2. Universidad Cardenal Herrera-CEU

Although the effects of sperm microbiota and sperm quality have been described previously, recent studies provide evidence that female genital modifications triggered by seminal components could be of significant importance to identify some disturbances associated with fertility. So, sperm microbiota could play a key role in sperm quality, contributing to fertilisation. To understand how sperm microbiota diversity is influenced by the host genetics, the symbiotic bacteria in four inbred lines raised in the same animal facility and their effects on sperm quality and fertility were analysed. Forty healthy rabbits from four selected Spanish commercial lines were used in this research (three based on litter performance, designated A, V and LP, and one selected for daily body weight gain, called R). Significant variations in the seminal concentration, morphology and some motion parameters were found among inbred lines, but sperm motility and viability were similar among inbred lines. After mating, inbred lines selected for litter size had the same fertility rate, significantly higher than inbred line selected for body weight (82±3.3%, 79±3.5% and 89±4.5% versus 61±3.7%, for the A, V and LP vs R lines, respectively, p<0.05). Bacteria belonging to Proteobacteria, Firmicutes, Fusobacteria and Bacteroidetes were identified in sperm microbiota. At genus level, the bacterial community composition in the sperm microbiota was influenced by host genetics. A total of 35, 16, 34, and 51 genera were accurately detected in the A, V, LP, and R lines, respectively. Moreover, Enhydrobacter, Ferruginibacter, Myroides Paracoccus, Rheinheimera, Tepidiphilus, Tetradesmus obliquus and Thauera genera were present only in the inbred lines selected for litter size. Moreover, the discriminant analysis revealed Lysinibacillus and Flavobacterium genera as potential biomarkers for fertility. Thus, these two genera may play a key role in fertility. Our results demonstrated the 3 existence of a rabbit inbred line-specific variation in bacterial occurrence in sperm microbiota. Moreover, fertility differentials among inbred lines that were not predicted by routine semen analysis could be partly explained by the symbiotic state of the semen microbiota.

Although great attention is paid to hygiene during semen collection and processing, bacteria are commonly found in the semen of healthy fertile males of different species. As the storage of extended semen might facilitate bacterial growth, extenders are commonly supplemented with antibiotics. This study aimed to evaluate the antibacterial activity of ethylenediaminetetraacetic acid (EDTA), bestatin and chitosan-based nanoparticles added to rabbit semen extender and their effect on reproductive performance under field conditions. Four different extenders were tested, supplemented with antibiotics (TCG+AB), with EDTA and bestatin (EB), with EDTA, bestatin and chitosan-based nanoparticles (QEB) or without antibiotics (TCG-AB). Extended semen was cooled at 15 C for three days. Cooled samples were examined for bacterial growth and semen quality every 24 h for 3 days. The enterobacteria count increased considerably during storage at 72 h in semen extended with TCG+AB and TCG-AB, while extenders EB and QEB showed a bacteriostatic effect over time. After 24, 48 and 72 h, quality characteristics were retained in all groups, with no significantmotility differences, either in acrosome integrity, membrane functionality or the viability of spermatozoa. Additionally, bacterial concentration present in fresh semen did not affect reproductive performance. In conclusion, EDTA and bestatin exerted a potent bacteriostatic effect over time and could be used as an alternative to conventional antibiotics in rabbit semen extenders.

In recent decades, gamete and embryo cryopreservation have become routine procedures in livestock and human assisted reproduction. However, the safe storage of germplasm and the prevention of disease transmission continue to be potential hazards of disease transmission through embryo transfer. This study aimed to demonstrate the potential risk of cross-infection of embryos from contaminated liquid nitrogen, and cross-contamination of sterile liquid nitrogen from infected embryos in naked and closed devices. Additionally, we examined the e ects of antibiotic-free media on culture development of infected embryos. The study was a laboratory-based analysis using rabbit as a model. Two experiments were performed to evaluate both cross-infection (liquid nitrogen to embryos) and cross-contamination (embryos to liquid nitrogen) of artificially inoculated Salmonella Typhimurium, Staphylococcus aureus, Enterobacter aerogenes, and Aspergillus brasiliensis. Rapid cooling through vitrification was conducted on rabbit embryos, stored for a year, thawed, and cultured. In vivo produced late morulae–early blastocyst stages (72 h) embryos were used (n = 480). Embryos were cultured for 1 h in solutions with and without pathogens. Then, the embryos were vitrified and stored in naked and closed devices for one year in two liquid nitrogen biobanks (one pathogen-free and the other artificially contaminated). Embryos were warmed and cultured for a further 48 h, assessing the development and the presence of microorganism (chromogenic media, scanning electron microscopy). Embryos stored in naked devices in artificially contaminated liquid nitrogen became infected (12.5%), while none of the embryos stored in closed devices were infected. Meanwhile, storage of artificially infected embryos incurred liquid nitrogen biobank contamination (100%). Observations by scanning electron microscopy revealed that all the microorganisms were caught in the surface of embryos after the vitrification-thawed procedure. Nevertheless, embryos cultured in antibiotics and antimycotic medium developed to the hatched blastocyst stage, while artificially infected embryos cultured in antibiotic-free medium failed to develop. In conclusion, our findings support that both cross-contamination and cross-infection during embryo storage in liquid nitrogen biobanks are plausible. So, to ensure biosafety for the cryogenic storage, closed systems that avoid direct contact with liquid nitrogen must be used. Moreover, it seems essential to provide best practice guidelines for the cryogenic preservation and storage of gametes and embryos, to define appropriate quality and risk management procedures.