Kaurova Svetlana A, Uteshev Victor K, Gapeyev Andrew B, Shishova Natalia V, Gakhova Edith N, Browne Robert K, Kramarova Ludmila I
Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia.
Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia; and Moscow Region State University, Mytishchi, Moscow Region, 141014, Russia.
Reprod Fertil Dev. 2021 May;33:588-595. doi: 10.1071/RD20336.
Cryopreserved spermatozoa offers a reliable, efficient and cost-effective means to perpetuate the genetic variation of endangered amphibian species in concert with conservation breeding programs. Here we describe successful cryopreservation of testicular spermatozoa of the common frog Rana temporaria , preliminarily stored in the carcasses of decapitated animals at +4°C for 0, 1 and 4 days. The motility, membrane integrity and fertilisation capability of fresh testicular spermatozoa treated with cryoprotective medium supplemented with 15% dimethylformamide (DMF) or 15% dimethylsulfoxide (DMSO) were examined. DMSO had a significantly greater toxic effect on fresh frog spermatozoa than DMF. Low levels of DNA fragmentation were seen in spermatozoa stored in the testis for different times and then treated with DMF (mean (±s.e.m.) 8.2±0.7% and 18.2±1.8% after 0 and 4 days storage respectively). After 1 day of storage in frog carcasses, the quality of spermatozoa cryopreserved with DMF was not significantly different from that of control spermatozoa (0 days of storage). After 4 days of storage, the quality of frozen-thawed spermatozoa was significantly lower in the DMF-treated than control group: 35% of the spermatozoa cryopreserved with DMF retained motility, 25% maintained the ability to fertilise fresh oocytes and 80% of fertilised oocytes survived to hatch.
冷冻保存的精子提供了一种可靠、高效且具有成本效益的方法，可与保护育种计划协同维持濒危两栖动物物种的遗传变异。在此，我们描述了对普通青蛙（林蛙）睾丸精子的成功冷冻保存，这些精子预先在斩首动物的尸体中于+4°C保存0、1和4天。检测了用补充有15%二甲基甲酰胺（DMF）或15%二甲基亚砜（DMSO）的冷冻保护培养基处理的新鲜睾丸精子的活力、膜完整性和受精能力。DMSO对新鲜青蛙精子的毒性作用明显大于DMF。在不同时间储存在睾丸中然后用DMF处理的精子中观察到低水平的DNA片段化（分别在储存0天和4天后，平均值（±标准误）为8.2±0.7%和18.2±1.8%）。在青蛙尸体中储存1天后，用DMF冷冻保存的精子质量与对照精子（储存0天）没有显著差异。储存4天后，DMF处理组的冻融精子质量明显低于对照组：用DMF冷冻保存的精子中35%保持活力，25%保持使新鲜卵母细胞受精的能力，80%的受精卵存活至孵化。
