稀释液添加精氨酸对猪精子活力、一氧化氮合酶和一氧化氮含量的影响

Effects of semen diluent supplemented with L-Arg on sperm motility, nitro oxide synthase and nitric oxide content of boars

试验旨在探讨39℃体外培养时,常温稀释液中添加不同浓度L-精氨酸(L-Arg)对猪精子活力、NO含量及一氧化氮合酶(nitric oxide synthase,NOS)的影响。首先根据已有专利和文献报道设计了3种稀释液配方:高糖+青霉素钠+硫酸链霉素(M+N组),高糖+庆大霉素硫酸盐(M+Q组),低糖+青霉素钠+链霉素(L+N组),并将商用稀释液设为对照组。使用稀释液重悬后,17℃恒温箱中孵育精子,每隔24 h测定一次活力指标。其次,取成年健康大约克公猪精液(精子活力>70%)离心重悬于自配精液稀释液,分别添加不同浓度L-Arg(0.0、0.5、1.0、2.0 mmol/L)置于39℃下孵育0、2、4 h,分别收集稀释液和精子,利用计算机辅助精子分析方法(computer-assisted sperm analysis,CASA)测定精子活力参数,评估精液稀释液中L-Arg的最适添加浓度。用最适浓度L-Arg处理猪精子,39℃孵育0、2、4 h,收集稀释液和精子,使用CASA分析测定精子活力参数,测定NO含量,利用蛋白免疫荧光定位方法测定精子中诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和内皮型一氧化氮合酶(endotheliale nitric oxide synthase,eNOS)蛋白表达位置,并检测其酶活力,利用qPCR方法测定精子中iNOS mRNA表达水平,明确NOS在精子稀释和复温过程中不同时间点的变化特点。试验结果显示:1)与对照组相比,M+Q组可有效提高精子活力、向前运动比例、快速运动比例及原地摆动比例,降低精子不运动比例,保存效果相对较好。2)39℃体外培养精子2、4 h时,与对照组相比,精液稀释液中添加1 mmol/L和2 mmol/L L-Arg可显著提高精子细胞活力、向前运动比例、快速运动比例及慢速运动比例,显著降低精子原地摆动和不运动比例(P<0.05)。因此,选取1 mmol/L浓度的L-Arg进行后续研究。3)39℃孵育精子2、4 h时,和对照组相比,1.0 mmol/L L-Arg组NO含量呈升高趋势,总一氧化氮合酶(total nitric oxide synthase,TNOS)活力增强,iNOS酶活力显著增强(P<0.01)。试验结果显示,热孵育精子0、4 h时,L-Arg组精子细胞iNOS基因的相对表达显著高于对照组(P<0.01),iNOS和eNOS蛋白表达位置未发生改变。表明L-Arg可能通过减缓猪精子iNOS基因降解,增强NOS酶活力,促进NO的生成,维持精子活力。

The aim of the experiment was to investigate the effects of different levels of L-Arginine(L-Arg) added to the normal temperature dilution of boar semen during in vitro culture at 39 ℃ on sperm motility, NO content and nitric oxide synthase(NOS) in the animals. Firstly, three diluent formulations were designed based on the existing patents and reports: high sugar + penicillin sodium + streptomycin sulfate(the M + N group);high sugar + gentamicin sulfate(the M + Q group);and low sugar + penicillin sodium + streptomycin(the L + N group). After resuspending with the diluent we had prepared, the incubated sperm samples in a 17 ℃ incubator were collected and their motility indexes were measured every 24 hours. Secondly, healthy boar semen(motility>70%) were taken, centrifuged and added with semen diluents without L-Arg, or different concentrations of L-Arg(0.0, 0.5, 1.0, 2.0 mmol/L), respectively. After incubating at 39 ℃ for 0, 2 or 4 h, both the diluent and the sperms were collected. The sperms motility parameters were determined using the CASA method,and the optimal addition ofL-Arg to the semen diluent was evaluated. Boar sperm were treated with the optimal concentration ofL-Arg and incubated at 39 ℃ for 0, 2 or 4 h. Semen dilutions and sperms were collected and used to measure sperm motility and the NO content. The protein immunofluorescence localization method was used to determine the protein expression positions of inducible nitric oxide synthase(iNOS) and endotheliale nitric oxide synthase(eNOS) in the sperms, and their enzymatic activities were detected too. The expression level of the iNOS gene in the sperms was measured using the qPCR method to clarify the NOS change at different time points during sperm dilution.The results showed as follows: 1) Among the three kinds of the pig semen dilutions we had preapred, the M + Q group effectively improved their sperm motility and increased their progressive motility, fast motility and local motility ratios. The non-exercise proportion was reduced.2) After culturedin vitrofor 2 or 4 h at 39 ℃, adding 1. 0 mmol/L and 2. 0 mmol/LL-Arg to the semen diluent significantly improved their sperm motility, progressive motility ratio, fast motility ratio and slow motility ratio compared with the control group. The local motility and immotile ratios of the sperms were decreased(P<0. 05). Therefore, 1. 0 mmol/L concentration ofL-Arg was selected for the follow-up studies. 3) After the sperms were incubated at 39 ℃ for 2 h or 4 h, the NO content and the activity of total nitric oxide synthase in the 1. 0mmol/LL-Arg group showed an increasing trend compared with the control group. After adding 1. 0 mmol/LL-Arg, the iNOS activity was increased, too(P<0. 01). The relative mRNA expression ofiNOSin the sperms of theL-Arg group was significantly higher than that of the control group at 0 and 4 h time points(P<0. 01), but the protein expression positions of iNOS and eNOS did not change. The above results indicated thatL-Arg might promote NO production and maintain sperm motility by slowing down the degradation ofiNOSmRNA.