Department of Organismic Biology, Faculty of Natural Sciences, University of Salzburg, Salzburg, Austria.
Theriogenology. 2010 Sep 15;74(5):724-32. doi: 10.1016/j.theriogenology.2010.03.025. Epub 2010 May 26.
Motility and cryopreservation of testicular sperm of European common frog, Rana temporaria were investigated. Collected testicular spermatozoa were immotile in solutions of high osmolalities: 300 mmol/l sucrose and motility inhibiting saline solution-MIS. Full sperm motility could be activated in distilled water or in a solution of 50 mmol/l NaCl, = 90 mosmol/kg, with 75-90% motility and 14-16 microm s(-1) swimming velocity. Spermatozoa activated in distilled water and kept at room temperature ceased the motility within a period of 1 h. But when they were kept at 4 degrees C, no significant decrease in sperm motility and velocity occurred over a period of 1 h. Incubation of testicular sperm diluted 1:2 with MIS containing 10% DMSO, 5% glycerol, 10% methanol, or 10% propandiol for a period of 40 min at 4 degrees C showed that propandiol was the most toxic cryoprotectant for spermatozoa of European common frog R. temporaria. However, methanol was not toxic to spermatozoa during the 40 min incubation period, it failed to protect spermatozoa during the freezing and thawing process. DMSO and glycerol were useful penetrating cryoprotectants that interacted with sperm diluents in cryodiluent efficacy. In combination with the sucrose diluent, DMSO was a better cryoprotectant than glycerol, while in combination with MIS, DMSO and glycerol were similarly useful. Sperm was frozen at two freezing levels above the surface of liquid nitrogen. Sperm frozen 5 cm above the surface of liquid nitrogen resulted in immotile and non-viable spermatozoa. However, sperm frozen at 10 cm above the surface of liquid nitrogen showed 40-45% viability and 30-35% motility, compared to the untreated freshly collected testicular sperm. Addition of hen egg yolk had no positive effect on the post-thaw sperm motility, viability and hatching rate when added to sucrose cryodiluents. However, addition of 5% egg yolk to the MIS containing 5% glycerol and 2.5% sucrose significantly improved the hatching rate than all other treatments. Therefore, we conclude that, MIS and 300 mmol/l sucrose are suitable diluents for immotile storage of testicular semen. For cryopreservation, dilution to a final concentration of 5-6 x 10(6)/ml in MIS with 5% glycerol, 2.5% sucrose and 5% egg yolk, frozen in liquid nitrogen vapour at 10 cm above its surface, and thawed at 22 degrees C for 40 s is a useful cryopreservation protocol for R. temporaria sperm. Further research is needed to determine the motility parameters and cryopreservation of spermatic urine of R. temporaria.
研究了欧洲普通蛙（Rana temporaria）睾丸精子的运动性和冷冻保存。收集的睾丸精子在高渗透压溶液中无运动性：300mmol/l 蔗糖和运动抑制盐溶液-MIS。在蒸馏水或 50mmol/l NaCl 溶液中，渗透压为 90mosmol/kg，可完全激活精子的运动性，运动性为 75-90%，游动速度为 14-16μm/s。在蒸馏水中激活的精子在室温下放置 1 小时内停止运动。但当它们在 4°C 下保存时，在 1 小时内精子运动性和速度没有明显下降。将睾丸精子用 MIS 稀释 1:2，MIS 中含有 10%DMSO、5%甘油、10%甲醇或 10%丙二醇，在 4°C 下孵育 40 分钟，结果表明丙二醇是欧洲普通蛙 R. temporaria 精子最有毒的冷冻保护剂。然而，甲醇在 40 分钟的孵育过程中对精子没有毒性，但在冷冻和解冻过程中不能保护精子。DMSO 和甘油是有用的渗透冷冻保护剂，与冷冻保护剂中的精子稀释剂相互作用，以提高冷冻保护剂的效能。与蔗糖稀释剂结合时，DMSO 比甘油更适合作为冷冻保护剂，而与 MIS 结合时，DMSO 和甘油同样有效。精子在高于液氮表面的两个冷冻水平上冷冻。精子在距离液氮表面 5 厘米处冷冻导致精子无运动性和无活力。然而，与未经处理的新鲜收集的睾丸精子相比，在距离液氮表面 10 厘米处冷冻的精子显示出 40-45%的活力和 30-35%的运动性。添加鸡蛋黄对添加到蔗糖冷冻保护剂中的精子解冻后运动性、活力和孵化率没有积极影响。然而，与所有其他处理相比，在含有 5%甘油和 2.5%蔗糖的 MIS 中添加 5%鸡蛋黄可显著提高孵化率。因此，我们得出结论，MIS 和 300mmol/l 蔗糖是适合储存睾丸精液的无活力储存的合适稀释剂。对于冷冻保存，在含有 5%甘油、2.5%蔗糖和 5%鸡蛋黄的 MIS 中最终浓度稀释至 5-6×10(6)/ml，在液氮蒸气中于 10cm 处冷冻，在 22°C 下解冻 40s，这是一种有用的 R. temporaria 精子冷冻保存方案。需要进一步研究确定 R. temporaria 精子的运动参数和精液的冷冻保存。
