Análise da integridade da cromatina espermática de garanhões por meio de SCSA e teste de Nicoletti

Abstract

The integrity of chromatin, a structure composed of DNA and nucleoproteins, is crucial for preserving the genetic information of the male gamete. During spermiogenesis, chromatin undergoes a compaction process involving the replacement of histones with protamines to enhance DNA stability. However, alterations in this compaction process can compromise fertility, even in semen that appears to be of good quality. Several factors can lead to sperm DNA fragmentation, which is associated with fertility issues and impaired embryonic development. In this context, flow cytometry has emerged as a prominent tool for analyzing sperm chromatin instability. It allows for the assessment of chromatin integrity and, consequently, the identification of stallions with potential fertility problems. The Sperm Chromatin Structure Assay (SCSA), considered the gold standard, uses in situ acid-induced denaturation and acridine orange staining to quantify DNA damage through the DNA Fragmentation Index (DFI), offering reliable and rapid results. The Nicoletti method, originally developed to detect apoptosis in somatic cells, uses propidium iodide and features a simpler protocol. In this study, a Pearson correlation was performed between routine andrological parameters—such as motility, concentration, and vigor—and DFI data obtained from both the Nicoletti test and the SCSA, along with pregnancy outcomes following the insemination of healthy mares. The results showed that the Nicoletti method correlates more closely with the SCSA when high fluorescence events are interpreted as indicators of chromatin fragmentation. This behavior may be explained by the cellular differences between spermatozoa and the somatic cells originally analyzed with the Nicoletti technique. Therefore, we recommend that the analytical parameters of the Nicoletti method be revised to improve its accuracy in assessing stallion fertility.