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

## TL;DR

This study explores antibiotic-free methods for preserving horse semen, aiming to reduce antimicrobial resistance while maintaining sperm quality.

## Contribution

The study introduces antibiotic-free techniques for equine semen cryopreservation that maintain microbial control and sperm quality.

## Key findings

- Filtration and Colloidal Centrifugation achieved microbial load reduction comparable to antibiotic use.
- Sperm motility and viability were similar across methods, except for lower total motility in antibiotic-free treatments.
- Antibiotic-free methods combined with hygiene practices offer a viable alternative to reduce antimicrobial use.

## Abstract

Cryopreserved equine sperm is vital for global trade and genetic preservation. However, the antimicrobials included in semen extenders for this processing may be exacerbating the development of antimicrobial resistance, which is recognized by the World Health Organization as a major global health threat. This research evaluated three different processing methods—Simple Centrifugation (with/without antibiotics), Filtration and Colloidal Centrifugation, both without antibiotics—evaluating their effect on microbial load, motility and viability in frozen–thawed equine sperm. Post-freezing microbial load analysis showed a similar reduction across all methods, with Filtration and Colloidal Centrifugation without antibiotics achieving comparable values to the antibiotic control group. The results revealed no significant differences in progressive motility, average path velocity, straight-line velocity or wobble parameters between protocols. However, total motility was lower in the antibiotic-free Filtration and Colloidal Centrifugation methods compared to Simple Centrifugation with antibiotics. Viability was significantly higher in the control group with antibiotics than in the other antibiotic-free treatments. These findings suggest that the proposed alternative processing techniques, when combined with optimized hygiene practices, could control microbial load and maintain semen quality in cryopreserved sperm without antibiotics, offering a promising strategy to reduce antimicrobial resistance. Further research is recommended to validate these results and support their broader application.

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+ (p < 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 (p < 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.