Ministry of Agriculture and Rural Affairs, Lalahan Livestock Central Research Institute, Lalahan, Ankara, Turkey.
Cryobiology. 2010 Aug;61(1):89-93. doi: 10.1016/j.cryobiol.2010.05.005. Epub 2010 May 31.
The aim of the present study was to determine the effects of different doses of raffinose and methionine on post-thawed semen quality, lipid peroxidation and antioxidant enzyme activities of Angora buck (Capra hircus ancryrensis) sperm following cryopreservation. Ejaculates collected from three Angora bucks were evaluated and pooled at 37 degrees C. Semen samples, which were diluted with a Tris-based extender containing the additives raffinose (2.5, 5, 10mM) and methionine (2.5, 5, 10mM) and an extender containing no antioxidants (control), were cooled to 5 degrees C and frozen in 0.25 ml French straws. Frozen straws were thawed individually at 37 degrees C for 20s in a water bath for evaluation. The freezing extender supplemented with 2.5 and 5mM methionine led to higher percentages of CASA motility (63.6+/-7.0; 63.4+/-3.1%, respectively), in comparison to the controls (P<0.01) following the freeze-thawing process. The addition of antioxidants did not provide any significant effect on the percentages of post-thaw subjective and CASA progressive motilities as well as sperm motion characteristics (VSL and VCL), compared to the control groups (P>0.05). The freezing extender with raffinose (5 and 10mM) and methionine at three different doses (2.5, 5 and 10mM) led to lower percentages of acrosome abnormalities, in comparison to the controls (P<0.001). In the comet test, raffinose (5 and 10mM) and methionine (10mM) gave scores lower than those of the controls, and thereby reduced DNA damage (P<0.05). Malondialdehyde formation was found to be lower (1.8+/-0.1 nmol/L) in the group of 5mM raffinose, compared to the controls following the freeze-thawing process (P<0.01). The additives did not show any effectiveness on the maintenance of SOD, GSH-PX and GSH activities, when compared to the controls (P>0.05). In conclusion, methionine and raffinose play a cryoprotective role against sperm CASA motility, acrosome abnormality and DNA damage. Raffinose 5mM exhibited antioxidative properties, decreasing MDA levels. Further studies are required to obtain more concrete results on the characterization of microscopic parameters and antioxidant activities in cryopreserved goat sperm with different additives.
本研究旨在确定棉子糖和蛋氨酸不同剂量对冷冻解冻后安哥拉山羊精子活力、脂质过氧化和抗氧化酶活性的影响。在 37°C 下评估并混合来自三只安哥拉山羊的精液。将精液样品用含有添加剂棉子糖（2.5、5、10mM）和蛋氨酸（2.5、5、10mM）的 Tris 基础稀释液和不含抗氧化剂的稀释液（对照）冷却至 5°C 并在 0.25ml 法国 straws 中冷冻。将冷冻 straws 在 37°C 的水浴中单独解冻 20s 进行评估。与对照组相比（P<0.01），添加 2.5 和 5mM 蛋氨酸的冷冻稀释液可提高解冻后 CASA 活力的百分比（分别为 63.6+/-7.0%和 63.4+/-3.1%）。与对照组相比（P>0.05），添加抗氧化剂对解冻后主观和 CASA 渐进运动以及精子运动特征（VSL 和 VCL）的百分比没有任何显著影响。与对照组相比（P<0.001），添加 5mM 和 10mM 棉子糖和三种不同剂量（2.5、5 和 10mM）的蛋氨酸的冷冻稀释液可降低顶体异常的百分比。在彗星试验中，棉子糖（5 和 10mM）和蛋氨酸（10mM）的评分低于对照组，从而降低了 DNA 损伤（P<0.05）。与冷冻解冻过程后的对照组相比（P<0.01），棉子糖 5mM 组的丙二醛形成量较低（1.8+/-0.1 nmol/L）。与对照组相比（P>0.05），添加剂对维持 SOD、GSH-PX 和 GSH 活性没有任何效果。综上所述，蛋氨酸和棉子糖对精子 CASA 活力、顶体异常和 DNA 损伤具有保护作用。棉子糖 5mM 表现出抗氧化特性，降低 MDA 水平。需要进一步研究以获得关于不同添加剂冷冻山羊精子微观参数和抗氧化活性特征的更具体结果。
