University of Szczecin, Department of General Zoology, 71-412 Szczecin, Poland.
Theriogenology. 2011 Jul 15;76(2):300-11. doi: 10.1016/j.theriogenology.2011.02.007. Epub 2011 Apr 14.
The cryopreservation of salmonid sperm is a complex process involving the interplay of many factors. Although cryopreservation protocols can be evaluated through a range of responses at various stages in the process, the number of progeny is the ultimate indicator of success. We compared reproductive success from freezing Atlantic salmon (Salmo salar L.) sperm using the eight combinations of (1) the penetrating cryoprotectants, 10% dimethyl sulfoxide (DMSO) or methanol (MeOH); (2) the nonpenetrating cryoprotectants glucose (0.3 M) or sucrose (0.6 M), and freezing in 0.1 mL pellets or 0.25 mL straws. All cryodiluents were supplemented with 10% (v/v) of hen's egg yolk. Response variables were the percentage and degree of motility of thawed and activated sperm using computer assisted sperm analysis (CASA), and rates of eyed embryos, hatch and egg sac larvae. Growth rates of alevins were assessed to two months post hatch. Atlantic salmon milt cryopreserved in straws had higher spermatozoa motility and fertilization success than milt cryopreserved in pellets (P < 0.05). Type of sugar tested did not significantly affect the response variables. In the MeOH treatment, thawed spermatozoa achieved higher speed and a higher fertilization rate evaluated at the eyed embryo stage than spermatozoa subjected to the DMSO treatment. Higher mortality rate (especially before hatching) of MeOH offspring than DMSO offspring led to equal numbers of progeny for the two treatments from the swimming stage to the end of the study. Moreover, during feeding fish from the MeOH group produced significantly lower weight larvae than the DMSO and control groups. Even so, the weight of the MeOH group was satisfactory. Length and the condition factors did not differ significantly among the larvae groups. Significant positive correlations were found between fertilization success (measured in number of eyed eggs) and both motility (rs = 0.81), and velocity (rs = 0.49). Freezing in straws gave betters results than freezing in pellets for cryopreservation of salmon milt; whereas type of sugar tested (glucose vs sucrose) did not have significant effects. Penetrating cryoprotectants DMSO and MeOH differed in their effect on post-thawed sperm velocity, fertilization rate and mortality rate of progeny, suggesting the need for further research on the influence of these cryoprotectants on frozen sperm and and post-fertilization devopmental processes.
三文鱼精子的冷冻保存是一个复杂的过程，涉及许多因素的相互作用。虽然可以通过在过程的各个阶段评估冷冻保存方案的一系列反应来评估冷冻保存方案，但后代的数量是成功的最终指标。我们比较了使用 10%二甲基亚砜（DMSO）或甲醇（MeOH）（1）穿透性冷冻保护剂，0.3M 葡萄糖或 0.6M 蔗糖（2）非穿透性冷冻保护剂的组合冷冻大西洋三文鱼（Salmo salar L.）精子的生殖成功率，冷冻在 0.1 毫升颗粒或 0.25 毫升吸管中。所有冷冻稀释液均添加 10%（v/v）的鸡卵黄。解冻和激活精子的运动百分比和运动速度使用计算机辅助精子分析（CASA）评估，卵眼胚胎，孵化和卵囊幼虫的比率。孵化后两个月评估幼鱼的生长速度。吸管中冷冻的三文鱼精液的精子运动能力和受精成功率高于颗粒中冷冻的精液（P<0.05）。测试的糖类型对反应变量没有显著影响。在 MeOH 处理中，与 DMSO 处理相比，解冻的精子在达到眼睛胚胎阶段时具有更高的速度和更高的受精率。MeOH 后代的死亡率（特别是在孵化前）高于 DMSO 后代，导致两种处理方法从游泳阶段到研究结束时的后代数量相等。此外，在喂养期间，来自 MeOH 组的鱼产生的幼虫体重明显低于 DMSO 组和对照组。尽管如此，MeOH 组的体重还是令人满意的。幼虫组之间的体长和条件系数没有显著差异。发现受精成功率（以卵眼数衡量）与运动能力（rs=0.81）和速度（rs=0.49）之间存在显著正相关。与颗粒相比，吸管中冷冻保存三文鱼精液的效果更好；然而，测试的糖类型（葡萄糖与蔗糖）没有显著影响。DMSO 和 MeOH 等渗透性冷冻保护剂对解冻后精子速度、后代受精率和死亡率的影响不同，这表明需要进一步研究这些冷冻保护剂对冷冻精子和受精后发育过程的影响。
