Calcein Violet, a validated marker of membrane integrity in bulls and stallions.

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 its use as been reported in bulls, Calcein violet (CaV), a violet fluorochrome, has never been validated as a marker of semen viability in domestic animals.
Using SYBR/PI as reference dye, heat-treated samples as negative control, and serial staining combinations under fluorescence microscopy. The same operator repeated the observations twice. One straw per species per day was thawed to run the analysis. Images were collected with a digital microscope camera. 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 single stained with CaV, or from the tail only when CaV was combined with SYBR. We show that CaV can be used for acrosomal status evaluation. Unreacted acrosomes showed intense violet fluorescence irrespective of the plasma membrane integrity as spermatozoa with intact acrosomes emitting violet fluorescence even if the head of dead spermatozoa is marked in red by PI.
This is the first study that establishes CaV as a reliable marker of membrane integrity by fluorescence microscopy. It is a decisive first step towards further development with flow cytometry. As CaV fluoresces in violet, it frees the green and red channels and broadens the possibilities of combinations to expand the range of parameters simultaneously evaluated in a multiple parameters semen analysis.