Functional assessment and comparison of different methods of genomic DNA extraction from fresh ejaculated and cryopreserved caprine sperms for enhanced quality and long-term DNA banking

Acquiring high-quality DNA from spermatozoa is crucial for reliable downstream biotechnological applications, but obtaining intact genomic DNA (gDNA) from sperm can be challenging due to issues with cell lysis and DNA fragmentation. In this study, we evaluated six gDNA extraction methods from fresh ejaculated and cryopreserved caprine sperm, aiming to improve organic thiol (β-Mercaptoethanol; β-ME)- or commercial kit-based methods for high-quality gDNA recovery suitable for molecular analyses after long-term storage. We standardized a modified β-ME-based extraction method and compared its performance with in-house or commercial gDNA extraction kits using Dithiothreitol (DTT) or a β-ME-DTT combination. Following six months of storage at −80°C, functional sequence verification was performed by qRT-PCR and DNA sequencing of purified PCR products. The method combining β-ME and DTT resulted in higher gDNA yields from both fresh and cryopreserved sperm. When considering cost per extraction, the modified in-house method (β-ME and DTT) was the most efficient for both sperm types. These modified protocols enabled the extraction of significantly higher amounts of pure, degradation-free gDNA with minimal or no protein content, making them suitable for long-term DNA banking and downstream applications. Our findings provide economical strategies for efficient gDNA extraction from buck sperms, regardless of preservation status.