谷胱甘肽对绵羊精液低温保存效果的影响

doi:10.16431/j.cnki.1671-7236.2023.06.023

摘要/Abstract

摘要： 【目的】研究不同浓度谷胱甘肽（glutathione，GSH）对绵羊精液低温保存效果的影响。【方法】采集8只湖羊精液，用含0、2、4、8和16 μmol/L GSH的精液稀释液进行稀释，5 ℃保存。分别于保存24和120 h时，检测精子运动参数指标（精子活力、精子活率、路径速度（average path velocity，VAP）、曲线运动速度（curvilinear velocity，VCL）和直线运动速度（straight line velocity，VSL））、精子质量指标（精子质膜、顶体和DNA完整性、线粒体活性）以及精浆中总抗氧化能力（total antioxidant capacity，T-AOC）、超氧化物歧化酶（superoxide dismutase，SOD）活性和丙二醛（malondialdehyde，MDA）含量。【结果】绵羊精液低温保存24 h时，4 μmol/L GSH处理组精子活力、DNA完整性、线粒体活性及SOD活性均显著高于0和16 μmol/L GSH组（P<0.05）。低温保存120 h时，4 μmol/L GSH处理组的精子活力、精子活率、VAP、VCL、VSL、质膜完整率、顶体完整率、DNA完整率、线粒体活性及T-AOC水平均显著高于0和16 μmol/L GSH组（P<0.05）。此外，低温保存24和120 h时，4 μmol/L GSH处理组MDA含量显著低于8和16 μmol/L GSH组（P<0.05）。【结论】在精液稀释液中添加GSH可提高绵羊精子活力、质膜完整性、顶体完整性、线粒体活性、精浆抗氧化能力等，显著改善绵羊精液低温保存效果，其最适添加浓度为4 μmol/L。

关键词: 绵羊精液; 谷胱甘肽; 低温保存

Abstract: 【Objective】 The purpose of this study was to study the effect of glutathione (GSH) at different concentrations on cryopreservation of sheep semen.【Method】 The semen were collected from 8 Hu sheeps and diluted with 0, 2, 4, 8 and 16 μmol/L GSH and stored at 5 ℃.Sperm motility parameters including sperm viability, sperm motility, average path velocity (VAP), curvilinear velocity (VCL) and straight line velocity (VSL), sperm quality index including plasma membrane integrity, acrosome integrity, DNA integrity and mitochondrial activity, total antioxidant capacity(T-AOC), superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in the seminal plasma were measured at 24 and 120 h, respectively.【Result】 After 24 h cryoppreservation of sheep semen, sperm motility, DNA integrity, mitochondrial activity and SOD activity of 4 μmol/L GSH group were significantly higher than those of 0 and 16 μmol/L GSH groups (P<0.05).The sperm motility, sperm viability, VAP, VCL, VSL, plasma membrane integrity, acrosome integrity, DNA integrity, mitochondrial activity and T-AOC levels of 4 μmol/L GSH group were significantly higher than those of 0 and 16 μmol/L GSH groups after 120 h (P<0.05).In addition, MDA content in 4 μmol/L GSH group was significantly lower than that in 8 and 16 μmol/L GSH groups after 24 and 120 h cryopreservation (P<0.05).【Conclusion】 The addition of GSH in semen dilution could improve sperm motility, plasma membrane integrity, acrosome integrity, mitochondrial activity and seminal plasma antioxidant capacity, and significantly improve the cryoppreservation effect of sheep semen.The optimal concentration of GSH was 4 μmol/L.

Key words: sheep semen; glutathione; cryopreservation

中图分类号:

S826

刘锦钰, 张杨, 马旭梅, 周千, 周扬, 张春香, 石磊, 任有蛇. 谷胱甘肽对绵羊精液低温保存效果的影响[J]. 中国畜牧兽医, 2023, 50(6): 2388-2394.

LIU Jinyu, ZHANG Yang, MA Xumei, ZHOU Qian, ZHOU Yang, ZHANG Chunxiang, SHI Lei, REN Youshe. Effects of Glutathione on Cryopreservation of Sheep Semen[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2388-2394.

