Methods Used to Assess Bull Sperm Chromatin Integrity and Its Correlation with In Vitro Embryo Production Efficiency

Sperm chromatin analysis is a crucial tool for investigating fertility in bulls, both in the field and in in vitro fertilization. This study aimed to identify efficient, low-cost, and easy-to-apply methods for detecting chromatin integrity alterations. Frozen semen samples from four bulls with varying results in in vitro embryo production (IVEP) were used. The sperm chromatin from these samples was evaluated using different methods: differences in the staining intensity with toluidine blue and the Feulgen reaction (FR), assessed both visually and computationally; visual evaluation of smears stained with acridine orange (AO) using epifluorescence and confocal microscopy; and evaluation using transmission electron microscopy (TEM) of sections positively stained with uranyl and lead. The same samples were utilized for IVEP. The results obtained using each method were correlated with one another and with the cleavage and embryonic development rates achieved in IVEPs. The odds ratio was employed to compare the chromatin alteration identification rates of the studied methods. Overall, chromatin integrity alterations identified using the AO and TEM methods exhibited correlations with the cleavage (r = -1.00) and embryonic development rates (r = -1.00), respectively. Among the methods tested, TEM and AO were the most effective for identifying chromatin integrity alterations that interfere with IVEP efficiency.