Interaction of semen smear preparation method and slide region evaluated affects the probability of detecting morphologically normal sperm

Abstract

Bull breeding soundness examination (BSE) is important in identifying subfertile bulls, with sperm morphology being a key determinant. It is recommended that eosin-nigrosin stained semen smears are used for sperm morphology assessments. These smears are often made on glass slides with either another slide or a wood applicator. However, evaluation within only 1 smear region may not account for variations across regions that may be influenced by smear method. This may affect sperm evaluation and BSE classification, depending on smear region evaluated. Objective of our study was to determine if normal sperm morphology percentage varies across semen smear and whether smearing method modifies such variation. Coverslipped, stained semen smears were divided into 3 equal zones. Each zone was evaluated independently by experienced evaluators, with nontarget sections masked and each zone assigned a unique identifier to ensure blinding. Of 124 slides assessed, 33 (27%) contained at least 1 zone having a different classification category using a cutoff of 70% morphologically normal sperm. A linear mixed model with percentage of normal sperm as the outcome identified that sperm normality probability decreased from smear initiation site to smear end, with more pronounced effect (p < 0.001) for smears made with wood applicators. Decompositional models were applied to determine the probability of independent defect categories contributing to decreases in normal sperm morphology; most was from midpiece defects. These findings highlighted potential sources of variation in sperm morphology assessment and a need for standardized smear preparation methods.