Center of Biotechnology in Reproduction, La Frontera University, P.O. Box 54-D, Temuco, Chile.
Anim Reprod Sci. 2011 Mar;124(1-2):125-31. doi: 10.1016/j.anireprosci.2011.02.023. Epub 2011 Feb 19.
The aim of the present investigations was to test a novel technology comprising cryoprotectant-free vitrification of the spermatozoa of rainbow trout and to study the ability of sucrose and components of seminal plasma to protect these cells from cryoinjuries. Spermatozoa were isolated and vitrified using five different mediums: Group 1: standard buffer for fish spermatozoa, Cortland(®)-medium (CM, control); Group 2: CM+1% bovine serum albumin (BSA); Group 3: CM+1% BSA+0.125 M sucrose; Group 4: CM+1% BSA+40% seminal plasma; and Group 5: CM+1% BSA+40% seminal plasma+0.125 M sucrose. For cooling, 20 μL suspensions of cells from each group were dropped directly into liquid nitrogen. For warming, the spheres containing the cells were quickly submerged in CM+1% BSA at 37 °C with gentle agitation. The quality of spermatozoa before and after vitrification was analysed by the evaluation of motility, cytoplasmic membrane integrity (SYBR-14/propidium iodide staining technique), and mitochondrial membrane integrity (JC-1 staining). Motility (86%, 71%, 80%, 81%, and 82%, for Groups 1, 2, 3, 4, and 5, respectively) and cytoplasmic membrane integrity (90%, 82%, 83%, 84%, and 87%, respectively) of spermatozoa in all the 5 groups were not decreased significantly. All tested solutions can be used for vitrification of fish spermatozoa with good post-warming motility and cytoplasmic membrane integrity. However, mitochondrial membrane potentials of the spermatozoa in Groups 1, 2, 3, 4, and 5 were changed significantly (6%, 50%, 37%, 55%, and 34%, respectively) (P(1,2,3,4,5)<0.001; P(2,3,4,5) <0.01)(P(3-5)>0.1). This rate was maximal in Group 4 (CM+1% BSA+40% seminal plasma). In conclusion, this is the first report about successful cryoprotectant-free cryopreservation of fish spermatozoa by direct plunging into liquid nitrogen (vitrification). Vitrification of fish spermatozoa without permeable cryoprotectants is a prospective direction for investigations: these cells can be successfully vitrified with 1% BSA+40% seminal plasma without significant loss of important physiological parameters.
本研究旨在测试一种新的技术，即对虹鳟鱼精子进行无冷冻保护剂的玻璃化冷冻，并研究蔗糖和精液成分在保护这些细胞免受冷冻损伤方面的能力。精子是通过五种不同的培养基分离和玻璃化的：第 1 组：鱼精子的标准缓冲液，考尔德兰（®）-培养基（CM，对照）；第 2 组：CM+1%牛血清白蛋白（BSA）；第 3 组：CM+1%BSA+0.125M 蔗糖；第 4 组：CM+1%BSA+40%精液；第 5 组：CM+1%BSA+40%精液+0.125M 蔗糖。冷却时，将每组 20μL 的细胞悬浮液直接滴入液氮中。为了升温，将含有细胞的球体在 37°C 下迅速浸入含有 1%BSA 的 CM 中，并轻轻搅拌。通过评估运动性、细胞质膜完整性（SYBR-14/碘化丙啶染色技术）和线粒体膜完整性（JC-1 染色），分析了玻璃化前后精子的质量。第 1、2、3、4 和 5 组精子的运动性（分别为 86%、71%、80%、81%和 82%）和细胞质膜完整性（分别为 90%、82%、83%、84%和 87%）均无显著降低。所有测试溶液都可用于鱼类精子的玻璃化，具有良好的升温后运动性和细胞质膜完整性。然而，精子的线粒体膜电位在第 1、2、3、4 和 5 组中发生了显著变化（分别为 6%、50%、37%、55%和 34%）（P(1,2,3,4,5)<0.001；P(2,3,4,5)<0.01）（P(3-5)>0.1）。第 4 组（CM+1%BSA+40%精液）的变化率最大。总之，这是首次成功报道了通过直接浸入液氮（玻璃化）对鱼类精子进行无冷冻保护剂的冷冻保存。无渗透冷冻保护剂的鱼类精子玻璃化是一个有前途的研究方向：这些细胞可以成功地用 1%BSA+40%精液玻璃化，而不会显著损失重要的生理参数。
