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Monoxenous  Eimeria  species are widespread enteropathogenic apicomplexan protozoa with a high economic impact on livestock. In cattle, tenacious oocysts shed by  E. bovis- infected animals are ubiquitously found and making infection of calves almost inevitable. To become infectious oocysts, exogenous oxygen-dependent  E. bovis  sporogony must occur leading to the formation of sporulated oocysts containing four sporocysts each harboring two sporozoites. Investigations on sporogony by live cell imaging techniques of ruminant  Eimeria  species are still absent in literature as commonly used fluorescent dyes do not penetrate resistant oocyst bi-layered wall. Sporogonial oocysts were daily analyzed by a 3D Cell Explorer Nanolive microscope to explore ongoing aerobic-dependent sporogony as close as possible to an in vivo situation. Subsequently, 3D holotomographic images of sporulating  E. bovis  oocysts were digitally stained based on refractive indices (RI) of oocyst bi-layered wall and sub-compartments of circumplasm using STEVE software (Nanolive), and the cellular morphometric parameters were obtained. Overall, three different  E. bovis  sporogony phases, each of them divided into two sub-phases, were documented: (i) sporoblast/sporont transformation into sporogonial stages, (ii) cytokinesis followed by nuclear division, and finally (iii) formation of four sporocysts with two fully developed sporozoites. Approximately 60% of sporulating  E. bovis  oocysts accomplished aerobic sporogony in a synchronized manner.  E. bovis  sporogony was delayed (i.e., 6 days) when compared to an in vivo situation where 2–3 days are required but under optimal environmental conditions. Live cell 3D holotomography analysis might facilitate the evaluation of either novel disinfectants- or anti-coccidial drug-derived effects on ruminant/avian  Eimeria  sporogony in vitro as discrimination of sporogony degrees based on compactness, and dry mass was here successfully achieved. Main changes were observed in the oocyst area, perimeter, compactness, extent, and granularity suggesting those parameters as an efficient tool for a fast evaluation of the sporulation degree.

Morphometric analysis of aerobic Eimeria bovis sporogony using live cell 3D holotomographic microscopy imaging