牛精子原位杂交DTT去凝集条件优化

【目的】确定牛精子原位杂交DTT去凝集处理的优化浓度及时间。【方法】牛精子解冻后经密度梯度离心优选并制片,采用05、、10、20、40、80和100 mmol/L六个DTT浓度以及15、304、5和60 min 4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45 min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。

Optimization of Sperm Decondensation Treatments with Dithiothreitol in Situ Hybridization in Bulls

【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) μm2.In the 16 treatments with DTT concentration<40 mmol / L and in another 6 treatments with concentration ≥40 mmol /L DTT and time<45 min,sperm head area was less than(52.32 ± 4.68) μm2(P <0.01).In 5 treatment groups with 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.