Validation of Calcein Violet as a new marker of semen membrane integrity in Apis mellifera drones

Membrane integrity, most commonly assessed by double staining with SYBR®14 (SYBR) and propidium iodide (PI), is considered to reflect viability, which is routinely included in semen evaluation. Many fluorochromes emit in the green and red channels, limiting their possible combinations for multiple parameters analysis. Although previously suggested for that purpose, Calcein violet (CaV), a permeant viability dye that emits fluorescence after esterification of the non-fluorescent calcein violet acetoxymethyl into fluorescent calcein violet, has never been validated as a marker of semen viability in honeybee drones.
This is the first study that establishes CaV as a reliable marker for viability of drone semen using SYBR/PI as reference dye, heat-treated samples as negative control, and serial staining combinations. In all samples, dead spermatozoa were marked in red with PI and none of them showed violet fluorescence within the head and the tail. Live spermatozoa showed a decreasing violet emission from head to tail when single stained with CaV, or from the tail only when CaV was combined with SYBR.
Establishing CaV as a marker of membrane integrity by fluorescence microscopy is a decisive first step towards further development with flow cytometry. As CaV fluoresces in violet, it frees the green and red light spectrum channels and broadens the possibilities of combinations to expand the range of parameters simultaneously evaluated in a multiple parameters analysis of semen. This will be particularly beneficial for honeybee drones that have a very small volume of ejaculate and where sample size is an issue.