Nicotinic acid addition to equine frozen semen extender influences the number of sperm bound to bovine oviduct explants

Usually, equine non-capacitated sperm bind to the isthmus region of the oviduct before ovulation occurs, a process believed essential to prepare them to fertilize the oocyte. However, during semen freezing most of the seminal plasma, a rich source of antioxidants is discarded, resulting in a higher sperm vulnerability to oxidative damage. Thus, the aim of this study was to investigate the influence of adding nicotinic acid, an antioxidant, to equine frozen semen extender to improve the binding capacity of frozen equine sperm to bovine oviduct cell explants, as an indirect way to investigate the sperm fertilizing capability. To obtain bovine oviducts, reproductive systems of abattoir slaughtered cows were transported in saline solution at 35°C to the laboratory. After sectioning the uterus-tubal junction ipsilateral to the ovaries without a corpus luteum, the isthmus region was pressed towards the uterus and its contents expelled in a Petri dish with culture medium. The oviduct contents with medium were decanted into a tube and after 5 minutes, disaggregated using a needle and syringe three times. Then, the contents were deposited in Petri dishes and cultured for 24 hours in an incubator with 5% CO2 at 38°C. Semen from ten stallions was frozen in INRA96 (control, no nicotinic acid) and with 10mM and 20mM nicotinic acid. Thawed spermatozoa(30 × 106 sperm/mL) were co-incubated with 20 oviduct explants (aggregated oviduct cells) for 30min in an incubator with 5% CO2 at 38°C. Subsequently, the explants were washed with medium to remove the loosened sperm, placed on a glass slide, and covered with a coverslip. The number of sperm bound per nm of oviduct was observed with phase contrast microscopy (40x) and counted with ImageJ software. For statistical analysis, mean and standard error were calculated. When the data did not show normal distribution, logarithmic transformation was performed and the log mean values were evaluated with variance analysis and compared with Duncan's test. The probability of P<0.05 was considered significant. The addition of 10mM nicotinic acid to equine frozen semen extender increased the number of sperm bound to the explants (79.9 ± 9.5 sperm/nm explant) compared to the control (49.1 ± 6.8 sperm/nm explant, P<0.05) and 20mM nicotinic acid treatment (65.6 ± 8.3 sperm/nm explant, P<0.05). In conclusion, the addition of 10mM nicotinic acid to equine freezing extenders improved the number of sperm bound to bovine oviduct explants. Hence, nicotinic acid may be a useful addition to freezing extenders.