Unit for Reproductive Medicine, Clinic for Pigs and Small Ruminants, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
J Anim Sci. 2013 Oct;91(10):5018-25. doi: 10.2527/jas.2013-6287. Epub 2013 Aug 29.
Spermatozoa, especially those of the porcine species, are highly susceptible to in vitro chilling and ageing. Extenders are continuously developed to protect boar spermatozoa from chilling injury. New semen extenders and other modified preservation strategies require sensitive testing for essential sperm functions. The key process on the pathway of fertilization is capacitation. The aim of the present study was to examine whether the specific response to capacitating stimuli is sensitive enough to indicate different preservation capacities of extenders during hypothermic storage of boar spermatozoa. Semen was diluted in Beltsville Thawing Solution (BTS) and Androstar Plus and kept for 3 h at 22°C or stored at 17°C, 10°C, and 5°C. Semen was analyzed at 24 and 96 h of storage. Motility and membrane integrity remained at high levels, except for lower values when stored in BTS at 5°C. Washed subsamples were incubated in capacitating medium (Tyrode) and control medium and were assessed for intracellular calcium concentration and integrity of plasma membranes using a flow cytometer. On the basis of the loss of low-calcium live cells in a kinetic approach, the specific response to capacitation stimuli was determined. There was a higher loss of response in semen stored hypothermically in the standard extender BTS compared to Androstar Plus. Assessment of the extent of phospholipid disorder under capacitating and control conditions by use of merocyanine staining did not reveal any significant extender-related differences. A field insemination trial with 778 sows was performed to relate in vitro results to fertility. Fertility parameters did not differ in semen stored up to 48 h at 10°C in Androstar Plus compared to controls stored at 17°C in BTS. In conclusion, assessment of specific reactivity to capacitating stimuli appears to be a sensitive tool for detection of extender-dependent alterations in functionality of chilled boar spermatozoa.
精子，特别是猪种的精子，在体外冷却和老化过程中非常敏感。人们不断开发 extender 来保护公猪精子免受冷却损伤。新的精液 extender 和其他改良的保存策略需要对基本精子功能进行敏感测试。受精途径中的关键过程是获能。本研究的目的是检验特定的获能刺激反应是否足够敏感，以表明在猪精子低温储存期间不同 extender 的保存能力。精液在 Beltsville 解冻液（BTS）和 Androstar Plus 中稀释，并在 22°C 下保持 3 小时或在 17°C、10°C 和 5°C 下储存。在储存 24 和 96 小时时分析精液。除了在 5°C 下用 BTS 储存时值较低外，运动性和膜完整性仍保持在较高水平。洗涤后的亚样本在获能培养基（Tyrode）和对照培养基中孵育，并使用流式细胞仪评估细胞内钙浓度和质膜完整性。基于动力学方法中低钙活细胞的损失，确定了对获能刺激的特定反应。与 Androstar Plus 相比，在标准 extender BTS 中低温储存的精液中，反应丧失的程度更高。在获能和对照条件下通过使用甲烯蓝染色评估磷脂无序程度，没有发现任何与 extender 相关的显著差异。进行了一项 778 头母猪的现场授精试验，将体外结果与生育能力相关联。与在 BTS 中 17°C 储存的对照相比，在 Androstar Plus 中储存至 48 小时的精液在 10°C 时的生育参数没有差异。总之，评估对获能刺激的特定反应似乎是检测冷藏公猪精子功能中 extender 依赖性变化的敏感工具。
