Validation of the volumetric flow cytometry for bovine sperm concentration

Sperm 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 it is subjective and time-consuming. The present study was designed to validate the volumetric flow cytometry (volFC) in order to estimate bovine sperm concentration, comparing it with the performances of haemocytometer, NucleoCounter, and flow cytometry with the use of fluorospheres. Compared with other methods, volFC appeared less affected by large dilution of the sample, with similar concentrations calculated in the range of dilution 1:200-1:800. Using volFc the population detected on the basis of morphological criteria and fluorescence of DNA better represents the real concentration of sperm in the sample. The volFC showed high repeatability compared with the haemocytometer (coefficient of variation 1.85% and 4.52%, respectively) and stable performances with cryopreserved samples, with negligible effects of the medium components. The present study showed that volFC is as accurate and precise as other techniques to estimate sperm concentration in bovine fresh and frozen semen, but it is less affected by operative conditions, such as sample dilution. The possibility to quantify sperm functional subpopulations by volFC could potentially implement the study of the relationship between sperm attributes and fertility.