Many fluorochromes routinely used in semen quality analysis emit in the green and red channels, limiting their possible combination for multiple parameter analysis. The use of fluorophores emitting in different light channels broadens the possibilities of combination to expand the range of simultaneously evaluated criteria. This is of great interest in cases of small ejaculated volumes, such as those naturally occurring in roosters, small dog breeds and drones (Apis mellifera). The purpose of this experiment is to establish Calcein Violet (CaV), a blue fluorochrome, as a marker of viability and acrosomal integrity in domestic animals in order to free the red and green channels. SYBR^®14/Propidium Iodide (PI) was used as reference dye, heat-treated samples as negative controls, serial staining combination for validation and epifluorescence microscopy for observation. Dead spermatozoa marked in red with PI showed no blue fluorescence either from the head or the tail. Live spermatozoa showed a decreasing blue emission from head to tail when single stained with CaV. Unreacted acrosomes showed intense blue fluorescence irrespective of plasma membrane integrity. This needs to be further confirmed for species with small and difficult to observe heads. Establishment of CaV as a marker of membrane integrity by fluorescence microscopy is a decisive first step towards further technical development and use with flow cytometry.

Characterizing the microbiota is essential to improve the understanding and management of urogenital disorders. Using 16S rDNA sequencing, this study investigated the urogenital microbiota, including urine, vaginal, and prostatic communities, in a litter of 10 healthy beagle puppies from 4 months of age until the completion of the first estrous cycle in females and 18 months in males. A further objective was to compare these microbial profiles with those of their dam. Significant differences were observed between urinary and genital microbiota in both sexes, evolving over time. Notably, in females, puberty and the first estrous cycle were associated with marked changes in the vaginal microbiota, outweighing individual variability. In contrast, urinary microbiota remained stable during female growth. In males, microbiota showed time-dependent and individual-specific progression, with distinct microbial communities identified in the urinary bladder and prostate. Shared genera between the dam and her offspring were observed, but inter-individual variability suggested a limited maternal influence. Further investigation is needed to clarify vertical transmission patterns. In conclusion, the urogenital microbiota of healthy dogs undergoes dynamic and distinct site-specific changes during early life.