Adjusting cryodiluent composition for improved post-thaw quality of rabbit spermatozoa

Improved fertility following artificial insemination with frozen-thawed spermatozoa would offer rabbit producers faster genetic improvement. Previous work investigating cryoprotectants for rabbit spermatozoa have reported inconsistent results. Semen was collected from three rabbit bucks by artificial vagina and frozen using a standard procedure with varied cryodiluent components. Post-thaw analysis encompassed motility, sperm kinematic parameters and acrosome and membrane integrity. Spermatozoa were evaluated at 0, 2 and 4 h after thawing. Experiment 1 compared diluents with 3.5% dimethyl sulfoxide (DMSO), 1.5% acetamide, 1.75% DMSO + 0.75% acetamide or 3.5% DMSO + 1.5% acetamide. The treatment that resulted in the highest post-thaw motility (P<0.001) and acrosome integrity (P<0.001) was DMSO alone. Experiment 2 compared 3.5, 7 and 10% DMSO in the cryodiluent. The best post-thaw sperm motility (P<0.001) and linearity (P=.002) was in 3.5% DMSO, while 10% DMSO afforded higher acrosome/membrane integrity at this last time point (P<0.05). Experiment 3 varied the cryodiluent to contain either 9 or 17% egg yolk or 9 or 17% low density lipoproteins extracted from whole egg yolk. The treatment with the best post-thaw result was 17% egg yolk (motility, P=0.01; acrosome/membrane integrity, P<0.001). Experiment 4 compared different carbohydrates in the cryodiluent; 50 mM glucose (TCG), 25 mM glucose with 25 mM sucrose (TCGS low), or 50 mM glucose with 50 mM sucrose (TCGS high). When data were pooled across time points, TCG had significantly higher motility than TCGS high (P=0.021), but was not different from TCGS low. However, TCG had fewer spermatozoa with intact acrosomes and membranes than both TCGS low and TCGS high (P=.002). Put together, these results indicate that the best cryodiluent for rabbit spermatozoa frozen under the conditions used in this paper is with 7% DMSO and 17% egg yolk in a base medium containing 25 mM glucose and 25 mM sucrose.

采用冻融精子进行人工授精后受精率的提升，可助力兔养殖者实现更快速的遗传改良。此前针对兔精子冷冻保护剂的相关研究，所得结果并不一致。本研究通过人工阴道法从3只公兔采集精液，并采用添加不同冷冻稀释液组分的标准程序进行冷冻保存。解冻后分析指标涵盖精子活力、精子运动学参数、顶体完整性与膜完整性，并分别于解冻后0、2、4小时对精子进行评估。试验1对比了含3.5%二甲基亚砜（dimethyl sulfoxide, DMSO）、1.5%乙酰胺、1.75% DMSO + 0.75%乙酰胺，以及3.5% DMSO + 1.5%乙酰胺的稀释液，结果显示仅添加DMSO的处理组获得了最高的解冻后精子活力（P<0.001）与顶体完整性（P<0.001）。试验2对比了冷冻稀释液中3.5%、7%及10%的DMSO添加比例，结果表明解冻后精子活力（P<0.001）与运动线性度（P=0.002）最优的组为3.5% DMSO组；而在解冻后4小时这一时间点，10% DMSO组的顶体/膜完整性更高（P<0.05）。试验3调整了冷冻稀释液组分，分别添加9%或17%的蛋黄，或是9%或17%从全蛋黄中提取的低密度脂蛋白，其中解冻后表现最优的处理组为17%蛋黄组（活力：P=0.01；顶体/膜完整性：P<0.001）。试验4对比了冷冻稀释液中不同碳水化合物组分：50 mM葡萄糖（TCG）、25 mM葡萄糖+25 mM蔗糖（TCGS low），以及50 mM葡萄糖+50 mM蔗糖（TCGS high）；当合并所有时间点的数据进行分析时，TCG组的精子活力显著高于TCGS high组（P=0.021），但与TCGS low组无显著差异，不过TCG组中顶体与膜完整的精子数量显著低于TCGS low与TCGS high组（P=0.002）。综合来看，本研究所用条件下，兔精子冷冻的最优冷冻稀释液配方为：基础培养基添加25 mM葡萄糖与25 mM蔗糖，并加入7% DMSO及17%蛋黄。