Protocol for cryopreservation of goat sperm using mesenchymal stem cells from adipose tissue and bone marrow

This study aims to develop and evaluate a new approach to cryopreservation of goat semen to improve the efficiency of preserving their valuable genetic material. The relevance of this work is driven by the growing demand for goat milk and the need for import substitution in breeding, which necessitates the creation of highquality domestic sperm banks. Since goat semen does not tolerate freezing well and the availability of foreign cryoprotectants is limited, the aim of the study was to evaluate the effectiveness of mesenchymal stem cells (MSCs) isolated from adipose tissue (AT) and bone marrow (BM) as a biologically active component of a cryoprotective medium based on OptiXcell extender. Semen from Saanen and Alpine goats was used in the experiment. After isolation and cultivation of MSCs from adipose tissue and bone marrow, three groups were formed: control (sperm + OptiXcell) and two experimental groups (sperm + OptiXcell + BM MSCs or AT MSCs). Sperm quality was assessed at five stages: after collection, cooling, immediately after thawing, and after 2 and 4 hours of incubation, analyzing sperm motility, viability, and morphology. Analysis of variance (ANOVA) results revealed a statistically significant positive effect of MSC addition on key parameters. The experimental groups retained a significantly higher percentage of progressively motile sperm and recorded fewer immobile cells and cells with cervical defects compared to the control group. Furthermore, adiposederived MSCs tended to perform somewhat better in maintaining motility and reducing structural damage than bone marrow-derived cells, especially at late stages after thawing. Thus, the study demonstrates that the use of autologous MSCs in a cryoprotective medium significantly improves the post-thawing quality of goat sperm. The obtained data opens up the prospect of creating effective domestic cryopreservation environments, which is of great importance for the development of livestock farming and the implementation of genetic improvement programs in the current economic environment.