、15和20 min后投入液氮保存,37℃×30 s水浴解冻,确定最优冷冻方法;在选择最优冷冻方法的基础上,分别采用37℃×30s(对照组)、42℃×25 s、47℃×20 s、52℃×15 s、57℃×10 s和62℃×5 s共6种方法水浴解冻,确定最佳解冻方法。【结果】采用3 cm×10 min冷冻方法,解冻0、8 h精液中精子活率、质膜完整率和顶体完整率均最高。采用相对“高温短时”解冻,精子活率、质膜完整率、顶体完整率和线粒体膜电位均较高,同时丙二醛浓度降低;57℃×10 s和62℃×5 s解冻的精子各项指标无显著差异(P>0.05),但前4项指标显著高于对照组(P<0.05),丙二醛浓度显著降低(P<0.05)。【结论】优化猪精液0.5 ml细管快速冷冻的冷冻和解冻方法可以显著提高解冻质量,采用3 cm×10 min冷冻、57℃×10 s或62℃×5 s解冻方法效果最佳。

关键词: 优化, 冷冻-解冻方法, 快速冷冻, 0.5 ml细管, 猪精液

Abstract:

【Objective】 This study was performed to optimize freezing and thawing protocol of rapid boar seminal cryopreservation in 0.5 ml straws. 【Method】 0.5 ml straws were placed at the height of 3, 5, 7, 9 and 11 cm above liquid nitrogen (LN) for 3, 5, 10, 15 and 20 min in polyethylene box, respectively. And the optimal freezing protocol was determined after straws were frozen in LN and thawed in water bath at 37℃ for 30 s. The optimal thawing protocol was determined from six basal protocols, including 37℃ for 30 s, 42℃ for 25 s, 47℃ for 20 s, 52℃ for 15 s, 57℃ for 10 s and 62℃ for 5 s. 【Result】 In twenty-five freezing protocol, both at post-thawed 0 h and 8 h, the percentages of TMS, PMI and NAR of spermatozoa were highest in semen frozen at 3 cm for 10 min. In six thawing protocols, the percentages of TMS, PMI, NAR and Mt-MP of spermatozoa were higher, and the concentration of MDA was lower in semen thawed at higher temperature. Moreover, compared with the control, the percentages of TMS, PMI, NAR and Mt-MP were increased (P<0.05) and the concentration of MDA was decreased significantly (P<0.05) in semen thawed at 57℃ for 10 s and 62℃ for 5 s, and there was no difference between thawed at 57℃ for 10s and 62℃ for 5s (P>0.05). 【Conclusion】 For the rapid freeze-thawing method of this experiment, adopting optimized freezing and thawing protocol increased the quality of thawed semen, meanwhile, freezing 0.5 ml straws at 3 cm for 10 min and thawing at 57℃ for 10 s or 62℃ for 5 s were the optimal protocol.

Key words: optimization, freezing and thawing protocol, rapid freezing, 0.5 ml straw, boar semen