Enhanced bovine ICSI outcomes with sex-sorted spermatozoa: Controlled chromatin decondensation by thioglycolic acid

The efficiency of intracytoplasmic sperm injection (ICSI) in cattle remains limited, particularly when sex-sorted spermatozoa are used, partly due to insufficient chromatin decondensation associated with the highly compact protamine structure of bovine sperm. This study evaluated thioglycolic acid (TGA) as an alternative reducing agent to dithiothreitol (DTT) to promote controlled chromatin decondensation while preserving sperm DNA integrity and improving ICSI outcomes. Sex-sorted and conventional bovine spermatozoa were treated with different TGA concentrations (0.5-5 mM), and sperm functional parameters and chromatin integrity were assessed. Low TGA concentrations (0.5-1 mM) increased CMA3 positivity, indicating increased protamine-DNA accessibility, without compromising sperm viability or chromatin integrity, whereas higher concentrations impaired sperm function. Based on these findings, 1 mM TGA was selected for subsequent ICSI experiments. In contrast, DTT treatment increased DNA fragmentation and chromatin instability. Following ICSI, sperm treated with 1 mM TGA exhibited a higher normal fertilization rate (two pronuclei; 70.4%) compared with the DTT-treated (36.8%) and control groups (8.3%). Moreover, embryos derived from TGA-treated spermatozoa showed high cleavage competence (79.0%) and improved blastocyst development (46.0%). Gene expression analysis of Day 8 blastocysts revealed increased IGF2 and OCT4 expression, reduced Survivin expression, and increased HSP70 expression, potentially reflecting altered cellular regulation in embryos derived from TGA-treated spermatozoa. In conclusion, optimized TGA treatment may facilitate controlled sperm chromatin decondensation while preserving DNA integrity, representing a promising strategy to improve bovine ICSI outcomes using sex-sorted spermatozoa.