Simple SummaryOne way to conserve biodiversity is to cryobank good quality semen. Unsurprisingly, analysing multiple parameters improves semen quality and fertility evaluation. Semen membrane integrity, which indicates sperm cell viability and acrosomal status that reflects sperm-oocyte fusion capacity, are some of the basic parameters evaluated, among many others. Specific fluorescent markers (fluorochromes) that emit mostly green and red light routinely assess these parameters. Color overlapping limits simultaneous evaluation of multiple parameters. Small volume ejaculates, which naturally occur in roosters, small dogs and honeybees, will preclude repetition of analysis, thus interfering with fertility estimation. The use of fluorescent semen quality markers emitting in different light channels is the most appropriate for multiple parameter analysis. The purpose of this experiment is to establish Calcein Violet, a blue fluorochrome, as a marker of viability and acrosomal status in domestic animals in order to free the red and green channels for concomitant analysis of other parameters. Our serial analyses validate Calcein Violet for evaluation of membrane integrity and acrosomal status. Its use in multiple parameters analysis should therefore be recommended.AbstractMany 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.