Métodos alternativos para análise de cromatina espermática de garanhões

Abstract

Sperm chromatin, composed of DNA and compacted nuclear proteins, ensures genomic stability. The importance of sperm DNA integrity extends to embryonic development, as failures in compaction or proper incorporation of nuclear proteins lead to DNA fragmentation and can result in miscarriages or fetal development failures. Animals with normal sperm motility and morphology may still have DNA damage. Although the Sperm Chromatin Structure Assay (SCSA) is considered the standard method for this assessment, its application is limited by high cost and the need for flow cytometry, which justifies the investigation of alternative methods. In this context, the present study aimed to evaluate the possible association between changes in sperm DNA compaction and stability, identified by staining tests with acridine orange (AO), toluidine blue (TB), and Feulgen reaction (FR), and the conception of inseminated mares. Ninety-seven cooled semen samples from Quarter Horse and Mangalarga Marchador stallions were analyzed, collected between August 2023 and December 2025, all previously approved by conventional semen evaluation. The evaluation included visual analysis under fluorescence microscopy for AO. For the TB and FR tests, computational analysis was performed on images obtained by transmitted light microscopy of stained smears. These analyses identified regional patterns of chromatin decompaction, heterogeneity, and the percentage difference in chromatin decompaction. Conception was verified by ultrasound fifteen days after ovulation in inseminated mares. The results showed no statistically significant correlation between most chromatin integrity variables and conception, regardless of the test used. Only the categories of base-apex decompaction and total decompaction showed a moderate negative correlation with conception in some tests, suggesting that more extensive chromatin changes may have greater potential for biological impact. However, in general, the changes detected were of low magnitude and possibly below the threshold necessary to compromise initial fertilization. It is concluded that sperm chromatin evaluation by alternative methods in semen smears can be used as a complementary tool to conventional semen analysis and should not be considered an isolated marker of fertility in stallions.