Comparative evaluation of sperm quality assessment methods in purebred spanish horses

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.