Francisco Crespo

This study evaluated the association of BCS and serum metabolic parameters with sperm quality during 72 h of refrigeration at 5 °C in 20 Purebred Spanish stallions. Motility, vitality, acrosome integrity, mitochondrial membrane potential, protamination and DNA fragmentation were assessed at 0, 24, 48 and 72 h. Storage time significantly affected most sperm parameters, confirming a progressive decline in quality during refrigeration. Two-way repeated measures ANOVA revealed that stallions with high BCS (7–8) showed a significantly lower percentage of sperm with normal acrosome integrity (p = 0.0426) than those with normal BCS (4–6), with no BCS × storage time interaction. Similarly, high triglyceride and cholesterol levels were each associated with reduced acrosome integrity throughout refrigeration. A few group × storage time interactions (vitality with glucose and cholesterol, progressive motility with cholesterol, acrosome integrity with leptin) were exploratory, without differences at individual times. These results indicate that the metabolic profile is associated with sperm membrane-related parameters during cooled storage. In contrast, the absence of fertility data and independent validation means that these results do not support metabolic profiling as a breeding soundness test and need confirmation in larger studies.

Bacterial contamination of donkey semen samples is a major concern due to its deleterious effects on sperm quality; this issue is exacerbated during semen storage. Therefore, evaluating the effect of antimicrobials on sperm is of utmost importance. This study identified microorganisms present in donkey semen and evaluated the antimicrobial activity of cinnamaldehyde, determined by the Minimum Inhibitory Concentration (MIC), and its effects on sperm quality. Fourteen semen samples from seven donkeys were analysed, identifying 31 bacterial isolates representing 21 different species. Cinnamaldehyde showed antimicrobial activity (MIC ₅₀ = 0.06 mg/mL; MIC ₉₀ = 4 mg/mL). Reactive oxygen species levels in sperm were decreased, suggesting an antioxidant effect. However, high concentrations of cinnamaldehyde reduced sperm motility. In conclusion, cinnamaldehyde is a promising antimicrobial for semen preservation; however, further studies are needed to ensure antimicrobial efficacy and sperm functionality.

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.

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.

The growing relevance of the Purebred Spanish horse has driven the extensive use of assisted reproductive technologies to preserve elite stallion genetics. Optimal reproductive performance is intrinsically linked to systemic health and high semen quality. This study aimed to evaluate the impact of body condition on the stallion’s metabolic and endocrine profiles and their seminal parameters. Semen and blood samples were collected from 26 stallions (February–May in 2024–2025) whose body condition score (BCS) was assessed using the Henneke scale. They were classified into two groups, low BCS with a score range of 4–6 (n = 14) and high BCS with a score range of 7–8 (n = 12). The low BCS is regarded as a normal condition, whereas the high BCS is regarded as obesity. Metabolic and endocrine parameters and semen quality variables were analyzed using ANOVA, and principal component analyses (PCA). Stallions with high BCS showed significantly increased insulin and triglycerides concentrations (p = 0.0263 and p = 0.0046, respectively), with a positive correlation between BCS and insulin levels (r = 0.576, p = 0.04), as well as between BCS and triglyceride levels (r = 0.525, p = 0.0049). A significantly lower sperm count was observed in stallions with high BCS (p < 0.0001), together with lower total sperm motility (p = 0.0055), when compared to stallions with low BCS. PCA of blood parameters revealed the first component (PC1) associated with glucose, insulin, triglycerides, and cholesterol. In semen’s PCA, PC1 was associated with quality parameters, while PC2 was influenced by total sperm count. No direct correlation was found between PCA components and BCS. However, integration of both PCA showed that increased insulin, triglycerides, and cholesterol were significantly associated with a reduced total sperm count. These findings suggest that reproductive changes in stallions with high BCS are primarily mediated by metabolic and endocrine dysfunction rather than adiposity per se.

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.

