Characterization of Whole, Single and Double Strand DNA Damage in Sex-Sorted Bovine Spermatozoa.

Abstract

The objective of the present investigation was to examine the dynamics of DNA damage after flow cytometry sex-sorted bovine sperm. The hypothesis tested was that during sperm sorting, the level of DNA damage in the sperm fraction to be used for insemination can be reduced due to a step included in the methodology that removes non viable sperm although the DNA quality could be compromised. Eight sperm samples were sex-sorted using a MoFlo SX (Beckman Coulter, Miami, FL) to produce purified sperm subpopulations bearing the X or Y chromosomes. These samples and conventional non sorted sperm from the same ejaculates were cryopreserved. Sperm DNA fragmentation (SDF) of each sample was assessed after thawing. Evaluations occurred immediately after thawing, (t0), 24 and 48 hours of incubation at 37°C using Halomax (Halotech SL, Madrid, Spain). SDF at t0 was lower in sex-sorted samples, and statistical differences were obtained after comparing both groups (P 0.01). However, a reduction in DNA longevity was observed in sorted sperm during the 24 and 48 hour incubation periods. In these samples, SDF increased between 24 and 48 hours of incubation while in conventional samples this tendency was delayed. The graphic representation of SDF showed a crossover point between both dynamic tendencies, the Crossover Positioning Time (CPT). This CPT can be used as an indicator of sperm quality for each bull, suggesting that higher values of CPT are indicative of bulls with more resistant chromatin to external stressors that affect DNA after sorting, while those exhibiting lower values may have a compromised chromatin structure. The increase in SDF in sorted samples was characterized to gain information about the type of DNA damage (single strand break -SSB- or double strand break -DSB-) using a 2D-comet assay. The SDF after thawing was a mixture of SSB and DSB. However, DNA damage produced during incubation was characterized as SSB. Differences in the kinetics for DSB production after a 24h incubation time period were obtained when the different individuals were compared (Kaplan-Meier estimator -log rank test statistic- Mantel-Cox; χ2 6.12; P: 0001). The conclusion to this research is that sex sorting sperm decreases the initial sperm DNA damage when compared to conventional semen samples, but it increases the rate of sperm DNA damage over time. The iatrogenic DNA damage produced is mainly SSB. Since the oocyte has a superb capacity for DNA repair, could be of interest to investigate which are the threshold levels of SSB that may induce lethal and sub-lethal effects in the progeny, as well as those which could be repaired by the female gamete. (poster)