Comparison of three sperm-FISH protocols to optimize population-scale screening in cattle

Abstract Fluorescence in situ hybridization (FISH) is widely used in chromosome structure and organization studies, playing a key role in diagnosing chromosomal aberrations in somatic and germ cells. A critical step in preparing spermatozoa for sperm-FISH analysis is effective nuclear chromatin decondensation. Sperm chromatin is highly condensed, posing a barrier to the proper hybridization of molecular probes. Insufficient or excessive decondensation can result in unclear fluorescent signals, reducing the reliability of analysis. Since optimizing decondensation is essential for achieving precise and reproducible results, this study aimed to optimize chromatin decondensation in domestic cattle spermatozoa using three different sperm-FISH protocols, to identify a protocol suitable for future population-scale sperm-FISH analyses. The research material consisted of semen from 10 bulls obtained in collaboration with the Malopolska Biotechnic Center in Krasne. Three separate protocols were applied: Protocol 1 involved decondensation in 1 M DTT in PBS; Protocol 2 used a papain–DTT solution; and Protocol 3 involved treatment with a 3 M NaOH solution. All three protocols resulted in a percentage of properly decondensed sperm, but Protocol 3 yielded a statistically higher proportion of this type. It provided well-decondensed sperm chromatin, clear fluorescent signals specific to the studied chromosomes, and allowed reliable detection of normal and abnormal signals. Alkaline denaturation may provide more stable chromatin decondensation in bovine spermatozoa, thereby improving hybridization efficiency and signal clarity.