Laboratory of Animal Germplasm Conservation-LCGA, Universidade Federal Rural do Semi-Árido-UFERSA, Mossoró, RN, Brazil.
Cryobiology. 2013 Aug;67(1):50-5. doi: 10.1016/j.cryobiol.2013.04.009. Epub 2013 May 9.
The objective of this study was to verify the effect of different freezing curves, straw sizes, and thawing rates on the cryopreservation of collared peccary semen. Twelve ejaculates were obtained from captive adult males by electroejaculation, and evaluated for sperm motility, kinetic rating, viability, morphology, and functional membrane integrity. The ejaculates were diluted in a coconut water extender (ACP-116c) with egg yolk and glycerol, packaged into 0.25 mL or 0.50 mL plastic straws and cryopreserved in liquid nitrogen following a slow (-10 °C/min) or a fast (-40 °C/min) freezing curve. After one week, samples were thawed at 37 °C/1 min or 70 °C/8s and evaluated as reported for fresh semen, and also for kinematic parameters (computerized analysis). A significant decrease in sperm motility and kinetic rating was observed after glycerol addition at 5 °C and also after thawing for all the treatments (P<0.05). Regarding post-thaw semen variables, no differences were verified between freezing curves when the same straw size and thawing rate were taken as reference (P>0.05). In general, values for sperm characteristics found after thawing at 37 °C were better preserved than at 70 °C (P<0.05), both in the use of 0.25 mL or 0.50 mL straws, which were similar for semen packaging (P>0.05). The evaluation of the kinematic parameters of sperm motility confirmed these results at values varying from 20% to 30% motile sperm for the samples tha wed at 37 °C, and values fewer than 12% motile sperm for samples thawed at 70 °C (P<0.05). In conclusion, we recommend the use of a fast freezing curve that reduces the time spent on the cryopreservation of collared peccary semen, which could be packaged both in 0.25 mL or 0.50 mL straws, but the thawing should be conducted at 37 °C/1 min.
本研究的目的是验证不同的冷冻曲线、 straw 大小和解冻速率对卷毛猪精液冷冻保存的影响。通过电刺激法从圈养成年雄性中获得 12 份精液，评估精子活力、运动评分、活力、形态和功能膜完整性。将精液用椰子水稀释剂（ACP-116c）与蛋黄和甘油混合，装入 0.25 mL 或 0.50 mL 塑料 straw 中，按照慢（-10°C/min）或快（-40°C/min）冷冻曲线在液氮中冷冻保存。一周后，样品在 37°C/1 min 或 70°C/8s 下解冻，并按照新鲜精液的报告进行评估，同时也评估运动参数（计算机分析）。在 5°C 下添加甘油后，以及所有处理的解冻后，精子活力和运动评分都显著下降（P<0.05）。关于解冻后精液的变量，当参考相同的 straw 大小和解冻速率时，冷冻曲线之间没有差异（P>0.05）。一般来说，与在 70°C 下解冻相比，在 37°C 下解冻时，解冻后精子特征的数值保存得更好（P<0.05），无论是使用 0.25 mL 还是 0.50 mL straw，包装精液的情况都相似（P>0.05）。精子运动的运动学参数评估证实了这些结果，在 37°C 下解冻的样本中，有 20%至 30%的精子有活力，而在 70°C 下解冻的样本中，有活力的精子少于 12%（P<0.05）。总之，我们建议使用快速冷冻曲线来减少卷毛猪精液冷冻保存的时间，可以使用 0.25 mL 或 0.50 mL straw 包装，但解冻应该在 37°C/1 min 进行。