The aim of this study was to determine the effect of silver nanoparticles (AgNPs) on donkey sperm parameters and ultrastructure. AgNPs were synthesized, purified and resuspended in the extender. Nine frozen–thawed donkey sperm samples were exposed to different concentrations of AgNPs (0, 1.25, 2.5, 5, 12.5, 25 and 50 μg/mL). Sperm parameters: total (TMOT, %) and progressive (PMOT, %) sperm motility, plasma (LIVE, %) and acrosomal membrane integrity (AIS, %), and sperm morphology (MORF, %) were evaluated immediately after AgNPs exposure (T0) and after 2 h of incubation (T2). The interaction beween AgNPs and spermatozoa was visualized by transmission electron microscopy (TEM). At T0, sperm motility and AIS were reduced (p < .05) when using concentrations ≥50 and ≥25 μg/mL, respectively. At T2, sperm motility and LIVE were significantly decreased (p < .05) in concentrations ≥25 and ≥50 μg/mL, respectively. TEM analysis revealed nanoparticle adhesion to the acrosomal region of the plasma membrane. In conclusion, AgNPs at concentrations ≥25 μg/mL impair motility, acrosome and plasma membrane integrity of donkey sperm, which may be mediated by adhesion to the acrosomal region of the sperm surface membrane.

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.

The Purebred Spanish Horse, according to our clinical experience, is characterized by having a high number of stallions that do not meet the international commercial recommendations for equine-sperm cryopreservation. This means that artificial insemination with frozen semen from these stallions is less widespread than in other breeds. In this study, we investigated if the incorporation of single-layer colloidal centrifugation prior to cryopreservation in clinical conditions could increase the number of ejaculates of Purebred Spanish stallions suitable for this processing, observing the influence of centrifugation and freezing extender protocol on post-thawed sperm motility. Using colloidal centrifugation, the percentage of ejaculates available to be frozen was increased from 35% (6/17) to 71% (12/17), doubling the number of samples that could have been subjected to cryopreservation. We only found significant differences in linearity (LIN) and lateral head displacement (ALH) after 5 min of incubation at 37 °C between colloidal and simple centrifugation processing techniques. No significant differences were found between the two different colloidal protocols in any of the variables considered. Colloidal centrifugation allowed us to obtain, from worse fresh-quality ejaculates, thawed sperm doses with similar quality to that of good-quality ejaculates. BotuCrio^® produced, in general, higher motility parameters and its characteristics than the other extenders analyzed, with significant differences found in comparison to Inra-Freeze^® and Lac-Edta in both total (MOT) and progressive motility (PMOT) when using colloidal centrifugation and only in PMOT when applying simple centrifugation. Colloidal centrifugation optimized the efficiency of cryopreservation, as it allowed us to increase the number of ejaculates of Purebred Spanish Horses suitable to be frozen. Including these semen processing techniques in the freeze test could help to optimize equine-sperm cryopreservation protocols, especially when dealing with individuals or breeds for which initially low sperm quality prevents or limits their inclusion in sperm cryopreservation programs.

Despite the advances in reproductive technology, there is still a considerable number of low sperm quality cases in stallions. Recent studies in humans have detected several seminal microflora–spermatozoa associations behind some idiopathic infertility cases. However, no studies are available on horses, and there is limited information on the microflora present in stallion ejaculates. Accordingly, the objective of this study was to examine associations to the presence of bacteria families with five sperm quality parameters: concentration, total number of spermatozoa, total and progressive motility, and DNA fragmentation. Samples were cryopreserved after their extraction. High-speed homogenization using grinding media was performed for cell disruption. Family identification was performed via 16S rRNA sequencing. Bacterial families were only considered if the relative abundance was higher than 1%. Only two families appeared to have a correlation with two sperm quality parameters. Peptoniphilaceae correlated positively with total sperm motility, whereas Clostridiales Incertae Sedis XI correlated negatively with progressive motility. No significant differences were found for the rest of the parameters. In conclusion, the seminal microbiome may affect spermatozoa activity. Our findings are based on statistical associations; thus, further studies are needed to understand the internal interactions between seminal flora and cells.