鲢鱼精子的精液学特征及其超快速冷冻的效果：首次超快速冷冻报告。
Spermatological characteristics and effects of cryopreservation in Lebranche mullet spermatozoa (Mugil liza Valenciennes, 1836): First report of ultra-rapid freezing.
机构信息
Laboratório de Piscicultura Marinha, Department of Aquaculture, Center of Agricultural Sciences, Federal University of Santa Catarina, Florianópolis, SC CEP 88061-600, Brazil.
Facultad de Medicina Veterinaria y Agronomía, Universidad de las Américas, Santiago, Chile.
出版信息
Anim Reprod Sci. 2022 Jun;241:106986. doi: 10.1016/j.anireprosci.2022.106986. Epub 2022 May 10.
The present study investigated the spermatological characteristics of raw semen of Lebranche mullet (Mugil liza), namely pH, and sperm density, and motility; and subsequently evaluated the effects of different times of exposure to cryoprotectants, and the application of an ultra-rapid freezing protocol, on sperm motility and plasma membrane integrity. Semen samples were analyzed undiluted (control) and diluted 1:50 v/v in CF-HBSS + 10% Dimethyl sulfoxide + 30% Ethylene glycol + 94.58 gL Trehalose dehydrate (n = 15). Two treatments - diluted semen samples in cryoprotective medium without ultra-rapid freezing (T1), and diluted semen in cryoprotective medium with ultra-rapid freezing (T2) - were evaluated at 0, 2, 4, 6 and 8 min. The frozen samples were thawed at 37ºC for 60 s. The spermatological characteristics recorded for the semen were: pH: 7.57 ± 0.21; sperm density: 30.4 ± 2.9 × 10 sperm mL; motility: 82 ± 4.9%. Sperm motility presented differences after 2 min exposure to cryoprotectants (70.0 ± 2.7%) and ultra-rapid freezing (66.5 ± 5.8%) compared to the control group (98.5 ± 1.9% and 98.5 ± 2.1%, respectively; p < 0.05). On the other hand, the plasma membrane integrity of the spermatozoa after 2 min exposure to cryoprotectants (64.0 ± 8.6%) and ultra-rapid freezing (62.5 ± 5.2%) presented no differences compared to the control group (69.5 ± 3.9% and 70.0 ± 3.5%, respectively p > 0.05); however, differences were observed in the parameters evaluated after longer exposure and cryopreservation times. This is the first report evaluating the effects of different times of exposure to cryoprotectants and direct ultra-rapid freezing in liquid nitrogen on Mugil liza sperm. Our results demonstrated the protocol of sperm ultra-freezing is safe within a time´s window of 2 min of exposure to cryoprotectants, after which a toxicity effect on sperm can be observed.
本研究调查了勒布兰奇鲻鱼（Mugil liza）原始精液的精子学特性，即 pH 值、精子密度和活力；随后评估了不同时间暴露于冷冻保护剂以及应用超快速冷冻方案对精子活力和质膜完整性的影响。精液样本未经稀释（对照）和以 1:50 v/v 稀释在 CF-HBSS+10%二甲亚砜+30%乙二醇+94.58 gL 海藻糖脱水剂中（n=15）。两种处理方式——未经超快速冷冻的稀释精液样品中的冷冻保护剂（T1）和含超快速冷冻的稀释精液样品中的冷冻保护剂（T2）——分别在 0、2、4、6 和 8 分钟时进行评估。冷冻样本在 37°C 下解冻 60 秒。记录的精液精子学特征为：pH 值：7.57±0.21；精子密度：30.4±2.9×10 个精子/mL；活力：82±4.9%。与对照组（98.5±1.9%和 98.5±2.1%）相比，暴露于冷冻保护剂 2 分钟后精子活力（分别为 70.0±2.7%和 66.5±5.8%）和超快速冷冻（分别为 70.0±2.7%和 66.5±5.8%）呈现差异（p<0.05）。另一方面，暴露于冷冻保护剂 2 分钟后精子质膜完整性（64.0±8.6%）和超快速冷冻（62.5±5.2%）与对照组（69.5±3.9%和 70.0±3.5%）无差异（p>0.05）；然而，在较长暴露和冷冻保存时间后评估的参数中观察到差异。这是首次评估不同时间暴露于冷冻保护剂和直接在液氮中超快速冷冻对勒布兰奇鲻鱼精子的影响的报告。我们的结果表明，在暴露于冷冻保护剂 2 分钟的时间窗口内，精子超冷冻方案是安全的，之后可以观察到精子的毒性作用。
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