Establishment of a rapid, cost-effective, and accurate method for assessing insect sperm viability

IF 2.3 2区 农林科学 Q1 ENTOMOLOGY Journal of insect physiology Pub Date : 2024-07-26 DOI:10.1016/j.jinsphys.2024.104682
Mika Takeshima , Ayako Gotoh
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Abstract

High-quality sperm cells are crucial to reproductive success for both males and post-mating females in animals. Sperm viability, defined as the proportion of viable sperm cells, is used as a sperm quality index and this method has provided new insights into research on reproductive strategies. Sperm viability has been assessed by fluorescent staining of sperm cells. However, current staining protocols could potentially underestimate viability due to cell damage caused by cell treatments such as high dye concentration and long time for post-mounting. In this study, we established a method that enables rapid sperm viability assessment, has low sperm cell toxicity, and provides precise results regardless of operator expertise, and cost-effective using sperm cells from an ant, Crematogaster osakensis (Hymenoptera). First, to shorten the time for observation of a sufficient number of sperm cells, the volume per field of view was increased by height elevation between the glass slide and the coverslip, thereby we increased the number of sperm cells in a field of view. Second, to reduce sperm cell toxicity, we optimized the minimum dye concentration and incubation time using acridine orange (AO) and Hoechst in addition to SYBR 14 and propidium iodide (PI), which has been used in most previous studies. We determined the optimal protocol to be 1 µg/mL AO and 150 µM PI without incubation. Besides, we automated counting sperm cells with ImageJ software and combined with manual correction for more accurate results. We employed the improved method for sperm samples from mealworm beetles (Tenebrio molitor) and silkmoths (Bombyx mori). This method, established through our study, will advance research on reproductive strategies, including sperm competition and sperm quality maintenance in females.

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建立快速、经济、准确的昆虫精子活力评估方法。
高质量的精子细胞对雄性动物和交配后的雌性动物的繁殖成功至关重要。精子存活率(定义为存活精子细胞的比例)被用作精子质量指标,这种方法为生殖策略研究提供了新的视角。然而,目前的染色方案可能会低估精子的存活率,原因是高浓度染料和长时间贴装等细胞处理方法会造成细胞损伤。在这项研究中,我们利用蚂蚁(膜翅目)Crematogaster osakensis的精子细胞,建立了一种能快速评估精子活力、精子细胞毒性低、结果精确且不受操作者专业知识影响的方法。首先,为了缩短观察足够数量精子细胞的时间,我们通过提高玻璃载玻片和盖玻片之间的高度来增加每个视野的体积,从而增加了视野中精子细胞的数量。其次,为了降低精子细胞的毒性,我们优化了最小染料浓度和孵育时间,除了使用 SYBR14 和碘化丙啶(PI)之外,还使用了吖啶橙(AO)和 Hoechst。我们确定最佳方案为 1 µg/mL AO 和 150 µM PI,无需孵育。此外,我们还利用 ImageJ 软件对精子细胞进行了自动计数,并结合人工校正以获得更准确的结果。我们对黄粉虫(Tenebrio molitor)和丝蛾(Bombyx mori)的精子样本采用了改进的方法。通过我们的研究建立的这一方法将推动生殖策略的研究,包括精子竞争和雌性精子质量的维持。
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来源期刊
Journal of insect physiology
Journal of insect physiology 生物-昆虫学
CiteScore
4.50
自引率
4.50%
发文量
77
审稿时长
57 days
期刊介绍: All aspects of insect physiology are published in this journal which will also accept papers on the physiology of other arthropods, if the referees consider the work to be of general interest. The coverage includes endocrinology (in relation to moulting, reproduction and metabolism), pheromones, neurobiology (cellular, integrative and developmental), physiological pharmacology, nutrition (food selection, digestion and absorption), homeostasis, excretion, reproduction and behaviour. Papers covering functional genomics and molecular approaches to physiological problems will also be included. Communications on structure and applied entomology can be published if the subject matter has an explicit bearing on the physiology of arthropods. Review articles and novel method papers are also welcomed.
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