Resveratrol addition to freezing equine semen extender influence the number of sperm bound to bovine oviduct explants

Abstract

During semen freezing, approximately 90% of the seminal plasma is discarded, resulting in higher sperm vulnerability to lipid peroxidation. Thus, the addition of antioxidants, such as resveratrol, to equine semen freezing extenders may increase the viability of cryopreserved sperm. Further, non- capacitated sperm usually bind to the isthmus region of the oviduct, which can be a way to identify the sperm able to fertilize the oocyte. The aim of the present study was to evaluate the binding capacity of cryopreserved equine sperm to bovine oviduct explants. To prepare the oviduct explants, the reproductive system of slaughtered cows was 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 were expelled in a Petri dish with culture medium. These oviduct content with the medium was deposited in 14 ml tube decanted and after five minutes it was disaggregated with a needle and syringe. After repeating this process three times, the contents were deposited in Petri dishes and cultured for 24 hours in an incubator in atmosphere with 5% CO 2 . The explants were distributed in six treatments according to the resveratrol concentration added to the INRA 96 freezing extender. Semen from sixstallions were frozen in the INRA96 (control, no resveratrol) and with 5, 10, 50, 100 and 150 μM resveratrol. The six treated semen samples were co-incubated with the explants for a min. Then, the explants were washed with a medium to withdraw the loosened sperm and placed on glass slide and covered with a coverslip. The number of sperm bound per nm of oviduct was observed with microscopy and analysed with the ImageJ software. For the statistical analysis, mean and standard error were calculated and when the data did not have a normal distribution, logarithmic transformation was performed, and the log means were evaluated with variance analysis and compared with Duncan's test. The probability of P<0.05 was considered significant. The addition of 5μM resveratrol to the equine semen freezing extender increased the number of sperm bound explants (64.6 ± 1.1 sperm/nm explant), while 50μM resveratrol reduced (21.4 ± 1.1 sperm/nm of explant) compared to the other treatments (control: 48.1 ± 1.0, 10 μM: 56.2 ± 1.0, 100 μM: 46.8 ± 1.0 and 150 μM resveratrol: 53.7 ± 1.1 sperm/nm of explant). In conclusion, the addition of 5 μM resveratrol in frozen equine semen extender was improved equine sperm binding ability to oviduct explants.