Association of seminal plasma and alternative cryoprotectants with the antioxidant and the fertilizing capacity of cryopreserved sperm in donkeys

Abstract

Seminal plasma (SP) in Equidae is involved in several events that precede sperm fertilization, such as motility activation, antimicrobial action, metabolite neutralization, and a mediating effect on sperm capacitation and postcoital uterine inflammatory response. In the donkey species, it has been shown that SP can provide to sperm subjected to cryopreservation, a greater antioxidant capacity, since SP contains various molecules that can protect spermatozoa from oxidative stress. The aim of this study was to evaluate the association between seminal plasma components (proteins and enzymes) and alternative cryoprotectants, with the antioxidant and the fertilizing capacity of cryopreserved sperm of Colombian Creole donkeys. Ten Colombian Creole donkeys (Equus asinus) were collected using the artificial vagina method to obtain three ejaculates per animal, for a total of 30 ejaculates. A fraction of each ejaculate was centrifuged to obtain the SP. Evaluation of some components of seminal plasma was performed. The enzymes catalase, superoxide dismutase and glutathione peroxidase were evaluated by spectrophotometry and the concentration of the cysteine-rich secretory 2 (CRISP-2) protein 2 by the ELISA assay. For semen cryopreservation, treatments with different combinations of dimethylformamide (DMF), sucrose (SAC) and homologous seminal plasma (SP) were used. In thawed semen, total antioxidant capacity was evaluated by the ABTS and ORAC assays; additionally, the reactive oxygen species (ROS) production was evaluated. Fertilizing capacity was evaluated using a sperm binding assay to the zona pellucida of oocytes. The semen quality index (SQi) was calculated for total motility, progressive motility, vitality and plasma membrane integrity. Correlation and regression analysis were performed between the SP components, TAC, ROS and fertilizing capacity. For the comparisons of means, a Tukey Test was carried out. For CRISP-2 a positive correlation coefficient of 0.17 with the seminal quality index (SQi) of thawed semen, was obtained (P< 0.05). The sperm binding to the zona pellucida was 265.34±11.03, 135.2±7.66, 268.37±9.01 and 299.14±10.22 for treatments of 5% DMF; 0.2M SAC and 1% BSA; 5%DMF, 0.2M SAC and 10% SP; and 5% DMF, 0.2M SAC and 10% SP, respectively. The last treatment being the one with a higher value (P< 0.05). For the combination 5% DMF and 10% SP; 0.2 SAC,1%BSA and 10% SP, it could be found that the antioxidant capacity measured by the (ABTS) method had a negative regression coefficient with total ROS (P<0.05). In conclusion, SP components, TAC and ROS are associated with semen quality and the fertilizing capacity of donkey sperm. Additionally, freezing of semen under a medium composed of dimethylformamide (DMF), bovine serum albumin (BSA) and seminal plasma (PS), improves the adherence of sperm to the zona pellucida, being a favorable indicator of the fertilizing capacity of donkey sperm.