Sperm DNA Extraction in Livestock: Structural Challenges, Methodological Advances, and Applications in Assisted Reproductive Technologies

Abstract

Assisted reproductive technologies (ART), including artificial insemination, in-vitro fertilization, intra-cytoplasmic sperm injection, and embryo transfer, are central to genetic improvement and reproductive management in livestock. Beyond conventional semen parameters, sperm DNA integrity has emerged as a critical determinant of fertilization success, embryo development, pregnancy establishment, and offspring viability. However, the extraction of high-quality DNA from spermatozoa remains technically demanding due to the highly compact chromatin structure formed by protamine-rich DNA packaging and extensive disulfide cross-linking, which renders sperm nuclei resistant to standard lysis procedures. This review synthesizes current knowledge on sperm DNA extraction methodologies across livestock species, with emphasis on the structural and biochemical challenges unique to spermatozoa. Conventional extraction approaches, including organic solvent- and salt-based methods, are evaluated alongside modern and modified techniques incorporating strong reducing agents, optimized enzymatic digestion, chaotropic lysis buffers, and solid-phase purification systems. Recent advances, such as TCEP-based reduction, magnetic bead- and silica column–based platforms, and automated high-throughput workflows, have improved DNA yield, purity, integrity, and reproducibility, making them increasingly suitable for downstream molecular applications. Species-specific differences in chromatin organization among bovine, buffalo, ovine, caprine, porcine, and equine spermatozoa are highlighted, underscoring the need for tailored extraction protocols. The review also emphasizes the importance of rigorous DNA quality assessment for applications in genomic selection, epigenetic profiling, fertility prediction, and ART outcome optimization. Finally, key research gaps are identified, including the need for protocol harmonization, inter-laboratory validation, and integration with high-throughput omics and data-driven analytical approaches to support sustainable livestock breeding.