Flow Cytometric Analysis of Fish Sperm Quality: A Case Study of Black Sea Turbot

Objective: the aim of this study was to develop a method for evaluating fish sperm quality based on the mitochondrial status of spermatozoa, which is linked to their functional motility. Methods: to assess sperm quality of sexually mature Black Sea turbot males of different ages from a spawning population, flow cytometric analysis with preliminary staining of live sperm samples using the fluorescent dye rhodamine 123, which characterizes the membrane potential of spermatozoa mitochondria, was applied for the first time. Results: individual variability in concentration, size composition, and fluorescence of rhodamine-stained spermatozoa was determined in the sperm of 27 sexually mature turbot males of different ages from the same spawning population. The concentration of spermatozoa (with a head size of 2.2 μm) in turbot seminal fluid averaged 2.8 ± 1.3 × 109 cells ml-1, and individually varied from 0.56 to 4.98 × 109 cells ml-1. The proportion of fluorescent spermatozoa (i. e., with functional mitochondria stained by rhodamine 123) was 86.3 ± 13.9% of all spermatozoa in the sperm samples. Within the large group of rhodamine 123-stained spermatozoa, a subgroup of larger cells (approximately 2.4 μm) with the most intense fluorescence and, apparently, the highest metabolic activity was clearly distinguished, comprising 16.2 ± 3.8%. Novelty and Practical significance: the developed cytometric protocol provides a more accurate assessment of spermatozoa functionality in the sperm of Black Sea turbot and other fish species. It can be applied for investigating reproductive characteristics of fish from natural populations, selecting the best breeders when creating broodstock for aquaculture, and evaluating fish sperm quality before and after cryopreservation.