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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
Author(s) -
Watts John
Publication year - 2021
Publication title -
reproduction in domestic animals
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.546
H-Index - 66
eISSN - 1439-0531
pISSN - 0936-6768
DOI - 10.1111/rda.13935
Subject(s) - staining , repeatability , semen , membrane filter , propidium iodide , biology , pi , andrology , membrane , microbiology and biotechnology , chemistry , medicine , chromatography , biochemistry , apoptosis , genetics , programmed cell death
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 < .05). Measurement of MIS percentage using red and red–green filter CASQ appeared to be the most reliable automated methods.