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.

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.

Evaluating semen quality in Purebred Spanish Horses is essential to determine reproductive potential and optimize assisted reproductive techniques. This study compared several sperm analysis methods in Purebred Spanish Horses to standardize protocols suitable for both laboratory and field conditions. Fifteen stallions were evaluated through three ejaculates, analyzing sperm parameters using different techniques. Sperm kinematics was assessed by Sperm Computer Analyzer (SCA-CASA) or iSperm mCASA. Viability was analyzed by eosin-nigrosin-Giemsa (ENG) staining or Hoechst 33258. Peanut agglutinin-FITC (PNA-FITC) or ENG were used for acrosome integrity. PNA-FITC or chlortetracycline (CTC) were employed for capacitation. Sperm protamination was evaluated by Chromomycin A3, Diff-Quik, toluidine or aniline blue. DNA fragmentation was assessed by sperm chromatin structure assay (SCSA). SCA-CASA and iSperm mCASA differed significantly in most kinematic parameters. Viability was higher with ENG (85.4 ± 2.28%) than Hoechst33258 (64.9 ± 3.70%; P=0.0015). Acrosomal integrity was more reliably assessed using PNA-FITC than ENG (P = 0.021). PNA-FITC results correlated with CTC (r=0.836) for capacitation . Protamination analysis showed a significant correlation between Chromomycin A3 and aniline blue (r=0.689). The SCSA detected higher DNA damage levels than Chromomycin A3, aniline blue, or toluidine blue (P = 0.0030, P=0.0005, and P=0.0145, respectively). SCA-CASA offers greater precision compared to iSperm mCASA. ENG is suitable for viability assessment, while PNA-FITC enables simultaneous evaluation of acrosomal status and capacitation patterns. Aniline blue is appropriate for chromatin maturation analysis under bright-field microscopy. However, for a highly sensitive and objective evaluation, DNA fragmentation should be assessed via SCSA, although cytochemical stains remain valid alternatives for routine screening.

The aim of this study was to evaluate the effect of silver nanoparticles (AgNPs) on donkey sperm parameters and their antimicrobial efficacy on bacteria isolated in the presence of antibiotics. Nine donkey ejaculates were collected and stored in a skim‐milk extender at different AgNPs concentrations (0, 0.01, 0.8, 1.6 and 3.12 mg/mL) at 5°C or 17°C. Sperm parameters, including total and progressive motility (CASA), plasma membrane integrity (PMI) and reactive oxygen species (rROS) production, were evaluated at 0, 24 and 48 h of storage. Additionally, bacteria isolated from donkey semen extended with antibiotics were exposed to AgNPs to determine the minimum inhibitory/bactericidal concentrations (MIC/MBC). Sperm motility parameters were higher at 17°C than 5°C after 48 h. AgNPs showed a dose‐ and time‐dependent cytotoxic effect, with concentrations ≥ 0.8 mg/mL significantly reducing motility, membrane integrity and increasing rROS after 24 h. AgNPs exhibited bactericidal activity at concentrations between 1.56 and ≥ 25 mg/mL. In conclusion, while liquid storage at 17°C is an effective strategy for preserving donkey semen, the use of AgNPs as a standalone antimicrobial is currently limited by their cytotoxicity at effective bactericidal doses.

Preservation protocols for donkey sperm are based on those developed for horses, despite potential interspecific differences in cooling tolerance. This study compared horse and donkey sperm during long‐term storage at 5°C and 17°C. Ejaculates from four stallions and four jacks ( n = 16) were centrifuged, resuspended in skimmed milk‐based extender, and stored at both temperatures. Sperm motility, plasma and acrosome membrane integrity, and morphology were evaluated after 1, 2 and 7 days. Fresh semen parameters showed no interspecific differences ( p > 0.05). At 5°C, donkey sperm showed reduced motility and membrane integrity from Day 1, whereas horse sperm parameters remained stable at both temperatures up to Day 2. After 7 days, horse doses stored at 5°C maintained significantly higher quality. In conclusion, donkey sperm was more sensitive to storage at 5°C than horse sperm, highlighting the need for species‐specific preservation strategies.