Development of a fluorescent computer‐assisted spermatozoal quantification method and a comparison of results for manual counting with a haemocytometer and computer‐assisted semen analysis in dogs

Abstract

The aim of this study was to develop and validate a novel, computer‐assisted spermatozoal quantification (CASQ) method of determining spermatozoal concentration in canine semen. In Experiment A, the spermatozoal concentration was measured (n = 28) with a haemocytometer using light microscopy, CASQ and computer‐assisted semen analysis (CASA; MMC sperm), following three independent dilutions. The limits of agreement between the haemocytometer and CASQ were −13.1% to 13.8% and −27.0% to 28.6% between the haemocytometer and CASA. The precision CVs (limits of agreement) were 5.7% (−7.8% to 8.9%) for the haemocytometer, 6.2% (−8.8% to 12.3%) for CASQ and 10.8% (−16.0% to 19.5%) for CASA. In Experiment B, spermatozoa were manually counted (n = 42) with the haemocytometer under fluorescent illumination using the CASQ sample. The limits of agreement between the CASQ and the haemocytometer were satisfactory (−4.6% to 4.6%) and the precision CVs (limits of agreement) were 6.2% (−9.0% to 11.4%) for the haemocytometer and 4.4% (−5.8% to 8.6%) for CASQ. The CASQ method was then clinically applied to compare the haemocytometer (light and fluorescent methods) with CASQ and CASA. Outlying data were removed. These studies demonstrated that CASQ was reliable and that the MMC sperm CASA was unreliable as methods for determining spermatozoal concentration in canine semen. Computer‐assisted spermatozoal quantification was also determined to be more precise than manual counting with the haemocytometer. Using the clinical protocol, the agreement between the haemocytometer and CASQ method was acceptable, but it was worse than in the experiments where duplicate samples and a larger volume of semen were analysed. The CASQ method may be a useful method to measure the membrane status of canine spermatozoa; however, further investigation is required. Counting spermatozoa using fluorescent microscopy and the haemocytometer may improve the efficiency of counting and the accuracy of the method.

Measurement of membrane integrity in canine spermatozoa using a fluorescent computer‐assisted spermatozoal quantification method and manual counting after eosin‐nigrosin staining compared with manual counting after CFDA/PI staining

“…The CASQ method can be used to measure the MIS percentage in canine semen, having been previously found a useful and accurate method for determining the concentration of canine spermatozoa (Watts, 2018 (Pintado, de la Fuente, & Roldan, 2000). The Hoescht 33258 staining produced similar results to eosin staining (Pintado et al, 2000).…”

“…The CASQ method can be used to measure the MIS percentage in canine semen, having been previously found a useful and accurate method for determining the concentration of canine spermatozoa (Watts, ). The CASQ had advantages over the other methods used.…”

“…As it does not disrupt the cellular membrane, a MDS percentage can be measured and the MIS percentage calculated (Chemometec, ). A similar method, computer‐assisted spermatozoal quantification (CASQ), has recently been developed (Watts, ) and may also be used to calculate the MIS percentage.…”

“…A similar method, computer-assisted spermatozoal quantification (CASQ), has recently been developed (Watts, 2018) and may also be used to calculate the MIS percentage.…”

Evaluation of the use of SYBR‐14 and propidium iodide stain in a fluorescent computer‐assisted spermatozoal quantification method in dogs

“…Recently, a new precise method of quantifying spermatozoa in canine semen was developed—computer‐assisted spermatozoal quantification (CASQ; Watts, 2019a). Spermatozoa were stained with propidium iodide (PI) and then examined under fluorescent microscopy, after which a photomicrograph was taken and automated counting of spermatozoa on the photomicrograph was performed using the open‐source software, ImageJ.…”