Zabala Argüelles, Sonsoles MercedesSerres Dalmau, María ConsolacionMontero Serra, NataliaCrespo Castejón, FranciscoLorenzo González, Pedro LuisPérez-Aguilera, VerónicaGalán López, CarmenDomínguez Gimbernat, MónicaOliet Palá, AgustínMoreno, SantiagoGonzález Zorn, BrunoGutiérrez Cepeda, Luna2024-04-022024-04-022024-01-05Zabala SM, Serres C, Montero N, Crespo F, Lorenzo PL, Perez-Aguilera V, Galan C, Dominguez-Gimbernat M, Oliet A, Moreno S, Gonzalez-Zorn B and Gutierrez-Cepeda L*. Strategies to Reduce the Use of Antibiotics in Fresh and Chilled Equine Semen. Animals, 14(2):179. 2024. (A). ISSN: 2076-2615. Impact factor 2022: 3.000. Category: Agriculture, dairy & animal science, Quartile: 1, Position: 12 of 62. DOI: 10.3390/ani140201792076-261510.3390/ani14020179https://hdl.handle.net/20.500.14352/102498Author Contributions: The study was conceived by L.G.-C. and C.S. who, together with S.M.Z., B.G.-Z., P.L.L., S.M. and F.C., participated in its design and coordination. S.M.Z., L.G.-C., V.P.-A., M.D.-G., C.G. and F.C. performed the semen collection, and processed the samples for refrigeration. Semen evaluation was executed by S.M.Z., V.P.-A., A.O., C.G., M.D.-G., F.C. and L.G.-C. Microbial load assessment was conducted by B.G.-Z. and N.M. with the assistance of S.M.Z. and V.P.-A. for sample preparation. L.G.-C. and C.S. performed the statistical analysis, with the support of Pedro Cuesta. C.S., L.G.-C., S.M.Z., B.G.-Z., P.L.L., F.C. and S.M. interpreted the data. The manuscript was written by L.G.-C., S.M.Z., C.S. and F.C., helped by the rest of authors, who revised it critically. All authors have read and approved the final version of the manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work have been appropriately investigated and resolved. All authors have read and agreed to the published version of the manuscript.The study assessed the impact of four equine semen processing techniques on sperm quality and microbial load immediately post-processing and after 48 h of refrigeration. The aim was to explore the potential reduction of prophylactic antibiotic usage in semen extenders. Semen from ten adult stallions was collected and processed under a strict hygiene protocol and divided into four aliquots: Simple Centrifugation with antibiotics (SC+), Simple Centrifugation (SC−), Single-Layer Colloidal Centrifugation (CC−), and Filtration (with SpermFilter®) (F−), all in extenders without antibiotics. Sperm motility, viability, and microbial load on three culture media were assessed. No significant differences were observed in the main in the sperm quality parameters among the four protocols post-processing and at 48 h (p < 0.05 or p < 0.1). Microbial loads in Columbia 5% Sheep Blood Agar and Schaedler vitamin K1 5% Sheep Blood Agar mediums were significantly higher (p < 0.10) for raw semen than for CS+, CC−, and F− post-processing. For Sabouraud Dextrose Agar medium, the microbial load was significantly higher (p < 0.10) in raw semen compared to CS+ and F−. No significant differences (p < 0.10) were found in 48 h chilled samples. Regardless of antibiotic presence, the evaluated processing methods, when combined with rigorous hygiene measures, maintained semen quality and reduced microbial load to the same extent as a traditional protocol using antibiotics.engAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/journal article10.3390/ani14020179open access636.09chilled equine semensemen microbial loadsimple centrifugationsingle-layer colloidal centrifugationfiltrationabsence of antibioticsantibiotics-free extenderVeterinaria3109 Ciencias Veterinarias