Evaluation of the effects of photostimulation on freeze-thawed bull sperm cells in terms of reproductive potential.

The aim of this study was to determine the time-dependent effectiveness of photo-stimulation against bovine sperm cells using a multi-wavelength LED (Light Emitting Diode). Spermatological parameters were evaluated for viability, acrosome structure and motility. In addition, the effect of photo-stimulation on frozen-thawed sperm cells subjected to in vitro capacitation was evaluated in terms of changes in mitochondrial membrane potential. The study consisted of two separate experiments and a total of 32 sperm samples obtained from separate bulls were used. All sperm samples were obtained from Holstein bulls using an artificial vagina. Semen was diluted to a final concentration of 92 x 106 spermatozoa per ml in 0.25 ml straws. The sperm cells were frozen using the conventional method. Straws were kept in a 37°C water bath for 20 seconds and diluted 1:4 in phosphate buffered saline (PBS) to eliminate the potentially deleterious effect of glycerol, the main permeable cryoprotectant in the freezing medium for bull sperm. This dilution also helped in the evaluation of sperm quality parameters. In the first experiment, whereas the 15-10-15 showed no differences with the control, other treatments such as 10-10-10, 5-5-5, and 3-1-3 exhibited significantly higher percentages of viable spermatozoa at 24h. The results obtained for acrosome integrity were pretty much similar to those observed in the sperm viability assessment. In effect, while the treatment consisting of 15-10-15 had no positive effects, shorter treatments exerted a much more positive effect. The percentages of acrosome-intact spermatozoa in 2-1-2 and 1-1-1 were significantly higher than those obtained in the control. The significant differences in mitochondrial membrane potential were observed at 0, 2, 4 and 24h post-photo-stimulation in all treatments, except 15-10-15. The highest increase in the percentage of spermatozoa exhibiting high mitochondrial membrane potential was found in 10-10-10, 5-5-5 and 3-1-3 treatments. With regard to total and progressive motility, whereas 10-10-10 was the best regime, 5-5-5 and 3-1-3 treatments also had a positive effect. However, 15-10-15 appeared to have a stimulating effect upon progressive motility at 2h and 4h but later declined and showed no significant differences with regard to the control at 24h. In the second experiment, not immediately after thawing but after having been kept at room temperature for up to 24h, it was observed that there was no statistical difference in terms of viability, acrosome integrity and total/progressive motility between photostimulation and the control group. This indicates that photo- stimulation is less able to exert a beneficial effect when post-thawed sperm are not immediately stimulated. As a result it was determined that photo-stimulation at a pattern of 10-10-10, 5-5-5, 3-1-3 and, to a lesser extent 2-1-2, increases the resilience of frozen-thawed bull sperm when applied upon thawing.