Objective To study the effects of two cryopreservation methods ( direct vapor nitrogen method and program freezing method) on the motility of post-thaw human sperm.Methods The 25 semen samples were taken from 25 donors from Shanghai Human Sperm Bank during May to July 2011,using straw as a frozen carrier, the semen samples contained modified glycerol-yolk-citrate ( GYC ) ( 1 ∶1 ) as cryoprotectant,after the liquefaction, each semen sample was divided into 2 groups,and separately cryopreserved by direct vapor nitrogen method and program freezing method.According to the recommended standard of the World Health Organization, semen samples were analyzed before and after cryopreserving.Density,total motility,PR,motility parameters ( including VAL,VSL,VCL,BCF) were determinated by the IVOS sperm analyzer.Semen samples were stained by modified papanicolaou stain and 200 sperms of each sample were counted under microscope by manual analysis to calculate the rate of normal morphology sperm.The paired t test was used to compare the above indictors before and after cryopreserving in one method and after thawing between two methods.Results Before cryopreserving,the total motility,PR,VAL,VSL,VCL,BCF were(91.36±4.96)％,(81.28 ±6.41)％,(59.29 ±9.30) μm/s,(49.80±8.40) μm/s, (83.39 ± 15.70) μm/s, (16.68 ± 3.36) Hz respectively, and after cryopreserving by direct vapor nitrogen method the results were (46.04 ± 20.14)％,(40.22 ± 18.42 )％,(48.00± 11.60)μm/s,(41.11 ±8.50) μm/s,(71.55 ±17.19) μm/s,(15.94 ± 14.30) Hz respectively,the differences of the indictors above were observed with statistic significance except BCF ( t =11.324,11.958,4.266,3.964,3.620,0.258,P of BCF ＞0.05,P of others ＜0.05); and after cryopreserving by program freezing method,the results were(48.05 ± 18.27)％,(42.47 ± 15.62)％,(45.30 ± 6.51) μm/s,(38.08 ± 4.99) μm/s,(64.23 ± 11.75) μm/s,(14.00 ± 2.99) Hz respectively,the differences of the indictors above showed statistic significance (t =10.337,10.874,7.154,5.510,6.494,3.244,P ＜0.05).No significant differences were observed in the revival rate,total motility,PR and normal morphology sperm rate by using different methods (t =0.425,0.364,0.622,1.106,P ＞0.05).The results of VAL,VSL,VCL,BCF in direct vapor nitrogen method were higher than that in program freezing method,when the differences of VSL and VCL were observed with statistic significance(t =2.489,2.822,P ＜ 0.05 ).Conclusions Using straw as carrier is practicable.Direct vapor nitrogen method is easier to operate than program freezing method.Post-thaw sperm cryopreseved by direct vapor nitrogen method has higher motility parameters,thus making this method an ideal one for fast freezing.%目的 比较一步熏蒸法和程序降温法对人类精子冷冻复苏后运动能力的影响.方法 25份精液标本取自2011年5至7月上海市人类精子库25名精液捐赠者,应用麦管为冷冻载体,按照1∶1的比例添加改良甘油-卵黄-柠檬酸钠作为冷冻保护剂,将液化后的每份精液标本平均分为2份,分别进行一步熏蒸法和程序降温法冷冻.按照世界卫生组织推荐标准对冷冻前后的标本进行精液常规分析,利用IVOS精子分析仪进行精子密度、总活动率、前向运动精子百分率、运动参数(包括平均路径速度、平均直线速度、平均曲线速度、平均鞭打频率)的测定.精子形态学染色使用改良巴氏染色,采用人工分析办法,显微镜下计数200条精子,计算其形态正常率.同一种冷冻方法进行冷冻前、后上述各指标的比较,以及两种不同冷冻方法精子复苏后上述各指标的比较均采用配对t检验.结果 冷冻前,精子总活动率、前向运动精子百分率、平均路径速度、平均直线速度、平均曲线速度、平均鞭打频率分别为(91.36 ±4.96)％、(81.28 ±6.41)％、(59.29±9.30) μm/s、(49.80 ±8.40) μm/s、(83.39±15.70) μm/s、(16.68 ±3.36) Hz,采用一步熏蒸法冷冻复苏后,分别为(46.04 ±20.14)％、(40.22±18.42)％、(48.00±11.60)μm/s、(41.11±8.50) μm/s、(71.55±17.19)μm/s、(15.94±14.30) Hz,与冷冻前比较,除平均鞭打频率差异无统计学意义外(t=0.258,P＞0.05),其余指标差异均有统计学意义(t=11.324、11.958、4.266、3.964、3.620,P均＜0.05).采用程序降温法冷冻复苏后,上述各指标分别为(48.05±18.27)％、(42.47±15.62)％、(45.30 ±6.51) μm/s、(38.08 ±4.99) μm/s、(64.23 ±11.75) μm/s、(14.00 ±2.99) Hz,与冷冻前比较,差异均有统计学意义(t=10.337、10.874、7.154、5.510、6.494、3.244,P均＜0.05).一步熏蒸法和程序降温法冷冻复苏后,精子复苏率、总活动率、前向运动精子百分率、形态正常率在两种方法间差异均无统计学意义(t=0.425、0.364、0.622、0.676,P＞0.05)；而一步熏蒸法的平均路径速度、平均直线速度、平均曲线速度、平均鞭打频率均高于程序降温法,其中平均直线速度和平均曲线速度的差异有统计学意义(t=2.489、2.822,P均＜0.05).结论 麦管可作为精子冷冻的载体.与程序降温法相比,一步熏蒸法操作简便,冷冻复苏后精子具有较高的运动能力,是一种值得推荐的精子快速冷冻方法.
展开▼