参考文献

[1] 孙福魁, 冉本康, 贾永宏, 等.影响猪精液常温保存效果的因素[J].畜牧兽医杂志, 2021, 40(4):99-101. SUN F K, RAN B K, JIA Y H, et al.Factors affecting the preservation of boar semen at room temperature[J].Journal of Animal Science and Veterinary, 2021, 40(4):99-101.(in Chinese)
[2] 赵尔军.绵羊冷冻精液人工输精方法及影响因素[J/OL].畜牧兽医科学(电子版), 2020, 7:32-33. ZHAO E J.Methods and influencing factors of artificial insemination with frozen semen of sheep[J/OL].Graziery Veterinary Sciences (Electronic Version), 2020, 7:32-33.(in Chinese)
[3] JANNATIFAR R, PARIVAR K, ROODBARI N H, et al.Effects of N-acetyl-cysteine supplementation on sperm quality, chromatin integrity and level of oxidative stress in infertile men[J].Reproductive Biology and Endocrinology, 2019, 17(1):24.
[4] JOHINKE D, DE GRAAF S P, BATHGATE R.Quercetin reduces the in vitro production of H₂O₂ during chilled storage of rabbit spermatozoa[J].Animal Reproduction Science, 2014, 151(3):208-219.
[5] UPADHYAY V R, RAMESH V, DEWRY R K, et al.Bimodal interplay of reactive oxygen and nitrogen species in physiology and pathophysiology of bovine sperm function[J].Theriogenology, 2022, 187:82-94.
[6] 郭良勇, 殷雨洋, 周俊波, 等.稀释液中添加维生素C对湖羊精液低温保存效果的影响[J].畜牧与兽医, 2021, 53(8):18-23. GUO L Y, YIN Y Y, ZHOU J B, et al.Effect of vitamin C addition to the diluent on fresh semen quality of Hu sheep[J].Animal Husbandry & Veterinary Medicine, 2021, 53(8):18-23.(in Chinese)
[7] 李方舟, 温飞, 杜烨青, 等.褪黑素对羊精液低温保存效果的影响[J].家畜生态学报, 2018, 39(2):50-53. LI F Z, WEN F, DU Y Q, et al.Effect of melatonin on sheep semen cryopreservation[J].Journal of Domestic Animal Ecology, 2018, 39(2):50-53.(in Chinese)
[8] 孙凤俊, 张丽, 薛建华, 等.谷胱甘肽对蒙贝利亚种公牛性控精液冻后效果的影响[J].中国奶牛, 2018, 8:24-27. SUN F J, ZHANG L, XUE J H, et al.The Effect of glutathione on the motility rate of frozen sexed semen from montbeliard bull[J].China Dairy Cattle, 2018, 8:24-27.(in Chinese)
[9] 邹家浩.还原型谷胱甘肽对奶山羊精液冷冻保存效果的研究[D].杨凌:西北农林科技大学, 2022. ZOU J H.Effects of reduced glutathione on dairy goat semen cryopreservation[D].Yangling:Northwest A&F University, 2022.(in Chinese)
[10] ZOU J, WEI L, LI D, et al.Effect of glutathione on sperm quality in Guanzhong dairy goat sperm during cryopreservation[J].Frontiers in Veterinary Science, 2021, 8:771440.
[11] 张柳明, 孙晓梅, 康言, 等.稀释倍数对湖羊精液低温保存效果的影响[J/OL].中国畜牧杂志, 1-7[2022-12-18]. ZHANG L M, SUN X M, KANG Y, et al.Effect of dilution factor on cryopreservation effect of lake sheep semen[J/OL].Chinese Journal of Animal Science, 1-7[2022-12-18].(in Chinese)
[12] 尤武林, 黄桂成, 王建伟.微囊化转基因骨髓间充质干细胞与成骨细胞共培养后的成骨分化潜能[J].中国组织工程研究, 2022, 26(24):3852-3857. YOU W L, HUANG G C, WANG J W.Osteogenic difffferentiation potential of microencapsulated transgenic bone marrow mesenchymal stem cells cocultured with osteoblasts[J].Chinese Journal of Tissue Engineering Research, 2022, 26(24):3852-3857.(in Chinese)
[13] 牛宏泽, 马政禹, 靳天宇, 等.蜂王浆对低温保存绵羊精子质量和糖转运蛋白GLUTs表达的影响[J].中国畜牧杂志, 2022, 58(5):142-146. NIU H Z, MA Z Y, JIN T Y, et al.Effects of royal jelly supplementation on sheep sperm quality during liquid storage at 5 ℃[J]. Chinese Journal of Animal Science, 2022, 58(5):142-146.(in Chinese)
