Validation of Calcein Violet as a New Marker of Semen Membrane Integrity in Domestic Animals

Abstract

Many 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.

Sperm Quality Assessment in Stallions: How to Choose Relevant Assays to Answer Clinical Questions

“…Its limitations are its incompatibility with cell fixation and its consumption of two channels to analyze one parameter, which restricts its use in multiple-parameter analyses. Recently, calcein violet fluorochrome has also been validated as a semen membrane integrity assay in different species, including horses [60]. The hydrolyzation of its permeant non-fluorescent form into an impermeable blue fluorescent form is detected in cells with intact membrane integrity.…”

“…The hydrolyzation of its permeant non-fluorescent form into an impermeable blue fluorescent form is detected in cells with intact membrane integrity. Using these kits in flow cytometry will enable the easy assessment of viability for the largest number of events that an operator could deal with [60].…”