Veterinary Teaching Hospital, Laboratory of Equine Reproduction, Faculty of Veterinary Medicine, University of Extremadura, Cáceres, Spain.
Theriogenology. 2012 Apr 15;77(7):1280-9. doi: 10.1016/j.theriogenology.2011.10.033. Epub 2012 Feb 10.
Glycerol is, to date, the most widely used cryoprotectant to freeze stallion spermatozoa at concentrations between 2% and 5%. Cryoprotectant toxicity has been claimed to be the single most limiting factor for the success of cryopreservation. In order to evaluate the toxic effects of the concentrations of glycerol used in practice, stallion spermatozoa were incubated in Biggers Whitten and Whittingham (BWW) media supplemented with 0%, 0.5%, 1.5%, 2.5%, 3.5%, and 5% glycerol. In two additional experiments, a hyposmotic (75 mOsm/kg) and a hyperosmotic (900 mOsm/kg) control media were included. Sperm parameters evaluated included cell volume, membrane integrity, lipid peroxidation, caspase 3, 7, and 8 activation, mitochondrial membrane potential, and integrity of the cytoskeleton. Glycerol exerted toxicity at concentrations ≥ 3.5% and the maximal toxicity was observed at 5%. The actin cytoskeleton was especially sensitive to glycerol presence, inducing rapid F actin depolymerization at concentrations over 1.5%. The sperm membrane and the mitochondria were other structures affected. The toxicity of glycerol is apparently related to osmotic and nonosmotic effects. In view of our results the concentration of glycerol in the freezing media for stallion spermatozoa should not surpass 2.5%.
甘油是迄今为止最广泛用于冷冻马精子的冷冻保护剂，浓度在 2%至 5%之间。有人声称，冷冻保护剂的毒性是冷冻保存成功的唯一最限制因素。为了评估在实践中使用的甘油浓度的毒性作用，将精子在含有 0%、0.5%、1.5%、2.5%、3.5%和 5%甘油的 Biggers Whitten 和 Whittingham (BWW) 培养基中孵育。在另外两个实验中，还包括了低渗（75 mOsm/kg）和高渗（900 mOsm/kg）对照培养基。评估的精子参数包括细胞体积、膜完整性、脂质过氧化、半胱天冬酶 3、7 和 8 的激活、线粒体膜电位和细胞骨架完整性。甘油在浓度≥3.5%时表现出毒性，在 5%时观察到最大毒性。肌动蛋白细胞骨架对甘油的存在特别敏感，在浓度超过 1.5%时会迅速引起 F-肌动蛋白解聚。精子膜和线粒体是受影响的其他结构。甘油的毒性显然与渗透和非渗透作用有关。鉴于我们的结果，马精子冷冻液中的甘油浓度不应超过 2.5%。
