The protective capacity of different diluents on stallion sperm during storage in a passive refrigeration system “BotuflexⓇ '' for 24 h at 5°C before freezing

Abstract

The refrigeration of semen at 5°C for 24 hours before freezing, using Equitainer^Ⓡ and Max-Semen Express^Ⓡ, resulted in fertility rates similar to conventional methods (Melo.et al. Animal Reproduction. 2006; v.94, p.78-81). The objective of this study was to compare conventional freezing and refrigeration prior to freezing, using different refrigeration diluents in Botuflex-Botupharma^Ⓡ, with the purpose of improving sperm motility, plasma membrane stability, mitochondrial potential, and sperm morphology rates. The study used three ejaculates from four stallions of different breeds with proven fertility, collected using a Botucatu^Ⓡ artificial vagina. After, the sperm parameters were measured and the ejaculates were diluted with Botu-Special^Ⓡ (BSP), Botu-Sêmen^Ⓡ (BSEM), Botu-Sêmen Gold^Ⓡ (BG) and Botu-Turbo^Ⓡ (BT), which were divided into two groups, with “C” being the control frozen according to the conventional methodology with Botucrio^Ⓡ and “R”, refrigerated at 5°C for 24 hours and subsequently frozen by the same methodology. After thawing, kinetics (CASA), plasma membrane stability, mitochondrial potential by flow cytometry and sperm morphology (DIC) were evaluated. Statistical analysis was performed by Kolmogorov-Smirnov, ANOVA and Tukey test, P〈 0.05 was considered. The results of the R BSP (36.9 ± 3.5ab) and R BG (43.5 ± 4.2a) groups showed similar stability to the control C BSP (41.3 ± 3.2ab) and C BG (47.9 ± 3.5a). Regarding mitochondrial potential, R BG (46.9 ± 4.0a) and R BSP (39.5 ± 3.5ab) showed similar values to the controls C BG (50.5 ± 3.3a) and C BSP (44.3 ± 3.1ab), suggesting no negative impact on mitochondrial function. As for total motility, R BG (73.9 ± 3.2a) and R BSP (70.8 ± 4.1ab) were similar to their respective controls C BG (79.2 ± 2.8a) and C BSP (79.6 ± 1.9a). Progressive motility was also preserved in R BG (41.9 ± 3.8a) and R BSP (40.0 ± 4a), when compared to the C BG (43.7 ± 3.6a) and C BSP (45.9 ± 3.4a) groups. In terms of fast sperm, the R BG (62.4 ± 4.7a) and R BSP (57.7 ± 5.4a) groups did not show significant reductions. The experimental groups R BSEM and R BT were not mentioned during the discussion as they did not have satisfactory results. The sperm morphology analysis revealed no statistical differences between the groups analyzed. The conclusion is the R BG and R BSP groups showed significant sperm function preservation similar to the controls. Thus, the Botu-Special^Ⓡ and Botu-Sêmen Gold^Ⓡ media can be used to refrigerate semen at 5°C for 24 hours before freezing. This technique allows veterinarians to optimize their work in the field, without having to freeze semen in establishments where conditions are not suitable for the cryopreservation of equine semen.