In vitro maintenance of drones and development of a new software for sperm quality analysis facilitate the study of honey bee reproductive quality

This study aimed to develop a laboratory method that allows the in vitro maintenance of honey bee drones for several days while preserving their reproductive capacity and to create a new open-source software for the automatic analysis of their sperm quality. Three experiments were performed. The first experiment was designed to validate the new open-source software named CASABee for sperm quality assessment specifically designed for the honey bee. The software was able to identify motile and static spermatozoa with high precision. Results showed a high correlation between the results of sperm quality obtained both manually and by the CASABee system (0.95 and 0.96 for sperm motility and concentration, respectively, p < 0.001). In the second and third experiments, the effect of in vitro maintenance of drones without attendant workers for four days on their ejaculatory capacity and sperm quality, respectively, was evaluated. Survival rate was 98.68%, 89.48%, 75.93%, and 60.97% on average on days 1, 2, 3, and 4 after capturing, respectively. A high proportion of the drones (80.37% on average) were able to ejaculate providing semen, and there were no significant differences in the ejaculatory capacity and sperm quality of drones on the different days of in vitro maintenance, except for sperm viability, which decreased slightly on day 4 (71.71% vs 82.8% on day 0, p < 0.05). It was concluded that the new CASABee system and the method for laboratory maintenance of honey bee drones facilitate the study of reproduction in this species.