Innovative Approaches to Avoid Antibiotic Use in Equine Semen Cryopreservation: Advancing Sustainable Reproductive Technologies

This study evaluated the impact of different processing techniques on microbial load and sperm quality in frozen-thawed equine semen to identify alternatives to reduce the preventive use of antibiotics. Semen was obtained and processed under rigorous hygiene measures from ten stallions, using four protocols: Simple Centrifugation with antibiotics (S+) and Simple Centrifugation (S-), Filtration (F-) and Single-Layer Colloidal Centrifugation (C-) in an antibiotic-free extender. Microbial load in different culture media, sperm viability and motility were assessed. Microbial load results were consistent across protocols, except in Columbia 5% Sheep Blood Agar media, where S- exhibited higher microbial load than S+ (<i>p</i> < 0.05). However, F- and C- showed similar microbial loads to S+. No significant differences were observed in progressive motility, average path velocity, straight-line velocity or wobble parameters between protocols. Total motility and viability were significantly higher in S+ compared to other treatments (<i>p</i> < 0.05). Thus, regardless of antibiotics, the proposed methods achieved results similar to the traditional antibiotic-inclusive protocol in terms of microbial load and the most relevant semen quality parameters. These findings suggest that the use of F- and C-, combined with optimized hygiene measures, offers an effective alternative to reduce the prophylactic use of antibiotics in semen extenders.