[14] 孙迎迎.稀释液中添加槲皮素和白藜芦醇对绵羊精液低温保存效果的影响[D].太谷:山西农业大学, 2020. SUN Y Y.Effects of quercetin and resveratrol supplementation in diluent on sheep semen quality during low temperature liquid storage[D].Taigu:Shanxi Agricultural University, 2020.(in Chinese)
[15] ESTRADA E, RODRíGUEZ-GIL J E, RIVERA DEL ÁLAMO M M, et al.Effects of reduced glutathione on acrosin activity in frozen-thawed boar spermatozoa[J].Reproduction, Fertility, and Development, 2017, 29(2):283-293.
[16] GADEA J, MOLLA M, SELLES E, et al.Reduced glutathione content in human sperm is decreased after cryopreservation:Effect of the addition of reduced glutathione to the freezing and thawing extenders[J].Cryobiology, 2011, 62(1):40-46.
[17] MATA-CAMPUZANO M, ÁLVAREZ-RODRíGUEZ M, TAMAYO-CANUL J, et al.Refrigerated storage of ram sperm in presence of Trolox and GSH antioxidants:Effect of temperature, extender and storage time[J].Animal Reproduction Science, 2014, 151(3-4):137-147.
[18] 张文举, 余四九.GSH对体外保存精子品质和受精力影响的研究进展[J].中国兽医科学, 2018, 48(4):518-527. ZHANG W J, YU S J.A review on effects of GSH on the quality and fertilizing ability of in vitro cryopreserved sperm[J].Chinese Veterinary Science, 2018, 48(4):518-527.(in Chinese)
[19] CHATTERJEE S, DE LAMIRANDE E, GAGNON C.Cryopreservation alters membrane sulfhydryl status of bull spermatozoa:Protection by oxidized glutathione[J].Molecular Reproduction and Development, 2001, 60(4):498-506.
[20] OGATA K, IMAI A, SATO S, et al.Effects of reduced glutathione supplementation in semen freezing extender on frozen-thawed bull semen and in vitro fertilization[J].The Journal of Reproduction and Development, 2022, 68(1):53-61.
[21] 柳小斌, 郭建华, 罗兰, 等.谷胱甘肽对杜洛克猪精子冷冻保存效果的影响[J].黑龙江畜牧兽医, 2022.15:52-57. LIU X B, GUO J H, LUO L, et al.Effect of glutathione supplementation on cryopreservation of spermatozoa in Duroc pigs[J].Heilongjiang Animal Science and Veterinary, 2022, 15:52-57.(in Chinese)
[22] ZHANDI M, SEIFI-GHAJALO E, SHAKERI M, et al.Effect of glutathione supplementation to semen extender on post-thawed rooster sperm quality indices frozen after different equilibration times[J].Cryo Letters, 2020, 41(2):92-99.
[23] ANSARI M S, RAKHA B A, ANDRABI S M, et al.Glutathione-supplemented tris-citric acid extender improves the post-thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa[J].Reproductive Biology, 2012, 12(3):271-276.
[24] 刘琦, 苏泽智, 王乐, 等.谷胱甘肽对猪精液常温保存效果的影响[J].畜牧与兽医, 2015, 47(6):59-62. LIU Q, SU Z Z, WANG L, et al.Effects of GSH on quality of boar semen stored at normal temperature[J].Animal Husbandry & Veterinary Medicine, 2015, 47(6):59-62.(in Chinese)
[25] DEL PRETE C, STOUT T, MONTAGNARO S, et al.Combined addition of superoxide dismutase, catalase and glutathione peroxidase improves quality of cooled stored stallion semen[J].Animal Reproduction Science, 2019, 210:106195.
[26] ALLAI L, BENMOULA A, MARCIANE DA SILVA M, et al.Supplementation of ram semen extender to improve seminal quality and fertility rate[J].Animal Reproduction Science, 2018, 192:6-17.