纳米锌水平对公羊精液品质、抗氧化酶活性及附睾Cu-ZnSOD表达的影响

Effect of Different Nano-Zinc Levels in Dietary on Semen Quality,Activities of Antioxidant Enzyme and Expression of Copper Zinc Superoxide in Epididymis of Ram Lambs

【目的】微量元素锌是动物正常繁殖所必需的元素,研究旨在了解日粮纳米锌对公羊精液品质、抗氧化酶和附睾Cu-Zn SOD蛋白表达的影响,分析公羊精液品质参数与精浆抗氧化酶活性的相关性。【方法】将16只9月龄健康状况良好体重相近的晋中绵羊公羊,随机分成4组,分别喂给基础日粮(对照组),基础日粮+50 mg·kg-1、100 mg·kg-1和150 mg·kg-1DM纳米锌。试验期90 d。在试验78 d和79 d连续两天采集精液,每次采集精液取出100μL评价精液品质参数,剩余精液离心分离精浆,测定精浆Cu-Zn SOD、GPxs活性及总抗氧化能力;试验结束时,用手术去势法采集附睾头、附睾体和附睾尾组织,采用免疫组化技术对附睾头、附睾体和附睾尾中Cu-Zn SOD进行定位,并用Image Pro Plus 7.0软件分析的平均光密度值进行Cu-Zn SOD蛋白定量。【结果】日粮纳米锌对公羊射精量影响差异不显著(P>0.05)。与对照组相比,添加50 mg·kg-1或100 mg·kg-1纳米锌组公羊的精子密度、精子活力和精子质膜完整性显著增加(P<0.05),精子畸形率显著降低(P<0.05);添加150 mg·kg-1组精子质膜的完整度显著增加(P<0.05),其精子活力和精子密度与对照组的差异不显著(P>0.05)。日粮添加50 mg·kg-1或100 mg·kg-1纳米锌组公羊精浆Cu-Zn SOD活性、GPxs活性和总抗氧化能力显著高于对照组(P<0.05),精浆MDA含量显著低于对照组(P<0.05);添加150 mg·kg-1组公羊精浆总抗氧化能力显著高于对照组(P<0.05),其精浆Cu-Zn SOD活性、GPxs活性和MDA含量与对照组差异不显著(P>0.05)。免疫组化定位结果显示Cu-Zn SOD在附睾头和附睾体假复层上皮、结缔组织和附睾尾的单层柱状上皮中均检测到阳性信号,试验处理组公羊附睾中Cu-Zn SOD表达量由高到低顺序是:附睾体>附睾头>附睾尾;然而对照组的顺序为:附睾头>附睾体>附睾尾。50 mg·kg-1或100 mg·kg-1纳米锌组公羊附睾头和附睾体阳性信号的平均光密度显著高于对照组和150 mg·kg-1纳米锌(P<0.05)。精浆Cu-Zn SOD活性与精子密度、精子活力和精子质膜完整性呈正相关,与精子畸形率呈负相关。【结论】日粮添加50 mg·kg-1或100 mg·kg-1纳米锌可改善精液品质,提高精浆抗氧化能力,增加附睾中Cu-Zn SOD蛋白表达量。精浆Cu-Zn SOD活性可以作为检测精液品质优劣的指标在羊生产中应用。微量元素锌对精液品质影响调控的分子机理还需进一步深入研究。

[Objective] The objectives of this study were to investigate the effects of nano-zinc supplementation in dietary on the semen quality parameters, the activities of antioxidant enzyme in seminal plasma and the expression of copper zinc superoxide dismutase in epididymis of ram lambs, and to analyze the correlation between semen quality parameters and the activities of antioxidant enzyme.[Method]Sixteen 9-month-old Jinzhong ram lambs with good health and approximate weight were randomly divided into 4 groups, fed with a basal diet with supplementation of 0, 50, 100 and 150 mg·kg^-1DM nano-zinc, respectively. The experimental period was 90 d. Semen was collected on 78 d and 79 d in consecutive two days, samples of 100 μL fresh semen was used to analyze semen quality parameters, the rest of fresh semen sample was centrifuged at 2 000 r/min for 10 min, the supernatant （seminal plasma） was collected for measurement of the activities of copper zinc superoxide dismutase, glutathione peroxidase and total anti-oxidation competence. All rams were castrated for collection of epididymis caput, corpus and cauda at the end of experiment. Expression of Cu-ZnSOD protein in epididymis was detected and located by immunohistochemistry. The mean optical density was analyzed with Image Pro Plus 7.0 software.[Result]The results showed that there was no significant difference in ejaculate volume between the treatment groups and control group （P〉0.05）. Compared with the control group, the groups with supplementation of 50 mg·kg^-1or 100 mg·kg^-1nano-zinc had higher sperm concentration, sperm motility and membrane integrity, and less percentage of abnormal sperm. The group with supplementation of 150 mg·kg^-1had higher membrane integrity than control group, while it had no significant difference in sperm concentration, sperm motility with control group. The rams in groups with supplementation of 50 mg·kg^-1or 100 mg·kg^-1nano-zinc also had higher activities of Cu-ZnSOD, GPxs and total anti-oxidation competence, and less MDA concentration in seminal plasma. The rams in group with supplementation of 150 mg·kg^-1had higher total anti-oxidation competence than the control group, while they had no significant difference in activities of Cu-ZnSOD, GPxs and MDA content with rams in the control group. The result of immunohistochemistry showed the positive signals were detected in pseudostratified columnar epithelium in epididymis caput and corpus, and simple columnar epithelium in epididymis cauda. The order of expression of Cu-ZnSOD protein in epididymis from rams in the treatment group was corpus〉caput〉cauda, while those in the control group was caput〉corpus〉cauda. The mean optical density of Cu-ZnSOD was higher in the group with supplementation of 50 mg·kg^-1or 100 mg·kg^-1nano-zinc in epididymis caput and corpus. The activity of Cu-ZnSOD in seminal plasma had a positive correlation with sperm concentration, sperm motility and membrane integrity, and a negative correlation with percentage of abnormal sperm.[Conclusion]The optimal supplementation of 50 mg·kg^-1or 100 mg·kg^-1nano-zinc in diet could improve semen quality and seminal plasma anti-oxidase activities, and the expression of Cu-ZnSOD in epididymis of rams. The activity of Cu-ZnSOD in seminal plasma could be regarded as index of evaluation of semen quality in sheep production. The further researchs need to be done in molecular mechanism of trace element zinc regulation on semen quality.