Quinolones Abolish Bacterial Presence in Holstein Bull Sperm Samples and Indirectly Modulate the Kinetics of Sperm DNA Damage.

Abstract

Commercial cryopreserved semen samples from Holstein bulls (n=47) were examined for bacterial presence and its effect on the dynamic of sperm DNA fragmentation (SDF). Commercial semen doses produced in Spain (six ejaculates per individual) were included in this analysis. SDF were assessed after thawing (T0) and at 4, 24 and 48 hours of incubation at 37°C, using the commercial variant of the sperm chromatin dispersion test for Bovine (Halomax; Halotech SL, Madrid, Spain). Two groups of individuals, based on the presence/absence of bacteria in semen samples, were created. Using fluorescence microscopy, one group (n=23) had a readily detectable bacterial presence during the incubation time while the other group (n=24) had an undetectable bacterial presence during an equivalent period of incubation time. The comparison of the dynamic loss of DNA quality was assessed using the nonparametric maximum likelihood Kaplan-Meier estimator. The log rank test statistic (Mantel-Cox) which compares estimates of the hazard functions between the two groups at each time interval was used (SPSS v.17.0 for Windows; SPSS Inc., Chicago, IL, USA).When both groups were compared, differences in the frequency of sperm with damaged DNA immediately after thawing were not observed. However, these differences became statistically different (P<0.05) after 24 hours of sperm incubation. The extenders used during the preparation of every sperm sample contained the standard antibiotics used to putatively prevent these situations. Within this scenario, a first experiment was conducted to test the direct effect of bacteria on SDF over time. To this purpose, sperm samples from 6 different bulls without bacteria were inoculated after thawing with 5µL of sperm from a sample exhibiting bacterial presence at 48 hours. Determination of SDF was conducted after 0, 4, 24, and 48 hours of incubation at 37°C. Significant differences (p<0.05) were observed between SDF dynamics from the same sample without bacteria (control) and the inoculated sample. In a second experiment, 10 semen samples from different bulls were treated with 1µg/mL of ciprofloxacin at T0 in addition to existing antibiotics in the extender media. Five samples belonged to the group of bulls that repeatedly presented bacteria in semen and the other five were samples from bulls with an undetectable presence of bacteria in sperm. After determination of SDF at 0, 4, 24, and 48 hours of incubation at 37°C for the control sample and the treated sample, significant differences in the rate of SDF (p>0.05) were not detected on samples without bacteria. However, significant differences (p<0.05) were observed when comparing the control and the quinolone-treated samples with bacterial presence. These results call attention to two main points: 1) under similar conditions of sperm handling the dynamic behaviour of SDF can be directly correlated with sperm bacterial presence and 2) sperm DNA fragmentation can be significantly reduced by incubating sperm samples with quinolones. (poster)