10 Evaluation of Frozen Sperm Quality After Single Layer Centrifugation with Percoll Plus® of Fresh Bull Semen

The single layer centrifugation (SLC) with Percoll Plus® (PP; GE Healthcare, Uppsala, Sweden) before freezing is not a common technique used for selection of spermatozoa in bovine. Thus, this study aimed to verify the effect of SLC with PP before freezing on integrity of plasma and acrosome membranes (IPAM), phospholipid translocation (PT), and mitochondrial membrane potential (MMP) of frozen–thawed bull sperm. Three Nellore bulls housed at the Tairana Artificial Insemination Station were used. The ejaculates (6/bull) were collected by artificial vagina and assessed for sperm motility, concentration, and morphology. Then, the sperm were pooled and ~1 billion spermatozoa, either diluted [D; 1:2 (v/v)] in freezing extender (FE; tris, fructose, citric acid, egg yolk and antibiotics, without glycerol) or undiluted (UN), were placed on top of a 9-mL column of PP (in 15-mL centrifuge tubes) at concentrations of 70% or 90%, to form the 70D, 70UD, 90D, and 90UD treatment groups. After centrifugation at 839 × g for 13 min, except for the control (C), the supernatant was discarded and the pellet diluted in FE (plus glycerol) to a final concentration of 50 × 106 spermatozoa mL−1. Afterward, 0.5-mL straws were filled, cooled for 5 h at 4°C, and frozen in a programmable freezer (Digitcool, IMV, L’Aigle, France) following the temperature/time curve: from 4°C to –10°C (5°C min−1), –10°C to –100°C (40°C min−1) and from –100°C to –140°C (20°C min−1), in a total of 8 min, when the straws were plunged into and stored in liquid nitrogen until evaluation. Thawed sperm (at 37°C/30 s) was diluted at 5 × 106 spermatozoa mL−1 in TALP-polyvinyl alcohol (PVA) plus Hoechst 342 (100 μg mL−1; Sigma Co., St. Louis, MO, USA). After that, samples were stained for membrane integrity with the association of fluorescent probes propidium iodide (PI, 50 μg mL−1; Sigma Co.), fluorescein thiocyanate (FITC)-Pisum sativum agglutinin (PSA, 1 mg mL−1; Sigma Co.) and Annexin V-APC (BD Pharmingen, Franklin Lakes, NJ, USA), and with MitoStatus Red (20 nM; BD Pharmingen) and YO-PRO-1 (7.5 μM; Molecular Probes Inc., Eugene, OR, USA) for MMP. Sperm samples were analysed by flow cytometer (BD LSR; Fortessa, Becton Dickinson, Mountain View, CA, USA) and the results expressed as percentage of intact cells or qualitative fluorescence expressed in arbitrary units (AU). Analysis of variance and Tukey’s test were used for statistical analysis with P < 0.05 taken as significant. There were no differences between groups for IPAM (values ranging from 45.9 ± 7.0% to 55.6 ± 8.5%). Similarly, results of PT translocation did not differ among the groups (range from 34.7 ± 7.0% to 47.6 ± 7.0%). However, there was a tendency of increasing MMP (P = 0.10) in 70UD (1789 ± 258 UA), 70D (1776 ± 162.1 UA), and 90UD (1757 ± 133.8 UA) compared with C (1368 ± 267.4 UA) and 90D (1356 ± 145 UA). In conclusion, SLC did not compromise sperm membrane functionality and it seemed to select spermatozoa with higher mitochondrial functional activity when centrifuged at the concentration of 70% and 90D. This research was funded by FAPESP # 2015/20986-3, Tairana Artificial Insemination Station, MasterFertility Ltda, Brazil.