Different staining techniques evaluation for the study of sperm morphology and morphometry in bats (Mammalia: Chiroptera)

Abstract

The study of sperm morphology involves the use of various staining techniques that allow visualization of different structures of the spermatozoon and their variability. Each technique varies depending on the dyes used and the necessary steps for execution. The aim of this work was to evaluate different staining techniques for sperm morphology and morphometry analysis in bats. Samples were collected from the epididymis of 57 adult specimens, which were macerated in Farmer’s solution and used to prepare smears. The smears were stained with Toluidine Blue, Giemsa, May Grünwald-Giemsa, Gram stain, Hematoxylin-Eosin, DAPI (4’,6-diamid-ino-2-phenylindole), Basic Fuchsin, and Janus Green. Criteria for comparing the techniques were complexity, time required, and associated costs., Microphotographs were taken using light and epifluorescence microscopy for morphometric analysis, and 50 spermatozoa were measured per individual. Spermatozoa were obtained from 16 specimens: Artibeus planirostris (n=5), Sturnira erythromos (n=3), Molossus molossus (n=4), Molossops temminckii (n=2), Histiotus laephotis (n=1), and Myotis albescens (n=1). Among the evaluated techniques, Toluidine Blue proved to be a quick, simple, and cost-effective method in order to determine the presence of spermatozoa and describe their general morphology. Dicromatic stains like Hematoxylin-Eosin and May Grünwald-Giemsa were more efficient in differentiating the nucleus and acrosome, although they were also more complex and costly to perform. In conclusion, we recommend the use of the aforementioned three techniques as optimal choices for initial studies of sperm morphology in bats.