Validation of Calcein Violet as a marker of Apis mellifera semen viability in flow cytometry : preliminary results.

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 fluoresce after esterification of the non-fluorescent calcein violet acetoxymethyl into fluorescent calcein violet, has recently been validated as a marker of semen viability in honeybee drones (1).
However, compared to flow cytometry, epifluorescence is suboptimal for semen evaluation as it is time consuming and only allows evaluation of a limited number of spermatozoa. This report shows preliminary results on the application of CaV in flow cytometry for viability evaluation of drone semen. Heat-treated samples and unstained samples were used as negative control. Untreated samples showed 84% (73.7-94.3%) of positive cells compared to heat treated samples with 2.5% (1.35-3.7%) of positive cells. Autofluorescence was marginally detected with 0.15% of cells detected as positive on unstained samples.
Although these are preliminary results, this 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. With artificial insemination becoming more and more popular, development of semen quality assessment tools, that have been scientifically validated for the drones, has become crucial for apidology.

(1) Egyptien et al. Validation of calcein violet as a new marker of semen membrane integrity in domestic animals. 2023. Animals. (Under review).