摘要
为了优化湖羊精液超低温冷冻保存技术,改善超低温冷冻后的湖羊精液品质,试验采用假阴道法采集6只纯种湖羊精液作为供试精液,用A、B、C 3种不同成分的基础稀释液(基础稀释液A中含0.3 g/L葡萄糖、0.3 g/L柠檬酸钠、20%卵黄、1%100×青霉素和链霉素混合液;基础稀释液B中含18.252 g/L柠檬酸、5.044 g/L葡萄糖、36.342 g/L Tris、20%卵黄、1%100×青霉素和链霉素混合液;基础稀释液C中含有13.75 g/L柠檬酸、9.6 g/L果糖、27 g/L Tris、20%卵黄、1%100×青霉素和链霉素混合液)稀释后冷冻,解冻后测定精子活率、顶体完整率和质膜完整率,筛选最优基础稀释液;然后在最优基础稀释液中添加0,0.05,0.10,0.20 mg/mL的具有抗氧化功能的植物提取物冷冻保护剂(黄芪提取物、针叶樱桃提取物和竹叶提取物按质量比2∶1∶1混合而成)进行精液冷冻,解冻后测定精子活率、顶体完整率、质膜完整率及抗氧化指标[活性氧(ROS)和丙二醛(MDA)含量、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性],筛选最优植物提取物添加量;最后选择96只发情母羊,分为鲜精组、植物提取物组、未添加植物提取物组,每组32只,鲜精组用新鲜精液输精,植物提取物组用筛选的最优植物提取物添加量下的冻精输精,未添加保护剂组用未添加植物提取物的最优基础稀释液下的冻精输精,统计每组母羊受胎率。结果表明:基础稀释液C的精子活率、顶体完整率均显著高于A、B基础稀释液(P<0.05),质膜完整率介于基础稀释液A和B之间,但与基础稀释液A和B均差异不显著(P>0.05)。相较于0,0.05,0.20 mg/mL添加量,0.10 mg/mL植物提取物冷冻保护剂提高了精子活率、顶体完整率、质膜完整率以及SOD和GSH-Px(除低于0.20 mg/mL添加量外)活性,降低了ROS和MDA含量。植物提取物组的母羊受胎率(51.9%)显著高于未添加植物提取物组(43.3%)。说明选用基础冷冻稀释液C,同时加入0.10 mg/mL植物提取物冷冻保护剂可以改善湖羊冷冻后精液品质,提高输精后母羊的受胎率,可以达到生产实践要求的标准。
The aim of the study was to optimize the ultra-low temperature freezing technology technology of Hu sheep semen and improve the quality of Hu sheep semen after the ultra-low temperature freezing. The semen of 6 purebred Hu sheep were collected by pseudovaginal method. Firstly, the semen was diluted with basic diluents of A, B and C basic diluent A contains 0.3 g/L glucose, 0.3 g/L sodium citrate, 20% egg yolk and 1% 100 × penicillin streptomycin mixture;basic diluent B contains 18.252 g/L citric acid, 5.044 g/L glucose, 36.342 g/L Tris, 20% egg yolk and 1% 100 × penicillin streptomycin mixture;basic diluent C contains 13.75 g/L citric acid, 9.6 g/L fructose, 27 g/L Tris, 20% egg yolk and 1% 100 × penicillin streptomycin mixture, and then all samples were frozen. After thawing, the sperm viability, acrosome integrity and plasma membrane integrity were measured to screen the optimal basic diluent. Then, 0, 0.05, 0.10, 0.20 mg/mL of plant extract cryoprotectant with antioxidant function(Astragalus extract, acerola cherry extract and bamboo leaf extract mixed at a mass ratio of 2∶1∶1) were added to the optimal basic dilution for semen freezing. After thawing, the sperm activity, acrosome integrity, plasma membrane integrity and antioxidant index(reactive oxygen species [ROS], superoxide dismutase [SOD], glutathione peroxidase [GSH-Px], malondialdehyde [MDA]) were determined to screen the optimal amount of plant extract. Finally, 96 estrous ewes were selected and divided into fresh semen group, plant extract group and no protective agent group with 32 ewes in each group. The fresh semen group was inseminated with fresh semen, the plant extract group was inseminated with frozen semen under the optimal amount of plant extract selected in 2.2.2, and the no protective agent group was inseminated with frozen semen under the optimal basic dilution without plant extract. The fertilization rate of each group was counted. The results showed that the sperm viability and acrosome integrity rate in the basic diluent C group were significantly higher than those in the basic diluents A and B(P<0.05), and the plasma membrane integrity rate was between basic diluents A and B, but there was no significant difference with basic diluents A and B(P>0.05). Compared with 0, 0.05, and 0.20 mg/mL additions, the addition of 0.10 mg/mL plant extract cryoprotectant increased the sperm viability, the acrosome integrity, the plasma membrane integrity, the activities of SOD and GSH-Px(except for those below 0.20 mg/mL addition), and decreased the contents of ROS and MDA. The conception rate of ewes in the plant extract group(51.9%) was significantly higher than that in the non-protective group(43.3%). In conclusion, adding 0.1 mg/mL cryoprotectant into basic freezing dilution C could improve the quality of Hu sheep semen after freezing and improve the conception rate of ewes after sperm implantation, which could meet the requirements of production practice.
作者
彭彩娥
高慧
张云超
王争光
PENG Caie;GAO Hui;ZHANG Yunchao;WANG Zhengguang(Huzhou Agricultural Science and Technology Development Center,Huzhou 313000,China;Zhejiang Animal Husbandry Technology Extension and Breeding Livestock&Poultry Testing Station,Hangzhou 310021,China;College of Animal Science,Zhejiang University,Hangzhou 310058,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2022年第6期58-62,共5页
Heilongjiang Animal Science And veterinary Medicine
基金
浙江省农业重大技术协同推广计划项目(2021XTTGXM05-01)
湖州市科技局公益性应用研究项目(2019GZ14)
湖州市科技局乡村振兴专项(2019ZD2026)
湖州市农业科技创新团队项目(2019HN01)。
关键词
湖羊
精液
基础稀释液
植物提取物
复合抗氧化剂
人工授精
受胎率
Hu sheep
semen
basic diluent
plant extracts
composite antioxidant
artificial insemination
conception rate
