Szász F, Sirivaidyapong S, Cheng F P, Voorhout W F, Marks A, Colenbrander B, Solti And L, Gadella B M
Graduate School of Animal Health, Department of Herd Health and Reproduction, Faculty of Veterinary Medicine, University of Utrecht, Utrecht, The Netherlands.
Mol Reprod Dev. 2000 Mar;55(3):289-98. doi: 10.1002/(SICI)1098-2795(200003)55:3<289::AID-MRD7>3.0.CO;2-K.
The sensitivity of dog sperm cells for extracellular Ca(2+)/Ca(2+)-ionophore challenge was compared to the detrimental effects of an optimized freeze/thawing protocol. Three sperm-rich fractions of ejaculates from 9 dogs were obtained, and one aliquot of each ejaculate was washed in a modified Tyrode's medium (HBT containing 0.1 mM Ca(2+)), without (control sample) and with 2.5 microM Ca(2+)-ionophore (induced sample) and incubated for 60 min at 38 degrees C in humidified atmosphere. Another aliquot from the same semen fractions was diluted, washed in a Tris buffer, and packed into 0.5-ml straws with a Tris buffer containing 7.5 vol % glycerol. The samples were stored for 1 week in liquid nitrogen after a computer-driven three-step freeze protocol and subsequently thawed for 50 sec in a 37 degrees C water bath and reconstituted into HBT. The acrosome integrity was determined using fluorescein-conjugated peanut agglutinin (PNA-FITC) as an acrosomal marker, while the vitality of the sperm cells was simultaneously assessed with the membrane impermeable DNA supravital stain ethidium homodimer 1 (EthD-1) using fluorescence microscopy and flow cytometry. The motility of frozen/thawed sperm samples was evaluated by microscopic as well as computerized motility analyses. Remarkably, the percentage sperm cells that underwent acrosome reactions induced by Ca(2+)-ionophore correlated very positively (r = 0.93) with the amount of acrosome damage observed in cryopreserved sperm samples. Furthermore, the degree of cellular damage induced by Ca(2+)-ionophore treatment correlated very negatively (r = -0.99) with the relative amount of sperm cells that remained motile after cryopreservation. Such clear correlations between Ca(2+)-ionophore induced acrosome reaction and motility parameters for frozen/thawed dog sperm cells were not found, suggesting that the generation of acrosome leakage and sperm immotility are two independent detrimental processes occurring during cryopreservation. From these results it can be concluded that Ca(2+)-ionophore treatment followed by simultaneous determination PNA-FITC and EthD-1 staining can be used to predict the cryopreservability of ejaculates from individual dogs used as donors.
将犬精子细胞对细胞外Ca(2+)/Ca(2+)离子载体刺激的敏感性与优化的冻融方案的有害影响进行了比较。从9只犬的射精中获得了三个富含精子的部分，每个射精的一份等分试样在改良的Tyrode培养基（含0.1 mM Ca(2+)的HBT）中洗涤，一份不添加（对照样品），另一份添加2.5 microM Ca(2+)离子载体（诱导样品），并在38℃、湿润气氛中孵育60分钟。从相同精液部分取另一份等分试样进行稀释，在Tris缓冲液中洗涤，然后装入含有7.5体积%甘油的Tris缓冲液的0.5毫升细管中。样品在计算机驱动的三步冷冻方案后在液氮中保存1周，随后在37℃水浴中解冻50秒，并重新悬浮于HBT中。使用荧光素偶联的花生凝集素（PNA-FITC）作为顶体标记物测定顶体完整性，同时使用膜不透性DNA超活染色剂乙锭同二聚体1（EthD-1）通过荧光显微镜和流式细胞术评估精子细胞的活力。通过显微镜以及计算机化运动分析评估冷冻/解冻精子样品的运动性。值得注意的是，由Ca(2+)离子载体诱导发生顶体反应的精子细胞百分比与冷冻保存精子样品中观察到的顶体损伤量呈非常强的正相关（r = 0.93）。此外，Ca(2+)离子载体处理诱导的细胞损伤程度与冷冻保存后仍具有运动能力的精子细胞相对量呈非常强的负相关（r = -0.99）。在冷冻/解冻的犬精子细胞中未发现Ca(2+)离子载体诱导的顶体反应与运动参数之间存在如此明显的相关性，这表明顶体泄漏的产生和精子运动能力丧失是冷冻保存期间发生的两个独立的有害过程。从这些结果可以得出结论，Ca(2+)离子载体处理后同时测定PNA-FITC和EthD-1染色可用于预测用作供体的个体犬射精的冷冻保存能力。
