Influence of Random Factors and Two Different Staining Procedures on Computer-assisted Sperm Head Morphometry in Bulls

Contents Computer-assisted sperm analysis has the potential to improve reproducibility and objectivity in the assessment of sperm morphology. The aim of this study was to evaluate the use of a computer-assisted sperm morphometry analysis system for the determination of sperm head dimensions in bulls. Two experiments were performed to determine the variability caused by random factors and the influence of two different staining procedures. In the first experiment, three ejaculates were collected from each of five clinically healthy bulls. Air-dried semen smears were stained using a modified Farelly staining. The slides were observed via bright field microscopy with green filter using a 100× oil immersion objective. A video camera attached to the microscope transmitted images to a personal computer. Each sperm head was identified and analysed by the computer software (Morphology Analyzer V. 1.5; Mika Medical GmbH, Ismaning, Germany). Area, length and width of each sperm head were calculated and stored in a database for further statistical analysis. A minimum of 100 sperm heads were evaluated per slide. In experiment 2, the influence of two different staining procedures (Farelly and Papanicolaou) on sperm head dimensions was determined. The mean spermatozoal head measurements across all slides for area, length and width were 40.49 μm², 9.70 μm and 5.30 μm, respectively. On the basis of these results, the variability between slides, ejaculates and bulls using variance component estimation was calculated. All random factors (bull, ejaculate and slide) had a significant effect (p < 0.001) on sperm head dimensions. However, the variability attributable to bull (18.89–51.72%) was considerably higher compared with that of slide and ejaculate (0.17–5.27%). Additionally, differences existed between bulls concerning the shape and normality of histograms of their sperm head dimensions. The minimum number of spermatozoa required for analysis of sperm head dimensions was found to be about 60 spermatozoa per sample. The use of Papanicolaou stain resulted in significantly smaller sperm head dimensions, e.g. sperm head area 31.48 versus 38.35 μm² (p < 0.001). In conclusion, computer-assisted sperm head morphometry provides an objective, precise and reproducible tool. Comparisons of results from different studies should consider the influence of random and experimental factors to avoid misinterpretation.