280 BOVINE SPERM DNA FRAGMENTATION RATES AND EXTENDER pH: A DYNAMIC EXPERIMENTAL APPROACH

Abstract

Flow cytometry technology for the sorting of X- and Y-chromosome bearing sperm is currently utilised in research and for commercial applications. During sample preparation before the sex-sorting process, sperm go through different pH treatments that may affect their quality. The hypothesis to test in this study is that sperm DNA damage could occur due to differences in the pH of sperm extenders. Bull semen doses from 15 dairy and 15 beef bulls (2 ejaculates per individual), ranging in age between 13 and 96 months, were randomly selected from a bull stud in Texas (Sexing Technologies, Navasota, TX, USA). Each semen sample was divided into 4 separate aliquots. One neat semen sample was kept as a control and the other 3 aliquots were treated with 16 μL of 8.1 mM Hoechst 33342 (Molecular Probes, Eugene, OR, USA). A calculated amount of modified Tyrode’s albumin lactate pyruvate (Clear TALP), pH 7.4, was added based on neat ejaculate concentration. For the separation of live and dead sperm during the sex-sorting process, 2 mL of red TALP (Red Food Dye FD&C #40; Sensient Technologies Corp., Milwaukee, WI, USA) at 3 different pH treatment levels (5.5, 6.4, and 7.4) were added to the sperm samples. The dynamics of sperm DNA fragmentation were assessed immediately following the addition of red TALP treatment and after 24 h of incubation at 34°C. Sperm DNA fragmentation was measured using the commercial variant of the sperm chromatin dispersion test, the bull Sperm-Halomax^® kit (Halotech DNA, Madrid, Spain), and counting 300 sperm cells under fluorescence microscopy. Analysis of variance was used to determine if there were statistical differences (α = 0.05) among mean values of the groups (SPSS v.17.0 for Windows, SPSS Inc., Chicago, IL, USA). Bonferroni post hoc tests were utilised to determine the pairwise directional differences between groups. Differences in DNA fragmentation were not observed at 0 h (P > 0.05); however, at 24 h this value was significantly higher (P < 0.05) in semen samples treated with red TALP, pH 6.4, v. samples treated with red TALP, pH 7.4, (44.0 ± 24.9% v. 34.8 ± 24.9%), and even higher when using red TALP pH 5.5 (50.8 ± 23.8%). Further experiments should be designed to demonstrate that the DNA molecule could be affected by pH fluctuations, which may be important in refining the sex-sorting process. Notably, the undiluted neat semen controls had greater levels of DNA fragmentation overall (57.5 ± 29.4%), suggesting a negative effect of high sperm concentrations and seminal plasma.