Measurement of membrane integrity in canine spermatozoa using a fluorescent computer-assisted spermatozoal quantification method after SYBR-14/PI staining compared with manual counting after CFDA/PI staining.

The aim of this study was to compare measurements of spermatozoal membrane status in dogs using computer-assisted spermatozoal quantification (CASQ) after staining with SYBR-14 and propidium iodide (PI) with manual counting after CFDA/PI staining. CASQ was performed on fresh (n = 11) and thawed cryopreserved canine semen (n = 91) using (1) a red long-pass (LP) filter on an untreated sample (membrane-disrupted spermatozoa, MDS count) and in a sample with all cellular membranes disrupted (total spermatozoal count, TC), (2) green LP filter for a TC and the red filter for an MDS count, and (3) a green short-pass filter to obtain a membrane-intact spermatozoa (MIS) count and the red filter to obtain the MDS count, which were added to give a TC (red-green filter CASQ, n = 50). Spermatozoa were also stained with CFDA/PI, manually examined and classified as MIS or MDS. All measurements were performed in duplicate. The percentage of membrane-intact spermatozoa (MIS) was calculated. The percentage of progressively motile spermatozoa (PMS) was determined subjectively. The data were analysed to measure the agreement between the CASQ and CFDA/PI methods, repeatability of the methods and correlation between the MIS and PMS percentage. Compared with the CFDA/PI method, the agreement of MIS percentage with red filter CASQ was -12% to 34%, green LP filter CASQ -42% to 47%, and red-green filter CASQ -23% to 29%. The repeatability of the CFDA/PI and red-green filter CASQ methods were the highest. The MIS and PMS percentages were always correlated (P < 0.05). Measurement of MIS percentage using red and red-green filter CASQ appeared to be the most reliable automated methods.