Comparative Evaluation of Sperm Quality Assessment Methods in Purebred Spanish Horses.
Abstract: Evaluating semen quality in Purebred Spanish Horses is essential to determine reproductive potential and optimize assisted reproductive techniques. Objective: 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). Conclusions: 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.
Copyright © 2026. Published by Elsevier Inc.
Publication Date: 2026-03-14 PubMed ID: 41839319DOI: 10.1016/j.jevs.2026.105851Google Scholar: Lookup
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Summary
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Overview
- This study compared various methods to evaluate sperm quality in Purebred Spanish Horses, aiming to identify the most reliable techniques for both lab and field use.
- The goal was to standardize sperm assessment protocols to better predict reproductive potential and improve assisted reproductive technologies in this horse breed.
Introduction and Purpose
- Assessing semen quality in Purebred Spanish Horses is crucial for understanding their fertility and optimizing reproductive interventions.
- The study focused on comparing different sperm analysis methods to establish standardized procedures adaptable to different settings (laboratory versus field conditions).
- Key sperm quality parameters included motility, viability, acrosome integrity, capacitation status, chromatin packaging (protamination), and DNA fragmentation.
Methodology
- Subjects: 15 Purebred Spanish stallions were evaluated.
- Sample Collection: Each stallion provided three ejaculates for analysis to ensure consistency and account for variability.
- Sperm Parameters and Associated Techniques:
- Motility/Kinematics: Assessed using two systems:
- Sperm Computer Analyzer (SCA-CASA)
- iSperm mobile CASA system (iSperm mCASA)
- Viability: Evaluated by:
- Eosin-nigrosin-Giemsa (ENG) staining
- Hoechst 33258 staining
- Acrosome Integrity: Measured via:
- Peanut agglutinin-fluorescein isothiocyanate (PNA-FITC) staining
- ENG staining
- Capacitation Status: Assessed using:
- PNA-FITC
- Chlortetracycline (CTC) staining
- Protamination (Chromatin Packaging): Analyzed through:
- Chromomycin A3
- Diff-Quik
- Toluidine Blue
- Aniline Blue
- DNA Fragmentation: Evaluated by the sperm chromatin structure assay (SCSA), a sensitive flow cytometry method.
- Motility/Kinematics: Assessed using two systems:
Key Findings
- Motility Analysis:
- SCA-CASA and iSperm mCASA showed significant differences in most sperm movement parameters, with SCA-CASA being the more precise system.
- Viability Assessment:
- ENG staining yielded a higher viability percentage (about 85.4%) compared to Hoechst 33258 (approximately 64.9%), indicating ENG’s better detection capability (significant at P=0.0015).
- Acrosome Integrity:
- PNA-FITC was more reliable than ENG staining for detecting intact acrosomes (P=0.021).
- Capacitation:
- Strong positive correlation (r=0.836) found between PNA-FITC and CTC staining, validating PNA-FITC as an effective method for detecting capacitation.
- Protamination Analysis:
- Chromomycin A3 and aniline blue stainings were significantly correlated (r=0.689), indicating their suitability for evaluating sperm chromatin maturation.
- DNA Fragmentation:
- Sperm chromatin structure assay (SCSA) detected higher levels of DNA damage compared to chromomycin A3, aniline blue, or toluidine blue stains (with P-values indicating strong significance).
- This suggests that SCSA is more sensitive and objective for detailed DNA integrity analysis.
Conclusions and Implications
- Motility: The SCA-CASA system is preferred for more accurate and precise sperm kinetics assessment compared to the portable iSperm mCASA.
- Viability: ENG staining is recommended as an effective method for routine viability evaluation under both lab and field conditions.
- Acrosome and Capacitation Status: PNA-FITC staining is versatile, allowing simultaneous evaluation of acrosomal integrity and capacitation, making it advantageous over other stains.
- Protamination (Chromatin Maturation): Aniline blue staining provides a practical bright-field microscopy technique suitable for chromatin evaluation during routine semen analysis.
- DNA Fragmentation:
- SCSA remains the gold standard for detecting DNA damage due to its sensitivity and objectivity.
- However, cytochemical stain techniques (Chromomycin A3, aniline blue, toluidine blue) provide valid, simpler alternatives for regular screening purposes.
- This standardized comparative evaluation helps improve semen quality assessment protocols, which can enhance reproductive management practices in Purebred Spanish horses.
Cite This Article
APA
Latorre N, Gómez-Cuétara C, Cañizares E, Crespo F, Pérez-Aguilera V, Cuerda MI, Laborda-Gomariz JA, Soler AJ, Roldan ERS, Sanchez-Rodriguez A.
(2026).
Comparative Evaluation of Sperm Quality Assessment Methods in Purebred Spanish Horses.
J Equine Vet Sci, 105851.
https://doi.org/10.1016/j.jevs.2026.105851 Publication
Researcher Affiliations
- Department of Biodiversity and Evolutionary Biology, National Museum of Natural Science (CSIC), C/ José Gutiérrez Abascal, 2, Madrid, 28006, Spain.
- Los Callejones del Duende Equine Reproduction Center, Ctra. de Colmenar a Aranjuez, Aranjuez, Madrid, 28300, Spain.
- Department of Biodiversity and Evolutionary Biology, National Museum of Natural Science (CSIC), C/ José Gutiérrez Abascal, 2, Madrid, 28006, Spain.
- Military Center for Horse Breeding (CCFAA), P.° Cementerio, 9, Ávila, Castilla y León, 05005, Spain.
- Military Center for Horse Breeding (CCFAA), P.° Cementerio, 9, Ávila, Castilla y León, 05005, Spain.
- SaBiO IREC (CSIC-UCLM-JCCM), ETSIAMB, University Campus, P.° de los Estudiantes, s/n, Albacete, 02006, Spain.
- SaBiO IREC (CSIC-UCLM-JCCM), ETSIAMB, University Campus, P.° de los Estudiantes, s/n, Albacete, 02006, Spain.
- SaBiO IREC (CSIC-UCLM-JCCM), ETSIAMB, University Campus, P.° de los Estudiantes, s/n, Albacete, 02006, Spain.
- Department of Biodiversity and Evolutionary Biology, National Museum of Natural Science (CSIC), C/ José Gutiérrez Abascal, 2, Madrid, 28006, Spain; Department of Animal Reproduction (INIA-CSIC), Carretera de la Coruña, km 7.5, Madrid, 28040, Spain.
- Department of Biodiversity and Evolutionary Biology, National Museum of Natural Science (CSIC), C/ José Gutiérrez Abascal, 2, Madrid, 28006, Spain. Electronic address: anasanchez@mncn.csic.es.
Conflict of Interest Statement
Declaration of competing interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.
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