Abstract :
[en] 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 dyes, including SYBR/PI, emit in the green channels, limiting their possible combinations with other fluorochromes for multiple parameters analysis of semen in flow cytometry. 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 dogs. Using SYBR/PI as reference dye, heat-treated samples as negative control, and serial staining combinations under fluorescence microscopy, this is the first study that establishes CaV as a reliable marker for membrane integrity of canine spermatozoa. In all samples, dead spermatozoa were marked in red with PI and none of them showed violet fluorescence within the head or the tail. Live spermatozoa showed a decreasing violet emission from the head to the tail when stained with CaV only or from the tail only when CaV was combined with SYBR as its bright green fluorescence from the head quenches that of CaV. Our experiment showed that CaV can be used for acrosomal status evaluation, with unreacted acrosomes showing intense violet fluorescence irrespective of the plasma membrane integrity. CaV allows for identification of spermatozoa with intact acrosomes emitting violet fluorescence even if the head of dead spermatozoa is marked in red by PI. 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 allows freeing the green and red channels and broadens the possibilities of combinations with other fluorochromes to expand the range of parameters simultaneously evaluated in a multiple parameters analysis of semen. This will be particularly beneficial for individuals with a very small volume of ejaculate, such as toy breeds, where sample size may be an issue.
