Simonik Ondrej, Bubenickova Filipa, Tumova Lucie, Frolikova Michaela, Sur Vishma Pratap, Beran Jan, Havlikova Katerina, Hackerova Lenka, Spevakova Daniela, Komrskova Katerina, Postlerova Pavla
Laboratory of Reproductive Biology, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Prumyslova 595, 25250 Vestec, Czech Republic.
Department of Veterinary Sciences, Faculty of Agrobiology, Food and Natural Resources, University of Life Sciences Prague, Kamycka 129, 16500 Prague, Czech Republic.
Animals (Basel). 2022 Mar 30;12(7):868. doi: 10.3390/ani12070868.
The long-term storage of boar sperm presents an ongoing challenge, and the modification of the cryoprotective compounds in semen extenders is crucial for improving cryopreservation's success rate. The aim of our study was to reduce the percentage of glycerol in the extender by elimination or substitution with biocompatible, non-toxic polysaccharides. For boar semen extender improvement, we tested a novel modification with the polysaccharides dextran and pentaisomaltose in combination with unique in silico predictive modeling. We targeted the analysis of in vitro qualitative sperm parameters such as motility, viability, mitochondrial activity, acrosome integrity, and DNA integrity. Non-penetrating polysaccharide-based cryoprotective agents interact with sperm surface proteins such as spermadhesins, which are recognized as fertility markers of boar sperm quality. The in silico docking study showed a moderate binding affinity of dextran and pentaisomaltose toward one specific spermadhesin known as AWN, which is located in the sperm plasma membrane. Pentaisomaltose formed a hydrophobic pocket for the AWN protein, and the higher energy of this protein-ligand complex compared with dextran was calculated. In addition, the root mean square deviation (RMSD) analysis for the molecular dynamics (MD) of both polysaccharides and AWN simulation suggests their interaction was highly stable. The in silico results were supported by in vitro experiments. In the experimental groups where glycerol was partially or entirely substituted, the use of pentaisomaltose resulted in improved sperm mitochondrial activity and DNA integrity after thawing when compared with dextran. In this paper, we demonstrate that pentaisomaltose, previously used for cryopreservation in hematopoietic stem cells, represents a promising compound for the elimination or reduction of glycerol in extenders for boar semen cryopreservation. This novel approach, using in silico computer prediction and in vitro testing, represents a promising technique to help identify new cryoprotectants for use in animal breeding or genetic resource programs.
公猪精子的长期保存一直是个挑战，而改良精液稀释液中的冷冻保护剂对于提高冷冻保存成功率至关重要。我们研究的目的是通过用生物相容性、无毒的多糖消除或替代甘油，来降低稀释液中甘油的百分比。为了改良公猪精液稀释液，我们测试了用多糖葡聚糖和五异麦芽糖进行的新型改良，并结合了独特的计算机预测模型。我们的目标是分析体外精子的定性参数，如活力、生存力、线粒体活性、顶体完整性和DNA完整性。基于非穿透性多糖的冷冻保护剂与精子表面蛋白如精子黏附素相互作用，精子黏附素被认为是公猪精子质量的生育标志物。计算机对接研究表明，葡聚糖和五异麦芽糖对一种位于精子质膜上名为AWN的特定精子黏附素具有中等结合亲和力。五异麦芽糖为AWN蛋白形成了一个疏水口袋，并且计算出该蛋白-配体复合物与葡聚糖相比具有更高的能量。此外，对两种多糖和AWN模拟的分子动力学（MD）的均方根偏差（RMSD）分析表明它们的相互作用非常稳定。计算机模拟结果得到了体外实验的支持。在甘油被部分或完全替代的实验组中，与葡聚糖相比，使用五异麦芽糖解冻后精子线粒体活性和DNA完整性得到改善。在本文中，我们证明了先前用于造血干细胞冷冻保存的五异麦芽糖是一种有前景的化合物，可用于消除或降低公猪精液冷冻保存稀释液中的甘油。这种使用计算机预测和体外测试的新方法是一种有前景的技术，有助于识别用于动物育种或遗传资源项目的新型冷冻保护剂。
