Kabir M Salah Uddin, Sarder M Rafiqul Islam, Rahman M Matiur, Mollah M Fazlul Awal, Ryhan N Binte
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh, Bangladesh.
Biopreserv Biobank. 2022 Aug;20(4):357-366. doi: 10.1089/bio.2021.0055. Epub 2022 Jan 7.
This study dealt with the development of a sperm cryopreservation protocol of . Sperm was collected from hormone-induced males and the concentration and pH of sperm were estimated as 4.3 ± 0.1 × 10 cells/mL and 8.6 ± 0.2, respectively. Activation of sperm motility was evaluated in different osmolalities of NaCl solution where motility of sperm decreased with increasing osmolality of extenders, and was completely inhibited at 319 mOsmol/kg. Similarly, the swimming duration of activated sperm was affected as the osmolality of the extender increased. The duration of initial motility of sperm was recorded as 8.4 ± 0.4 minutes at 48 mOsmol/kg, while the highest motility was recorded as 68.0 ± 7.2 minutes at 128 mOsmol/kg. To evaluate the toxicity of cryoprotectants, sperm was incubated with dimethyl sulfoxide (DMSO) and methanol at 5%, 10%, and 15% concentrations, respectively, for 5-45 minutes. Alsever's solution with 5% and 10% DMSO produced better motility during 5-10 minutes of incubation and 15% DMSO seemed toxic to sperm. For the cryopreservation of sperm, Alsever's solution, egg yolk citrate, and urea egg yolk were used as extenders with DMSO and methanol. Alsever's solution with 10% DMSO provided the highest equilibration (90.0% ± 3.5%) and post-thaw (80.0% ± 3.5%) motility followed by that of 87.0% ± 2.0% and 79.0% ± 1.9% with egg yolk citrate plus DMSO, and 82.0% ± 2.6% and 78.0% ± 2.0% with urea egg yolk plus DMSO, respectively. The sperm cryopreservation protocol developed through this study can be applied for long-term preservation of genetic materials of the critically endangered , and eventually, it will be an effective tool for protecting them from extinction.
本研究涉及[物种名称]精子冷冻保存方案的制定。从激素诱导的雄性个体收集精子，精子浓度和pH值分别估计为4.3±0.1×10⁶个细胞/mL和8.6±0.2。在不同渗透压的氯化钠溶液中评估精子活力的激活情况，随着稀释液渗透压的增加，精子活力下降，在319毫摩尔/千克时完全被抑制。同样，随着稀释液渗透压的增加，激活精子的游动持续时间也受到影响。精子初始活力持续时间在48毫摩尔/千克时记录为8.4±0.4分钟，而在128毫摩尔/千克时最高活力记录为68.0±7.2分钟。为评估冷冻保护剂的毒性，精子分别与浓度为5%、10%和15%的二甲亚砜（DMSO）和甲醇孵育5 - 45分钟。含有5%和10%DMSO的阿氏液在孵育5 - 10分钟期间产生更好的活力，而15%DMSO似乎对精子有毒。对于精子冷冻保存，阿氏液、蛋黄柠檬酸盐和尿素蛋黄用作添加DMSO和甲醇的稀释液。含有10%DMSO的阿氏液提供了最高的平衡后（90.0%±3.5%）和解冻后（80.0%±3.5%）活力，其次是蛋黄柠檬酸盐加DMSO的为87.0%±2.0%和79.0%±1.9%，以及尿素蛋黄加DMSO的分别为82.0%±2.6%和78.0%±2.0%。通过本研究制定的精子冷冻保存方案可用于极度濒危[物种名称]遗传物质的长期保存，最终，它将成为保护它们免于灭绝的有效工具。
