9 Single Layer Centrifugation of Bull Semen Through Percoll Plus^® Before Cryopreservation

Abstract

We previously reported that single layer centrifugation (SLC) with Percoll^® (GE Healthcare, Uppsala, Sweden) of fresh bovine semen resulted in improved sperm progressive motility and movement, as evidenced by computer-assisted sperm analyzer (CASA) after freezing-thawing. However, no report has been found in the literature on the use of Percoll Plus^® (PP; GE Healthcare), a nontoxic colloid, for the same purpose. Thus, this study aimed to verify the effects of SLC-PP before bull sperm freezing on sperm kinematics after cryopreservation. Ejaculates were collected from 3 Nellore bulls (6 from each) using an artificial vagina. After collection, the semen was assessed and pooled, and then 1 billion spermatozoa either diluted [D; 1:2 (v/v)] in freezing extender (FE, without glycerol) or undiluted (UD) was layered 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. Following centrifugation for 13 min at 839 × g [except for the control (C) group], the supernatant was removed and the sperm pellet diluted to 50 × 10⁶ sperm mL⁻¹ in FE medium plus glycerol. Then, frozen–thawed sperm samples were analysed by CASA (MMC Sperm, St. Petersburg, Russia) for the following parameters: total motility (TM, %), progressive motility (PM, %), curvilinear velocity (VCL, µm⁻¹), straight line velocity (VSL, µm s⁻¹), average path velocity (VAP, µm s⁻¹), amplitude of lateral head displacement (ALH, µm), beat cross frequency (BCF, Hz), linearity (LIN, %), and straightness (STR, %). For statistical analyses, ANOVA and Student-Newman-Keuls test were used. Data are presented as mean ± SEM with P < 0.05 taken as significant. No difference was found among the groups for TM, VSL, BCF, and STR. However, the percentage of PM was higher (P < 0.05) in the SLC-selected sperm samples (values ranging from 42.0 ± 7.0 to 47.4 ± 11.4) than in C (28.8 ± 5.0), and ALH was lower in 70UD (1.6 ± 0.12) and 70D (1.7 ± 0.10) than in C (1.9 ± 0.2). Moreover, 70UD (49.0 ± 1.0), 90UD (50.0 ± 3.0), and 90D (50.0 ± 4.0) displayed higher percentage of LIN (P < 0.05) compared with C (45.0 ± 2.0) and 70D (48.0 ± 3.0). On the other hand, similar results were obtained for VCL (from 126.3 ± 8.0 to 130.0 ± 20.5) and VAP (from 82.7 ± 14.5 to 85.1 ± 6.9) in C, 70UD, and 70D, but these values differed (P < 0.05) from those for VCL in 90UD (104.6 ± 10.3) and 90D (97.2 ± 22.0) as well as for VAP in 90UD (72.2 ± 11.0) and 90D (71.8 ± 9.6). These are the first data demonstrating favourable influences of SLC with 70% Percoll Plus^® to select distinct sperm subpopulations as evidenced by enhanced PM, LIN, and ALH. Thus, SLC-PP could optimize the production of frozen bull semen by decreasing the number of sperm per insemination dose, and help to circumvent limitations associated with the poor semen quality sometimes found in bulls of high genetic merit.

This research was funded by FAPESP # 2015/20986-3, MasterFertility and Tairana Artificial Insemination Station, Brazil.