Evaluation of simple strategies to reduce the use of antibiotics in equine fresh sperm

According to the World Health Organization in 2021, the development of antimicrobial resistances is considered one of the top ten global public health threats. Microbial load negatively affects sperm quality and preservation (Ortega-Ferrusola and Peña, Reproduction in Domestic Animals. 2009; 44(3):518-522), which implies the use of antibiotics in semen extenders. The objective of this study was to study the effect of three semen processing techniques on quality and microbial load of fresh equine sperm to evaluate the possibility of reducing or eliminating the prophylactic use of antibiotics in the future. Ten adult stallions were included (one ejaculate per male). To avoid environmental contamination during sperm collection and processing, a protocol that maximizes hygiene measures was implemented. Aliquots of each ejaculate were processed following 4 protocols: Simple Centrifugation (450g x 7 min) in extender with antibiotics (SC+, as control group), Simple Centrifugation in extender without antibiotics (SC-), Single-Layer Colloidal Centrifugation (300g x 20 min) in extender without antibiotic (CC-) and Filtration (SpermFilter®) in extender without antibiotics (F-). Sperm motility (total and progressive motility, TM and PM, respectively, %), viability (Alives, %), and microbial load (on three different culture media (Columbia 5% Sheep Blood Agar, COS, general purpose medium for non-fastidious and fastidiousmicroorganisms, Sabouraud Dextrose Agar, SDA, for yeasts and molds, and Schaedler vitamin K1 5% Sheep Blood Agar, SCH, for fastidious anaerobic bacteria) were evaluated after processing. Statistical analysis was performed by Kruskal-Wallis, ANOVA and post hoc tests (p<0.05). Results are expressed as mean values ± SD. No significant differences were found between the control (SC+) and other groups (SC-, CC- and F-), neither in sperm characteristics (61,32a ± 20,84, 59,13a ± 21,84, 63,19a ± 18,87 and 52,54a ± 21,08 for PM and 84,60a ± 9,21, 85,05a ± 9,14, 84,90a ± 13,5 and 77,35a ± 13,27 for ALIVES respectively) nor in microbial load (216,50a ± 504,41, 348,30a ± 792,99, 731,30a ± 2180,11 and 51,70a ± 64,64 for COS; 1,00a ± 3,16, 11,00a ± 31,43, 4,00a ± 6,99 and 1,00a ± 3,16 for SDA and 25,00a ± 57,59, 41,00a ± 60,45, 10,00a ± 18,26 and 26,00a ± 42,48 for SCH respectively). The present processing methods, in combination with management measures that prevent environmental contamination, maintained sperm quality and microbial load of equine fresh semen to the same extent as traditional sperm processing protocols with antibiotics. Acknowledgements: Authors thank P. Cuesta for statistical assistance. This research was funded by Madrid Institute for Rural, Agricultural and Food Research and Development (IMIDRA).