Spotted wolffish Anarhichas minor reproduction in captivity is dependent on in vitro fertilization. However, low sperm volume with relatively low cell concentration and the lack of gametes synchronization (simultaneous availability of mature eggs and sperm) represent a challenge for the industry. Thus, the development of protocols for sperm storage are crucial. Four sequential experiments were conducted to optimize a sperm cryopreservation protocol for this species. First, three different cryoprotectants (DMSO; 1, 2-propanediol; and methanol) at different concentrations (5, 10, and 20%) were tested for their toxicity. No significant differences (p > 0.05) were detected between the control samples and cryoprotectants at concentration up to 10% DMSO, 10% propanediol, and 20% methanol in terms of motility parameters. Second, using the highest non-toxic concentrations of cryoprotectants, sperm was cryopreserved in 0.5 mL straws, at different distances from the liquid nitrogen (1.5, 2.5, 4.5, and 7.5 cm) that correspond to different freezing rates. Motility parameters after freezing/thawing decreased for all the cryoprotectants (p < 0.001), however, methanol had the lowest protective capacity while DMSO the highest. Afterwards, two different thawing rates (1 min at 5 °C; and 25 s at 10 °C) were tested using only 10% DMSO and 10% propanediol. Both for the DMSO and propanediol, there were no significant differences (p > 0.05) between the two thawing rates. The best results were obtained using 10% DMSO. Finally, the fertilization capacity of cryopreserved sperm (10% DMSO and thawed at 5 °C for 1 min) was tested against fresh sperm using two spermatozoa:egg ratios and 4 h gametes contact time. The ratio of eggs with normal cell cleavage, abnormal cleavage or undeveloped were counted at the 2-4 cell stage. Cryopreserved sperm showed lower fertilization capacity at a concentration of 5 × 104 spermatozoa:egg compared with fresh sperm (p < 0.001). At a concentration of 5 × 105 spermatozoa:egg, similar fertilizations rates to the fresh sperm were obtained. The presence of the cryoprotectant DMSO during the 4 h contact time did not affect the fertilization rate or the percentage of embryos with abnormal cleavage (p > 0.05). To cryopreserve spotted wolffish sperm it is recommended to use 10% DMSO, loaded in 0.5 mL straws, freeze at a height between 4.5 (-14.05 °C/min) and 7.5 cm (-5.9 °C/min) from liquid nitrogen for 10 min and thaw for 1 min at 5 °C (177.9 °C/min). In vitro fertilization with cryopreserved sperm should be performed with a concentration of at least 5 × 105 spermatozoa per egg.

中文翻译：

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斑点狼鱼精子冷冻保存方案的逐步优化（Anarhichas minor Olafsen，1772）

圈养的斑点狼鱼 Anarhichas 小规模繁殖依赖于体外受精。然而，精子体积低、细胞浓度相对较低以及缺乏配子同步（成熟卵子和精子的同时可用）对行业来说是一个挑战。因此，精子储存方案的开发至关重要。进行了四次连续实验以优化该物种的精子冷冻保存方案。首先，测试了不同浓度（5%、10% 和 20%）的三种不同冷冻保护剂（DMSO；1, 2-丙二醇和甲醇）的毒性。在运动参数方面，对照样品和浓度高达 10% DMSO、10% 丙二醇和 20% 甲醇的冷冻保护剂之间没有检测到显着差异 (p > 0.05)。第二，使用最高无毒浓度的冷冻保护剂，精子被冷冻保存在 0.5 毫升的吸管中，与液氮的距离不同（1.5、2.5、4.5 和 7.5 厘米），对应于不同的冷冻率。所有冷冻保护剂在冷冻/解冻后的运动参数均降低 (p < 0.001)，然而，甲醇的保护能力最低，而 DMSO 的保护能力最高。然后，仅使用 10% DMSO 和 10% 丙二醇测试了两种不同的解冻速率（5 °C 下 1 分钟；10 °C 下 25 秒）。对于 DMSO 和丙二醇，两种解冻速率之间没有显着差异 (p > 0.05)。最好的结果是使用 10% DMSO 获得的。最后，使用两种精子对冷冻保存的精子（10% DMSO 并在 5°C 下解冻 1 分钟）的受精能力进行了测试：卵比率和 4 小时配子接触时间。在2-4细胞期计算细胞分裂正常、分裂异常或未发育的卵的比例。与新鲜精子相比，冷冻保存的精子在 5 × 104 精子：鸡蛋的浓度下显示出较低的受精能力（p < 0.001）。在 5 × 105 精子：鸡蛋的浓度下，获得了与新鲜精子相似的受精率。在 4 小时接触时间内冷冻保护剂 DMSO 的存在不影响受精率或异常卵裂胚胎的百分比 (p > 0.05)。要冷冻保存斑点狼鱼精子，建议使用 10% DMSO，装入 0.5 mL 吸管中，在距离液氮 4.5 (-14.05 °C/min) 和 7.5 cm (-5.9 °C/min) 之间的高度冷冻 10分钟并在 5 °C (177.9 °C/min) 下解冻 1 分钟。