Validation of the volumetric flow cytometry for bovine sperm concentration

AbstractSperm concentration is a stronghold of the andrological evaluation and the production of insemination doses. The use of haemocytometers, although considered the gold standard, is difficult to apply in field conditions because subjective and time-consuming. The present study was designed to validate the volumetric flow cytometry to estimate sperm concentration, comparing it with the performances of haemocytometer, NucleoCounter, and flow cytometry using fluorospheres. Compared with other methods, volumetric flow cytometry appeared less affected by large dilution of the sample, with similar concentrations calculated in the range of dilution 1:200 to 1:800. Using volumetric flow cytometry the population detected using morphological criteria and fluorescence of DNA better represents the real concentration of sperm in the sample. Volumetric flow cytometry showed high repeatability compared with the haemocytometer (coefficient of variation 1.85% and 4.52%, respectively). Finally, volumetric flow cytometry showed stable performances in cryopreserved samples analysis, with negligible effects of the medium components and cryoprotectants. The present study showed that volumetric flow cytometry is an accurate and precise method to estimate sperm concentration in bovine fresh and frozen semen, making possible the quantification of sperm functional subpopulation. This implements the possibility to study the relationship between sperm attributes and fertility.