Laboratory of Animal Reproduction, Veterinary Medicine Faculty, Universidad Nacional Mayor de San Marcos, Lima, Perú.
Theriogenology. 2013 Mar 15;79(5):842-6. doi: 10.1016/j.theriogenology.2012.12.012. Epub 2013 Feb 1.
The main objective was to study the effects, on sperm function, of the addition of two superoxide dismutase (SOD) analogues (Tempo and Tempol) to alpaca semen extender for cryopreservation. Twelve alpaca semen samples were collected using an artificial vagina and then diluted at a 1:3 ratio in an extender based on skim milk, egg yolk, and fructose. Each semen sample was divided into three equal parts to form the following groups: control, Tempo (1 mM), and Tempol (1 mM). Groups were cooled to 5 °C in 90 minutes (-1 °C in 3 minutes); when samples reached approximately 10 °C, SOD analogues were added to the respective groups. At 5 °C, ethylene glycol (final concentration, 0.1 M) was added to each group. After 30 minutes at 5 °C, samples were loaded in 0.25 mL plastic straws, placed in liquid nitrogen vapor for 15 minutes, and then plunged. Percentages of sperm motility, functional sperm membrane integrity, and viable sperm with intact acrosomes were evaluated before and after freeze-thaw using visual analysis, the hypoosmotic swelling test, and the double-stain trypan blue/giemsa technique, respectively. The Terminal deoxymucleotidyl transferase dUTP Nick End Labeling assay was performed for evaluation of sperm DNA fragmentation of frozen-thawed sperm. Sperm motility was higher (P < 0.05) in the Tempol and Tempo groups than in the control group (mean, 22.1%, 19.7%, and 11.2%, respectively), with similar results for functional sperm membrane integrity. Additionally, DNA fragmentation was lower (P < 0.05) in the Tempol group (16.7%) than in the control group (38.8%). Viable sperm with intact acrosomes were not affected by the use of SOD analogues. There was a negative correlation (r = -0.58) between DNA fragmentation of alpaca sperm and sperm motility after freeze-thawing, but DNA damage was neither related to functional membrane integrity nor viable sperm with intact acrosomes. We concluded that DNA fragmentation and loss of motility during cryopreservation of alpaca sperm could be partially prevented by supplementation of the semen extender with 1 mM Tempo or Tempol.
本研究的主要目的是研究在 alpaca 精液冷冻保存中添加两种超氧化物歧化酶（SOD）类似物（Tempo 和 Tempol）对精子功能的影响。使用人工阴道采集 12 份 alpaca 精液样本，然后在基于脱脂奶、蛋黄和果糖的稀释液中以 1:3 的比例稀释。每个精液样本分为 3 等份，形成以下组：对照组、Tempo（1mM）和 Tempol（1mM）。各组在 90 分钟内冷却至 5°C（3 分钟内降温 1°C）；当样品达到约 10°C 时，将 SOD 类似物添加到各自的组中。在 5°C 时，向各组中加入乙二醇（终浓度为 0.1M）。在 5°C 下孵育 30 分钟后，将样品装入 0.25ml 塑料吸管中，在液氮蒸气中放置 15 分钟，然后骤冷。使用目测法、低渗肿胀试验和双染台盼蓝/吉姆萨技术分别评估冷冻-解冻前后精子运动率、功能精子膜完整性和具有完整顶体的存活精子的百分比。末端脱氧核苷酸转移酶 dUTP 缺口末端标记法用于评估冷冻-解冻后精子 DNA 片段化。与对照组相比（分别为 22.1%、19.7%和 11.2%），Tempo 和 Tempol 组的精子运动率更高（P<0.05），功能精子膜完整性也有相似的结果。此外，Tempol 组的 DNA 片段化（16.7%）低于对照组（38.8%）（P<0.05）。顶体完整的存活精子不受 SOD 类似物使用的影响。alpaca 精子冷冻解冻后的 DNA 片段化与精子运动率呈负相关（r=-0.58），但 DNA 损伤与功能膜完整性或顶体完整的存活精子均无关。我们得出结论，在 alpaca 精液的冷冻保存中，通过在精液稀释液中添加 1mM Tempo 或 Tempol，可以部分防止 DNA 片段化和运动能力的丧失。
