Assessment of Chromatin Damage in Bull Semen Utilizing an Acridine Orange Assay Adapted for Agilent 2100 Bioanalyzer

The objective of this study was to assess induced chromatin damage of sperm obtained from bulls consuming ergot alkaloids with an Acridine Orange (AO) staining procedure adapted to flow cytometry procedures using the Agilent 2100 Bioanalyzer Cell Chip technology. The AO staining technique was validated by exposing ≈1X10⁶ formalin fixed sperm (100 ml) to UV light at 254 nm, 302 nm, or 365 nm for 0, 30, 60, and 90 minutes, in triplicate, to induce acrosomal damage. Following UV exposure, cell membranes were permeabilized with 500 ml of a 0.1% triton solution. Samples were stained by incubating with 300 ml staining buffer and 2 ml of a 6 mg/ml Acridine Orange stain solution. Cells were pelleted, resuspended in 100 ml Agilent cell chip buffer and then analyzed through the Agilent Cell Chip Assay to evaluate percent gated cells, correlating to cells with chromatin damage. Angus bulls (n = 25), ≈12 mo of age and having passed a breeding soundness exam were stratified by BCS and BW and allotted to one of two treatments, non-toxic fescue, Texoma Max Q II (E-), or ergot alkaloid containing, Kentucky 31 (E+). Bulls were allowed to graze for 84 d (January to April). Semen was collected at days -28, 0, 28, and 56 (allotment to treatment = d 0) by electrojaculation. Samples were fixed in formalin until assessment of chromatin damage using the adapted AO assay. Results assessing UV induced chromatin damage were subjected to analysis using JMP (SAS Institute, Cary NC) using LS means procedures where UV wavelength, time of exposure and their interactions were the main effects. Data evaluating the effect on chromatin damage due to the consumption of E+ diets were subjected to LS means procedures where treatment, d, and their interaction were the main effects. Using the adapted AO staining procedure, an increase in chromatin damage was detected with exposure to 302 nm (P = 0.007). The highest percent gated was reached in cells exposed to UV for 90 min (P = 0.007). When comparing sperm for E+ and E- bulls, no significant differences in number of chromatin damaged cells due to treatment, d or the treatment by d interactions were detected. These data indicate that the AO assay adapted to the Agilent 2100 Bioanalyzer Cell Chip technology can detect sperm chromatin damage and that grazing E+ forage does not negatively impact sperm physiology through induction of chromatin damage.