Comparative evaluation of sperm quality assessment methods in purebred spanish horses

BACKGROUND: Evaluating semen quality in Purebred Spanish Horses is essential to determine reproductive potential and optimize assisted reproductive techniques. AIMS: This study compared several sperm analysis methods in Purebred Spanish Horses to standardize protocols suitable for both laboratory and field conditions. METHODS: 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). RESULTS: 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). CONCLUSION: 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.