Mustofa Imam, Susilowati Suherni, Wurlina Wurlina, Hernawati Tatik, Oktanella Yudit
Department of Veterinary Reproduction, Faculty of Veterinary Medicine, Universitas Airlangga, Kampus C Unair, Mulyorejo, Surabaya, 60115, East Java, Indonesia.
Department of Veterinary Reproduction, Faculty of Veterinary Medicine, Brawijaya University, Jl. Veteran, Ketawanggede, Lowokwaru, Malang, 65145, Indonesia.
Heliyon. 2021 Mar 8;7(3):e06372. doi: 10.1016/j.heliyon.2021.e06372. eCollection 2021 Mar.
The study aimed to determine the addition of green tea extract (GTE) in extender on the quality and DNA mutation of post-thawed Kacang buck sperm. The sperm DNA mutation was observed on nicotinamide adenine dinucleotide hydride (NADH) dehydrogenase 1 (ND1) of mitochondrial Deoxyribonucleic Acid (mtDNA). A pool of 12 Kacang buck ejaculates was diluted in skim milk-egg yolk extender contained 0, 0.05, 0.10, and 0.15 mg of GTE/100 mL for T0, T1, T2, and T3 group, respectively. Each of the aliquot groups was packaged in 0.25 mL French mini straw contained 60 million alive sperm and froze according to the protocol. The ND1 mtDNA amplification of samples was carried out Polymerase Chain Reaction machine, followed by DNA sequencing using the Sanger method. Meanwhile, the phylogenetic tree was constructed using the neighbor-joining (NJ) method with MEGA 7.0 software. The results showed that the T2 group maintained the highest quality for Kacang buck post-thawed semen. There was the highest percentages of sperms viability, motility, intact plasma membrane (IPM), the lowest of malondialdehyde (MDA) concentration, sperm DNA fragmentation (SDF), the total and types of ND1 mtDNA mutation frequency. The phylogenetic tree analysis revealed that the clade of the T2 group was most closely related to the sequence reference. However, there was no correlation between the semen quality parameters (sperm viability, motility, IPM, MDA concentration, and SDF) with ND1 mtDNA mutation of post-thawed Kacang buck semen. It could be concluded that GTE was useful as an antioxidant for Kacang buck semen extender for frozen sperm.
本研究旨在确定在稀释液中添加绿茶提取物（GTE）对解冻后卡苍公鹿精子质量和DNA突变的影响。在线粒体脱氧核糖核酸（mtDNA）的烟酰胺腺嘌呤二核苷酸还原酶1（ND1）上观察精子DNA突变。将12份卡苍公鹿的射精样本混合，分别用含有0、0.05、0.10和0.15mg GTE/100mL的脱脂乳-蛋黄稀释液稀释，分别对应T0、T1、T2和T3组。每个等分样本组包装在含有6000万个活精子的0.25mL法式小细管中，并按照方案进行冷冻。使用聚合酶链反应仪对样本进行ND1 mtDNA扩增，随后采用桑格法进行DNA测序。同时，使用MEGA 7.0软件的邻接法（NJ）构建系统发育树。结果表明，T2组解冻后卡苍公鹿精液的质量保持最高。精子活力、运动性、完整质膜（IPM）的百分比最高，丙二醛（MDA）浓度、精子DNA碎片率（SDF）、ND1 mtDNA突变频率的总数和类型最低。系统发育树分析表明，T2组的进化枝与序列参考最密切相关。然而，解冻后卡苍公鹿精液的精液质量参数（精子活力、运动性、IPM、MDA浓度和SDF）与ND1 mtDNA突变之间没有相关性。可以得出结论，GTE作为冷冻精子的卡苍公鹿精液稀释液中的抗氧化剂是有用的。
