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 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 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.