Evaluación de la capacidad fecundante de espermatozoides equinos previamente congelados con Dimetilformamida, glicerol y su combinación, mediante fecundación in vitro heteróloga

Abstract

Heterologous in vitro fertilization (IVF) using zona-intact bovine oocytes has been successfully used to assess the fertilizing capacity of stallion sperm. Therefore, this work evaluated the fertilizing capacity of equine spermatozoa previously frozen with DMF (5), GLY (5%), and their combination DMF-GLY (3%-3%) by heterologous IVF. In a first experiment thawed equine sperm samples (DMF, n=90, GLY, n=82 and DMF-GLY, n=84) and evaluated the kinetic parameters and integrity of the plasma and acrosomal membranes using a CASA system (SCA-Evolution® 2018) and the PI/PNA-FITC double fluorescent test, respectively. Subsequently, a heterologous IVF was used in a second experiment to evaluate the fertilizing capacity of the frozen-thawed semen measured in sperm-oocyte bound, penetration, and pronuclei formation. For this, frozen equine sperm with the three cryoprotectants and zona-intact bovine oocytes were used: DMF (n=258), GLY (n=214) and DMF-GLY (n=240) in 15 sessions. The results showed that the DMFGLY combination was more effective in cryoprotecting equine spermatozoa and producing higher values (P<0.05) of total (MT) and progressive (MP) motility, curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), beat-cross frequency (BCF), and integrity of plasma and acrosomal membranes compared to GLY. However, the fertilizing capacity of equine spermatozoa frozen with DMF-GLY was similar (P>0.05) to that obtained in those samples frozen with DMF or GLY. In conclusion, the combination of DMF-GLY is effective to cryopreserve equine sperm due to achievements obtained in kinetic and integrity of sperm membranes, but its fertilizing ability was similar to that obtained after using GLY or DMF. Further studies are recommended to validate the in vivo fertilization of these cryoprotective agents.