Uso das colorações de azul de toluidina e reação de Feulgen para avaliação de alterações cromatínicas em espermatozoides de garanhões

Abstract

Many stallions with sperm deemed normal in routine examinations still face fertility issues, which may be linked to alterations in sperm chromatin. Chromatin structure assessment methods, such as staining with toluidine blue 4N (TB) and the Feulgen reaction (FR), are used to identify these alterations, including DNA fragmentation and poor chromatin compaction. In this context, a computational image analysis of semen smears stained with TB and FR was proposed. In this study, 40 semen samples from stallions were stained with TB and FR and evaluated using computational image analysis. Parameters such as the percentage of sperm with decompaction areas, chromatin heterogeneity, differences in chromatin compaction relative to normal sperm, and the percentage of six types of decompaction based on the decompaction site in the sperm head were analyzed. All semen samples were used in artificial insemination, and pregnancy in mares was confirmed by ultrasound after two months. Spearman’s correlation test was performed between sperm variables and pregnancy outcomes. The results showed that none of the tested methods had a significant correlation with pregnancy rates. The FR staining method presented limitations, with 33 samples showing 100% of sperm with one type of chromatin alteration.This is the first work to employ algorithms for computational image analysis of equine sperm chromatin, both with toluidine blue and with Feulgen occurrence, which highlights its potential for future investigations in this area. Under these conditions, a negative, albeit non-significant, correlation between pregnancy and both total chromatin decompaction and dispersion, as well as the percentage difference when using Feulgen staining, and the percentage difference and heterogeneity when using TB staining, suggest that these variables have potential for evaluating stallion fertility.