Boar Sperm Cryopreservation Improvement Using Semen Extender Modification by Dextran and Pentaisomaltose

Simple Summary Cryopreservation of sperm is an important process used in livestock breeding. It is used to facilitate artificial insemination and helps to maintain genetic diversity by utilizing storage in cryobanks. Boar sperm are extremely sensitive to the conditions present during cryopreservation. Glycerol is the most used permeable cryoprotectant, showing both protective activity and some level of sperm toxicity. To improve the insemination rates, it is necessary to develop new cryoprotective agents. The goal of this paper is to evaluate the effect of the highly biocompatible, non-toxic polysaccharides dextran and pentaisomaltose on the cryopreservation of boar sperm. Additionally, we used computational modeling for the prediction of their cryoprotective action through interaction with selected sperm surface proteins. Our results show a lower impact of cryopreservation on sperm qualitative parameters when the extender is modified by pentaisomaltose. This approach represents a promising option for the complete removal of toxic glycerol in cryopreservation. 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.