Comparison of methods for morphological evaluation of sheep spermatozoa using the SPERM BLUE kit and Romanovsky-Giemse staining

Abstract

The purpose of this study is to test and comparatively evaluate the method of morphological examination of ram sperm using a differentiated staining Sperm Blue and Romanovsky-Giemsa staining. The sperm of Dorper rams was collected and examined. The obtained samples (n=10) were diluted in a ratio of 1:100 (PBS solution) to conduct a morphological assessment of sperm. Two methods were used for the morphological assessment of spermatozoa: a set of differentiated staining Sperm Blue (Microptic, exposure of the smear in the fixative – 10 minutes, in the main dye - 18 minutes) and Romanovsky-Giemsa staining (exposure of the smear in the fixative - 10 minutes, in the main dye – 20 minutes). After routine staining, the smears were washed with distilled water, dried, and microscopy was performed (magnification using a 100x10 immersion objective, oil immersion), followed by a manual method of counting the number of morphological forms of sperm. According to the results of a comparative analysis of stained smears, staining with Sperm Blue allows for a detailed morphological study and differentiation of teratozoospermic characteristics. When stained with Romanovsky-Giemsa, normal spermatozoa were clearly visible, but the acrosome was poorly visualized. Manual calculation of the percentage of normal sperm using both dyes resulted in the same decimal result. A statistically insignificant difference in head and tail defects was found between the dyes used (p = 0.41 and p = 0.77, respectively): Romanovsky-Giemsa staining (7.8% and 34.1%) detected these defects more often than Sperm Blue (6.3% and 32.9%). Considering the results obtained, the use of the Sperm Blue dyes dye in veterinary andrology is relevant, in particular for assesment the morphology of sperm of different animal species with the further development of appropriate protocols in terms of the duration of exposure of the smears themselves to the main dye.