Male subfertility is a global issue in human reproduction as well as in animal reproduction. Bacterial infection and semen contamination are still widely overlooked. As the collection of ejaculates is not a sterile process, it is necessary to add antimicrobial agents to avoid a possible depreciation of semen samples. As traditionally used antibiotics have been questioned because of an ever-increasing bacterial resistance, natural bioactive molecules could offer an alternative because of their antibacterial and antioxidant properties. As such, we decided to compare the effects of selected natural biomolecules (resveratrol-RES, quercetin-QUE and curcumin-CUR) with routinely used antibiotics in animal biotechnologies (penicillin-PEN, gentamicin-GEN and kanamycin-KAN) on the rabbit sperm vitality in the presence of Enterococcus faecalis. Changes in the sperm structural integrity and functional activity were monitored at 0, 2, 4 and 6 h. Computer-assisted sperm analysis (CASA) was used for the assessment of spermatozoa motility. Production of reactive oxygen species (ROS) was evaluated using chemiluminiscence, while the mitochondrial membrane potential (ΔΨm) was examined using the JC-1 dye. Finally, the sperm chromatin dispersion (SCD) test was used to assess DNA fragmentation, and changes to the membrane integrity were evaluated with the help of annexin V/propidium iodide. The motility assessment revealed a significant sperm motility preservation following treatment with GEN (p < 0.001), followed by PEN and CUR (p < 0.01). QUE was the most capable substance to scavenge excessive ROS (p < 0.001) and to maintain ΔΨm (p < 0.01). The SCD assay revealed that the presence of bacteria and antibiotics significantly (p < 0.05) increased the DNA fragmentation. On the other hand, all bioactive compounds readily preserved the DNA integrity (p < 0.05). In contrast to the antibiotics, the natural biomolecules significantly maintained the sperm membrane integrity (p < 0.05). The microbiological analysis showed that GEN (p < 0.001), KAN (p < 0.001), PEN (p < 0.01) and CUR (p < 0.01) exhibited the strongest antibacterial activity against E. faecalis. In conclusion, all selected biomolecules provided protection to rabbit spermatozoa against deleterious changes to their structure and function as a result of Enterococcus faecalis contamination. Therefore, administration of RES, QUE and/or CUR to rabbit semen extenders in combination with a carefully selected antibacterial substance may be desirable.

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Metschnikowia pulcherrima is common yeast on grape berries and can grow on special cultivation media with iron (e.g. FeSO4.7H2O) (max. 0.02 g/L) to produce reddish (maroon) pigment pulcherrimin. Many studies confirm that pulcherrimin has antimicrobial activity against postharvest pathogens, microscopic fungi and other yeast species. In this study, two strains of M. pulcherrima, isolated from grapes and identified by MALDI-TOF mass spectrometry, were used : VML and CBS 610NT. Sixteen yeast species: Aureobasidium (1), Candida (5), Hanseniaspora (1), Kregenvanrija (1), Pichia (2), Rhodotorula (2), Saccharomyces (1), Zygosaccharomyces (2) and Wickerhamomyces (1) were selected to test the antimicrobial capacity of these two M. pulcherrima strains. The results showed CBS 610NT had a stronger ability than VML to inhibit the tested yeasts. Among the yeast, Candida spp. and Pichia manshurica were the most sensitive to the pigment pulcherrimin.

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