361 INFLUENCE OF SEXING PROCEDURE ON BULL SPERM CHROMATIN STRUCTURE

Flow cytometry is the only reliable and relatively fast method allowing separation of live X and Y spermatozoa for sex regulation. Many thousands of animals of different mammalian species have been born after insemination with sexed semen during the past 20 years. Nevertheless, the question is still open: does the bull sperm sexing technology affect chromatin structure? A case of serious chromatin damage after sexing stallion semen was reported previously (Bochenek et al. 2006 Havemeyer Foundation Monograph Series No. 18, 13 –14). The aim of this work was to examine the effect of the sexing procedure and different UV laser powers on bull sperm chromatin structure. The ejaculates of 28 bulls (one ejaculate/bull) were used in the study. Each ejaculate was divided into 5 groups: (1) control, unprocessed; (2) sorted strictly according to XY Inc. protocol (Schenk et al. 1999 Theriogenology 52, 1375 –1391); (3) as group 2, but without the Red Food dye staining used for dead spermatozoa discrimination; (4) as group 2, but with double UV laser power (300 mW); and (5) as group 3, but with double UV laser power (300 mW). Sperm sorting was performed with a MoFLoSX flow cytometer at speeds of 3000 –5000 cells/s. Sorted fractions of X and Y spermatozoa were mixed again and stored for 24 h at 15 °C. A sperm chromatin structure assay (SCSA) was performed twice on each sorted sample, immediately after sorting and after 24 h. The chromatin of control samples was examined according to the same time schedule. The percentage of spermatozoa with damaged chromatin was calculated (COMP α-t) as well as standard deviation of the α-t parameter (SD α-t). The latter parameter, although less intuitive, is considered as even more precise than COMP α-t in chromatin investigations. The mean percentage of spermatozoa with abnormal chromatin was 1.12% (SD = 0.47) for control samples. The highest level of chromatin abnormality was noted for the 300 mW group with no dead cell discrimination (Red Food staining): 1.29% (SD = 1.05). After 24 h of storage, the mean level of chromatin abnormality increased to 1.97% (SD = 0.96) in control samples whereas that in all sorted samples was lower: from 1.06% (SD = 0.4) to 1.16% (SD = 0.62) in the 150 mW/non-Red Food-stained and the 300 mW/Red Food-stained groups, respectively. This difference appeared to be statistically significant (t; P ≤ 0.05). Interestingly, the percentage of abnormal spermatozoa decreased slightly after 24 h of storage in the 300 mW/Red Food-stained and the 300 mW/non-Red Food-stained groups ( –0.13% and –0.08%, respectively). Calculation of the SD α-t parameter showed statistically significant differences in chromatin abnormality between the control group vs. the 300 mW/non-Red Food-stained group immediately after sorting and the control group vs. the 150 mW/Red Food-stained group after 24 h of storage. In conclusion, although the statistically significant increase of chromatin damage was found after sexing in some investigated groups, it seems that the level of this abnormality is far too low to affect sexed semen fertility.