8 Effect of Single Layer Centrifugation with Percoll Plus^® of Fresh Bull Semen on Plasma Membrane Stability and Lipid Peroxidation After Cryopreservation

Abstract

The discontinuous density gradient with Percoll^® is routinely used for sperm selection before IVF in bovine in vitro embryo production. However, few studies have been addressed to investigate the use of this colloid in single layer centrifugation (SLC) before sperm freezing. Therefore, this study aimed to verify the effect of SLC with Percoll Plus^® (PP; GE Healthcare, Uppsala, Sweden) before freezing on plasma membrane stability (PMS) and lipid peroxidation (LP) of frozen–thawed bull semen. Three Nellore bulls housed at the Artificial Insemination Station were used. The ejaculates, 6 of each bull, were collected by artificial vagina. On the day of each collection, semen was first assessed for sperm motility, concentration, and morphology. Then, the ejaculates were pooled and ~1 billion spermatozoa either diluted [D; 1:2 (v/v)] in freezing extender (without glycerol) or undiluted (UN) was placed on top of a 9-mL column of PP at concentrations of 70% or 90%, resulting in the 70D, 70UD, 90D, and 90UD treatment groups. After centrifugation at 839 × g for 13 min, except for the control group, the supernatant was removed and the pellet of spermatozoa was resuspended in freezing extender (plus glycerol) to a final concentration of 50 × 10⁶ spermatozoa mL⁻¹. Then, 0.5-mL straws were filled, cooled, and frozen. Sperm samples of each group and different days of collection were thawed in a water bath at 37°C for 30 s and diluted at 5 × 10⁶ spermatozoa mL⁻¹ in TALP-polyvinyl alcohol (PVA) plus Hoechst 342 (Sigma Co., St. Louis, MO, USA). Then, samples were stained for PMS with the association of MitoSoxRed (Molecular Probes Inc., Eugene, OR, USA) and YO-PRO-1 (Thermo Fisher Scientific, Hampton, NH, USA) and with C11-BODYPY (Thermo Fisher Scientific) for LP. The sperm samples were analysed by flow cytometer (BD LSR, Fortessa, Becton Dickinson, Mountain View, CA, USA), with the data analysed through use of BD FACSDIVA^TM software (version 6.1). Analysis of variance and Tukey’s test were used for statistical analysis with P < 0.05 taken as significant. A lower percentage of cells displaying no destabilised membrane was found in 70D (27.6 ± 5.6) compared with the control group (43.1 ± 3.0), but no difference was observed among control group, 70UD (37.2 ± 5.1), 90UD (42.2 ± 7.5), and 90D (32.3 ± 6.5). There was a tendency (P = 0.08) for a higher proportion (P < 0.05) of cells showing LP in control group (48.2 ± 5.7), 70D (48.3 ± 8.5), and 90D (44.43 ± 7.3) compared with the undiluted groups. Thus, spermatozoa selection using SLC with Percoll Plus^® did not enhance membrane stability, but sperm LP might be diminished by using SLC with lesser dilution or concentration. It is suggested that lower generation of reactive oxygen species occurred when SLC with PP was used, indicating a possible protective effect on membrane phospholipids. Further studies, including anion superoxide, hydrogen peroxide, and mitochondrial membrane potential could elucidate these findings.

This research was supported by FAPESP (grant #2015/20986-3), Tairana AI Station and Master Fertility, Brazil.