370 ASSESSMENT OF SWIM-UP AND Percoll™ DENSITY GRADIENT FOR SPERM SEX PRESELECTION

Abstract

The low cost of sperm sexing methods combined with in vitro embryo production in genetic improvement programs can increase the profitability of cattle production, in particular when it does not decrease reproductive efficiency. The aim of this work was to evaluate the sex ratio deviation of thawed bovine semen processed by density gradient centrifugation and swim-up. Semen doses were collected from ten bulls of different breeds, and each experimental group was replicated ten times.A Percoll™ gradient was prepared by mixing Dulbecco’s Modified Eagle Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) isotonic solutions with Percoll™ (GE Healthcare Bio-Science AB, Uppsala, Sweden) stock solution with 0.3% BSA, resulting in densities ranging from 1.110 to 1.123 g mL^-1. The layers of discontinuous density gradient were disposed from the larger density (bottom of the tube) to the smaller into 15-mL conical centrifuge tubes. About 40 million sperm were overlaid on Percoll™ gradient and were centrifuged at 500 g for 15 min, at 22°C.The thawed semen samples were deposited in 15-mL conical centrifuge tubes containing 5 mLof DMEM and centrifuged twice at 300 g for 5 min for extender removal. After the second centrifugation, the supernatant was discarded and the sediment diluted in 1.0 mL of DMEM supplemented with 0.3% BSA. The tube was maintained in an incubator at 38.5°C and 5% CO² for 1 h. One milliliter of supernatant was recovered and evaluated for the sperm motility and vigor. Eighty million sperm were submitted to the swim-up method. For the association of density gradient and swim-up, the swim-up supernatant was recovered and overlaid on Percoll™ gradient. The gradients were centrifuged and the sperm pellet recovered. The recovered sperm were submitted to DNA extraction with phenol-chloroform. Quantitative Real Time PCR (Parati et al. 2006 Theriogenology 66, 2202-2209) was used for the determination of the proportion of X-chromosome-bearing sperm, after centrifugation through density gradient and after swim-up. The results of amplification were analyzed in 7500 Sequence Detection System Software (Applied Biosystems, Foster City, CA, USA), using Relative Quantification (Ct) Study assay. The results of X-sorted sperm samples analyzed by multiple pairwise comparisons (Tukey) were not different (P > 0.05). The percentage of X-chromosome-bearing sperm after the density gradient comprised 50.3% of the sample, after the swim-up was 49.9% and after swim-up combined with Percoll™ gradient centrifugation was 56.6%. Therefore, sperm sex selection using Percoll™ gradient centrifugation and swim-up was not effective.