Stepwise Proteinase K Digestion Improves Genomic DNA Yield and Purity from Goat Semen: A Comparative Pilot Study of Five Extraction Protocols

Abstract

Background: Genomic DNA of high quality is a prerequisite for molecular genetic analyses. However, DNA extraction from goat semen is frequently challenged by highly compacted sperm chromatin and the presence of enzymatic inhibitors within the seminal plasma. Objectives: This pilot study aimed to identify a reliable and cost-effective method for isolating high-quality genomic DNA from caprine semen. Methods: Five extraction protocols were evaluated: an unmodified blood protocol (Protocol B), two modifications of a sperm protocol (Modified Protocols 1 and 2), and a stool protocol (Protocol BS) using a commercial spin-column kit (Kit A), alongside a second commercial silica membrane kit (Kit B). Three technical replicates per protocol were processed from a single frozen semen straw of a Black Bengal buck (with one replicate evaluated for Protocol B). Extracted DNA was evaluated based on concentration, purity (absorbance ratios A260/A280 and A260/A230), cost per sample, and PCR amplification success of the mitochondrial hypervariable region 1 (HVR1). Results: Modified Protocol 2 utilizing 100 uL of semen and two sequential 1.5-hour proteinase K incubations at 55 degrees C yielded the highest normalized DNA concentration (mean +/- SD: 55.9 +/- 30.4 ng/uL semen), optimal purity ratios (A260/A280 = 1.78; A260/A230 = 2.06), and the lowest cost per nanogram of DNA (INR 0.073 +/- 0.037). All extracts from this protocol were successfully amplified via PCR. While the Kruskal-Wallis test for normalized yield did not reach statistical significance (p = 0.075), rendering yield differences exploratory, Modified Protocol 2 demonstrated the strongest overall performance. Conclusions: Modified Protocol 2 offers a promising, cost-efficient workflow for caprine sperm DNA isolation. Given that this pilot study sampled a single animal, these findings must be validated using biological replicates from multiple bucks prior to widespread implementation.