Upton Rose, Clulow Simon, Colyvas Kim, Mahony Michael, Clulow John
Conservation Biology Research Group, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW, Australia.
FAUNA Research Alliance, PO Box 5092, Kahibah, NSW, Australia.
Reproduction. 2023 Apr 26;165(6):583-592. doi: 10.1530/REP-22-0486. Print 2023 Jun 1.
Sperm cryopreservation has been recognised as a tool for preventing loss of genetic diversity in amphibians; however, the combined effect of penetrative and non-penetrative cryoprotectants in cryodiluents is poorly understood. We demonstrate a clear benefit of using low concentrations of non-penetrative cryoprotectants when cryopreserving sperm of Australian tree frogs.
Sperm cryopreservation protocols have been developed for an increasing number of amphibian species since the recognition of a global amphibian decline. Yet, the development of these protocols has neglected to elucidate the combined effect of the penetrative(PCP) and non-penetrative cryoprotectant (NPCP) on the recovery of live, motile sperm. The two-factor hypothesis of cryoinjury recognises a trade-off between cooling cells slowly enough to allow osmotic dehydration and therefore avoid intracellular ice formation, but fast enough to minimise effects from increasing extracellular osmolality as the frozen fraction of the media increases during freezing. We tested the effect of two concentrations of a PCP (10 and 15% v/v dimethyl sulfoxide (Me2SO)) and two concentrations of an NPCP (1 and 10% w/v sucrose) in various combinations on the sperm of six pelodryadid frogs. In all species, 15% v/v Me2SO with 1% w/v sucrose provided superior post-thaw recovery with high proportions of forward progressive motility, live cells and intact acrosomes (typically >50% for each). Theoretically, it has been suggested that increased NPCP concentration should improve cell survival by increasing the rate and extent of cell dehydration. We suggest, however, that the elevated osmolality in the unfrozen water fraction when 10% sucrose is used may be causing damage to cells via excessive cell shrinkage and solute effects as proposed in the two-factor hypothesis of cryoinjury. We showed this response in sperm across a range of frog species, providing compelling evidence for this hypothesis. We suggest protocol development using the PCP/NPCP ratios demonstrated in our study will be broadly applicable to many amphibian species.
精子冷冻保存已被视为防止两栖动物遗传多样性丧失的一种手段；然而，冷冻稀释液中渗透性和非渗透性冷冻保护剂的联合作用却鲜为人知。我们证明了在冷冻保存澳大利亚树蛙精子时使用低浓度非渗透性冷冻保护剂具有明显益处。
自全球两栖动物数量减少被认识以来，针对越来越多的两栖动物物种已制定了精子冷冻保存方案。然而，这些方案的制定忽略了渗透性冷冻保护剂（PCP）和非渗透性冷冻保护剂（NPCP）对存活、有活力精子复苏的联合作用。冷冻损伤的双因素假说认为，在缓慢冷却细胞以使其进行渗透脱水从而避免细胞内结冰，与快速冷却以将冷冻过程中随着培养基冷冻部分增加而导致的细胞外渗透压升高的影响降至最低之间存在权衡。我们测试了两种浓度的PCP（10%和15% v/v二甲基亚砜（Me2SO））和两种浓度的NPCP（1%和10% w/v蔗糖）以各种组合对六种姬蛙科蛙精子的影响。在所有物种中，15% v/v Me2SO与1% w/v蔗糖组合在解冻后能提供更好的复苏效果，具有高比例的向前运动性、活细胞和完整顶体（通常每种比例均>50%）。从理论上讲，有人提出增加NPCP浓度应通过提高细胞脱水的速率和程度来改善细胞存活。然而，我们认为，当使用10%蔗糖时，未冷冻水部分中升高的渗透压可能会如冷冻损伤双因素假说中所提出的那样，通过过度的细胞收缩和溶质效应导致细胞损伤。我们在一系列蛙类物种的精子中展示了这种反应，为该假说提供了有力证据。我们建议使用本研究中证明的PCP/NPCP比例来制定方案，这将广泛适用于许多两栖动物物种。
