Comparison of techniques for sperm evaluation in Purebred Spanish Horses

Abstract

The present study was designed to compare methods of sperm assessment in Purebred Spanish Horses, to evaluate different staining methods and to facilitate routine handling and work both in research laboratories and in the field. The parameters evaluated were acrosome integrity, capacitation, and sperm protamination. Semen was collected from February to May 2024 from 11 PRE stallions (three replicates of each). Immediately after collection, samples were diluted in INRA96 to a final concentration of 100 × 10⁶ sperm/mL and maintained at room temperature. For the examination of acrosome integrity, smears stained with Eosin-Nigrosin-Giemsa (ENG) were compared with smears stained with peanut agglutinin (PNA). Capacitation was evaluated by analyzing smears stained with PNA compared with samples fixed in 2% glutaraldehyde in cacodylate buffer and stained with Hoechst 33258 and chlortetracycline (CTC). Finally, for the analysis of protamination, smears stained with chromomycin A3 (CMA3), methylene blue (Diff-Quik), aniline blue, or toluidine blue were compared. Significant differences (p<0.05) between techniques were found using a paired t-test, when comparing two groups, or one-way-ANOVA, when comparing four groups; correlation tests were also performed (GraphPad Prism v9). The percentage of sperm with intact acrosomes identified with ENG was higher than with PNA, whereas sperm with damaged acrosomes with ENG was lower than with PNA. Conversely, the percentage of sperm with lost acrosomes was similar between the two techniques. Microscopic evaluation of sperm stained with ENG was more complex and time-consuming compared with PNA staining. The percentage of capacitated sperm analyzed with PNA and CTC, showed no significant differences between the two methods. For these two parameters, the correlations between the methods studied were not significant. Finally, chromatin compaction alterations showed significant differences between aniline blue (1.3±0.6%) and both Diff-Quik (4.0±0.7%) and toluidine blue (3.5±0.4%), whereas CMA3 (2.4±0.5%) were similar when compared with the other methods. The only significant correlation (0=0.019) appeared between CMA3 and aniline blue (Pearson r=0.69). It is noteworthy that these staining techniques evaluate different aspects of the chromatin, which may be the reason of the observed differences. Therefore, a reference technique, such as SCSA, would be a good option for the comparison of these 4 methods. Future studies should focus on the identification of a method of reference for each parameter to perform regression studies that allow us to model differences and generate an algorithm so the different staining techniques may be used in different laboratories or in the field and be comparable.