牛精子原位杂交DTT去凝集条件优化

摘要

[目的]确定牛精子原位杂交DTT去凝集处理的优化浓度及时同.[方法]牛精子解冻后经密度梯度离心优选并制片,采用0、5、10、20、40、80和100 mmol/L六个DTT浓度以及15、30、45和60 min4个时间段的28个组合进行去凝集处理,Motic Images Advanced 3.2软件测量精子解聚后头部面积.[结果]40 mmol/L DTT处理45min精子头部面积为(52.32±4.68 )μm2,浓度＜ 40 mmol/L的16个处理组及浓度≥40 mmol/L、处理时间＜45 min的6个处理组,精子头部面积小于(52.32±4.68) μm2(p＜ 0.01)；浓度＞40 mmol/L、时间≥45 min的5个处理组,精子头部面积与40 mmol/L DTT 45 min组差异不显著(P＞0.05),但精子形态不完整.[结论]40mmol/L DTT 45 min为较优处理组合.%[Objective] The purpose of this study is to determine the optimum concentration and preparation time of dithiothreitol (DTT) in sperm decondensation before the in situ hybridization. [Method] The frozen -thawed bull sperm was spread on glass slides after percoll gradient centrifugation and then treatments were performed by six DTT concentration (5, 10, 20, 40, 80, 100 mmol/L DTT) and four preparation times (15, 30, 45, 60 min), respectively. Decondensation of sperm head areas among them were measured by using Motic Images Advanced 3.2 software. [Result] After incubation for 45 min with 40 mmol/L DTT, the sperm head area was (52.32 ± 4.68) pun2. In the 16 treatments with DTT concentration 40 mmol / L DTT and time ≥45 min, the difference of sperm head area was not significant ( P > 0.05), but the sperm morphology was incomplete. [Conclusion] The 45 min incubation with 40 mmol/L DTT was selected as an optimal and eligible treatment for sperm depolymerization.