Arif Ani Atul, Maulana Tulus, Kaiin Ekayanti Mulyawati, Purwantara Bambang, Arifiantini Raden Iis, Memili Erdogan
Reproductive Biology Study Program, Faculty of Veterinary Medicine, IPB University, Bogor, Indonesia.
Biotechnology Livestock Research Group, Research Center for Biotechnology, Indonesian Institute of Science, Bogor, Indonesia.
Vet World. 2020 Nov;13(11):2422-2428. doi: 10.14202/vetworld.2020.2422-2428. Epub 2020 Nov 13.
Indonesia has two National Artificial Insemination centers and 17 Regional Artificial Insemination Centers. The frozen semen production techniques differed between the centers, including the type of diluent and semen dilution technique. The aim of the research was to compare the quality of frozen Limousin bull semen diluted using different techniques.
Semen was collected from three sexually mature Limousin bulls using an artificial vagina. Immediately after collection, the semen was evaluated macroscopically and microscopically. Semen that had >70% motile sperm and <20% sperm abnormality was divided into three tubes and diluted with skim milk-egg yolk (SMEY) using three different dilution techniques: One-step dilution (100% SMEY with 8% glycerol) at room temperature ([RT] 20°C until 25°C) two-step dilution (50% SMEY without glycerol at RT, stored at 5°C; and 50% SMEY with 16% glycerol after 1 h stored at 5°C); and three-step dilution (50% SMEY without glycerol at RT, stored at 5°C; and 50% SMEY with 16% glycerol added twice at 1 h and 1.5 h after being stored at 5°C). The diluted semen was loaded into 0.25 mL mini straws, equilibrated, and frozen using a freezing machine. Sperm motility, viability, membranes, DNA integrity, and concentrations of malondialdehyde (MDA) and aspartate aminotransferase (AST) enzymes were evaluated after thawing.
The results showed that there were no significant differences in sperm motility and DNA integrity between dilutions (p0.05). However, sperm viability and membrane intactness of one-step dilutions were higher than those of three-step dilutions. The concentrations of MDA and AST enzymes of sperm in one-step dilutions were lower than those of three-step dilutions (p0.05).
It was concluded that the one-step-dilution technique was better than three-step dilution for cryopreservation of Limousin bull semen.
印度尼西亚有两个国家人工授精中心和17个地区人工授精中心。各中心的冷冻精液生产技术有所不同，包括稀释剂类型和精液稀释技术。本研究的目的是比较采用不同技术稀释的利木赞公牛冷冻精液的质量。
使用人工阴道从三头性成熟的利木赞公牛采集精液。采集后立即对精液进行宏观和微观评估。精子活力>70%且精子异常率<20%的精液被分成三管，并用脱脂乳-蛋黄（SMEY）采用三种不同的稀释技术进行稀释：室温（[RT]20°C至25°C）一步稀释（含8%甘油的100%SMEY）、两步稀释（室温下50%不含甘油的SMEY，在5°C保存；1小时后在5°C保存，再加入含16%甘油的50%SMEY）；以及三步稀释（室温下50%不含甘油的SMEY，在5°C保存；在5°C保存1小时和1.5小时后分两次加入含16%甘油的50%SMEY）。将稀释后的精液装入0.25 mL的迷你细管中，平衡后使用冷冻机冷冻。解冻后评估精子活力、存活率、膜完整性、DNA完整性以及丙二醛（MDA）和天冬氨酸转氨酶（AST）酶的浓度。
结果表明，各稀释组之间精子活力和DNA完整性无显著差异（p>0.05）。然而，一步稀释的精子存活率和膜完整性高于三步稀释。一步稀释的精子中MDA和AST酶的浓度低于三步稀释（p<0.05）。
得出结论，对于利木赞公牛精液的冷冻保存，一步稀释技术优于三步稀释。
