Reduction in volume of Percoll gradients improves the quality and recovery of bovine sexsorting for IVP

The discontinuous Percoll gradient technique is one of the most used for animal sperm selection. However, in order to obtain a better use for conventional and/or sex-sorting semen pallets, besides reducing the costs of IVP and the IVF execution time, changes have been studied in the technic, such as different forces and time of centrifugation, number of Percoll gradients and volumes. This study aimed to evaluate the effect of different volumes of Percoll on the recovery membrane integrity and movement of bovine sexed sperm. Five replicates were conducted from a pool of sexed semen from two Bos taurus taurus bulls. The sperm selection was performed by discontinuous gradients (30, 60, 90%) Percoll (Folchini et al., Rev. Bras. Reprod. Anim., V.36, p.239-244, 2012), with volumes adjusted as treatments: Control: 300μL; Treatment 1 (T1): 100 μL and Treatment 2 (T2): 200μL for each gradient. At the first and second centrifugation a force of 2200 x g was used during 5’ and 1', respectively. The samples were evaluated by a computerized system (SCA®, Sperm Class Analyzer, New Route, Barcelona, Spain) immediately after treatment concerning curvilinear velocity (VCL, µm/s) straight-line velocity (VSL, µm/s), average path velocity (VAP, µm/s), amplitude of lateral head displacement (ALH, µm), beat cross-frequency (BCF, Hz), straightness (STR, %) and linearity (LIN, %), membrane integrity (staining with propidium iodide and 6-carboxyfluorescein diacetate) and sperm recovery rate obtained by the formula considering volumes and initial and final sperm concentrations (Machado et al., Theriogenology, v.71, p.1289-97, 2009). Data were analyzed by ANOVA (p<0.05). Reducing the volume of discontinuous Percoll gradients to 100 uL increased curvilinear velocity compared to the control and T2 (control: 34.4 um / s, T1: 70.2 um / s; T2: 43.9 um / s) and it was similar to T2 and higher than control as amplitude of lateral head displacement (Control: 1,3μm; T1: 3,5μm; T2: 1,8μm). About membrane integrity, it was observed that the Control (65.2%) and T2 (72.2%) were superior to T1 (55.8%). Evaluating the recovery rate, it was found that reducing the volume to 100 uL increased recovery rate (control: 28.3%; T1: 46.3%; T2: 28.8%) of sexed semen. These results demonstrate it is possible to obtain a higher recovery rate and selecting sperm motility with higher motility with the use of gradients 100 uL volumes.