Stepwise Proteinase K Digestion Improves Genomic DNA Yield and Purity from Goat Semen: A Comparative Pilot Study of Five Extraction Protocols
Year: 2026
Type: preprint
Abstract: ABSTRACT Genomic DNA of high quality is a prerequisite for molecular genetic analyses, but extraction from goat semen is difficult because sperm chromatin is highly compacted and seminal plasma contains inhibitors. This pilot study aimed to identify a reliable and cost-effective method for isolating genomic DNA from goat semen. Five extraction protocols were compared: the unmodified blood protocol (Protocol B), two modifications of a sperm protocol (Modified Protocols 1 and 2) and a stool protocol (Protocol BS) of a commercial spin-column kit (Kit A), and a second commercial silica membrane kit (Kit B). Three technical replicates per protocol were prepared from one frozen semen straw of a Black Bengal buck (one replicate for Protocol B). DNA concentration, purity (absorbance ratios at 260 and 280 nm, A 260 /A 280 , and at 260 and 230 nm, A 260 /A 230 ), cost per sample and amplification of the mitochondrial hypervariable region 1 (HVR1) by polymerase chain reaction (PCR) were measured. Modified Protocol 2, which used 100 µL of semen and two 1.5-h proteinase K incubations at 55 °C, gave the highest normalised yield (mean ± standard deviation, 55.9 ± 30.4 ng µL -1 semen), the best purity (A 260 /A 280 , 1.78; A 260 /A 230 , 2.06) and the lowest cost per ng of DNA (INR 0.073 ± 0.037), and its extracts amplified successfully. The Kruskal–Wallis test for normalised yield was not significant ( p = 0.075), so the differences in yield are exploratory. Because only one animal was sampled, the protocol must be confirmed with biological replicates from several bucks before wider use.
Language: English
Subfield: Reproductive Medicine
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W7222898095