Cellrox Deep Red® is effective for detecting oxidative stress in bovine sperm: preliminary studies

Reactive oxygen species (ROS) are produced physiologically by sperm cell. Cryopreservation is able to increase this production and it results in presence of antioxidants in composition of majority of extenders. The probe CellROX Deep RED® (Molecular Probes) was validated by our group to identify ROS in ram sperm. However, it has not been efficiently evaluated in bull sperm yet, which is the purpose of the study. For this, commercially available frozen semen from four bulls was used. Two straws of each bull (n=4) were thawed (37°C/30 seconds). The sample was centrifuged at 500 g/10 minutes to remove the supernatant (extender sperm free), stored at 5°C. The precipitate was suspended in 200 μL of Tyrode's albumin lactate pyruvate (TALP) and added to 50 µL of iron sulfate and 50 µL of ascorbic acid kept at 37°C/90 minutes maintained with open tube cap for oxidative stress induction. The sample was centrifuged at 500 g/10 minutes. The supernatant was removed and the precipitate was suspended in 300 μL of TALP. The concentration was adjusted to 20x106 sperm/mL, diluting one sample in TALP (control group), and another in the stored extender. It was performed that to evaluate if the probe would be able to identify sperm with ROS in control group, since in extender group probably it would be observe a little level of oxidative stress because of antioxidants proprieties of extender. Aliquots of 200 μL of the samples (control x extender) were added with 4 μL of CellROX Deep RED® 1 mM and 1μl of Hoescht 33342 0.5 mg/mL (Molecular Probes) and incubated at 37°C/30 minutes. The samples were centrifuged at 5000 g/5 minutes, the supernatant was removed and the samples were suspended in 200 μL of TALP. It was prepared a humid chamber with 4 μL of the samples and 200 cells per slide were counted. The cells were classified as absent or few presence of ROS (FEW), moderate level of ROS (MOD) and intense level of ROS (INT). FEW and INT variables were transformed and subjected to analysis of variance (ANOVA). For the variable MOD, an evaluation by nonparametric statistics was made. It was used SAS software (SAS Institute Inc., 2004) and the significance level was 5%. After the stress induction, control group showed a reduction (p=0.005) in FEW cells (5±3.84%) when compared to extender group (74.87±18.54%). There was no difference (p=0.24) between control group (68.87±4.14%) and extender group (24.50±18.26%) for MOD cells. However, control group (26.12±1.86%) showed higher (p<0.0001) INT amount of cells than the extender group (0.62±0.31%). Thus, it can be concluded that the CellROX® probe is able to identify ROS in bovine spermatozoa and that the extender is capable of neutralizing these species. However, more studies are being done by our group to confirm the efficiency of the ROS evaluation in bull criopreserved sperm.