<i>In vitro</i> 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 <i>in vitro</i> 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, <i>p</i> &lt; 0.001). In the second and third experiments, the effect of <i>in vitro</i> 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 <i>in vitro</i> maintenance, except for sperm viability, which decreased slightly on day 4 (71.71% vs 82.8% on day 0, <i>p</i> &lt; 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.本研究旨在开发一种可在体外（in vitro）维持蜜蜂雄蜂数日且保留其生殖能力的实验室方法，并构建一款全新的开源软件，用于自动分析雄蜂精液质量。本研究共开展三项实验：第一项实验旨在验证专为蜜蜂精液质量评估开发的开源软件CASABee。该软件可高精度识别运动与静态精子，结果显示人工手动分析与CASABee系统所得的精液质量结果具有极高相关性（精子活力与浓度的相关系数分别为0.95与0.96，p < 0.001）。第二项与第三项实验分别评估了无伴工蜂的雄蜂体外维持4天对其射精能力与精液质量的影响。雄蜂在捕获后第1、2、3、4天的平均存活率分别为98.68%、89.48%、75.93%与60.97%。多数雄蜂（平均占比80.37%）可成功射精并提供精液样本；除精子存活率在第4天略有下降（第4天为71.71%，第0天为82.8%，p < 0.05）外，不同体外维持天数下雄蜂的射精能力与精液质量均无显著差异。综上，全新的CASABee系统与蜜蜂雄蜂实验室维持方法，可有效推动该物种的生殖相关研究。