A 5-color flow cytometry panel to assess plasma membrane integrity, acrosomal status, membrane lipid organization and mitochondrial activity of boar and stallion spermatozoa following liquid semen storage

For a more practically applicable analysis of different sperm characteristics, this study aimed to develop a 5-color flow cytometry (FC) panel to concurrently analyze four sperm parameters in liquid boar and stallion semen, using also a DNA-marker for selecting sperm cell events. From each of thirty extended boar semen doses and twelve stallion semen doses, six aliquots were taken. For evaluating mitochondrial activity (A), degree of lipid disorder of plasma membrane (B), integrity of plasma membrane (C), acrosomal status (D) and marking DNA (E), five aliquots were individually stained with Rhodamine 123, Merocyanine 540, Propidium Iodide, PNA-Alexa Fluor 647, and Hoechst 33342, respectively. The sixth aliquot was stained with all the five fluorochromes simultaneously, whereas spectral overlap was corrected by a compensation matrix. Strong correlations were found between the single and 5-color staining assays for boar sperm (A: 0.99, B: 0.96, C: 0.93, D: 0.98, E: 0.99; P < 0.01). Furthermore, moderate and substantial Concordance Correlation Coefficients (CCC) were presented by all these parameters (0.99, 0.96, 0.92, 0.98, and 0.99, respectively). For stallion sperm, the correlation coefficients between the assays were also strong (A: 0.99, B: 0.98, C: 0.99, D: 0.99, E: 0.95; P < 0.01) and substantial CCC were observed for all of them (0.99, 0.97, 0.99, 0.99, and 0.90, respectively). For both species, the mean difference between the methods (d) did not overtake 0.84. The results confirmed that this 5 -color panel could be successfully implemented for analyzing boar and stallion sperm quality in a single, practical and quick FC assay.