Standardizing Sperm Vitality and Acrosomal Integrity Assays for the Conservation of Apis mellifera and Scaptotrigona mexicana

Reliable assessment of bee sperm quality is important for breeding programs, artificial insemination, and germplasm conservation. However, incubation conditions for combined fluorescence assessment remain poorly standardized across bee species. This study evaluated a sequential triple-staining protocol using Hoechst 33342, propidium iodide, and FITC-PNA in Apis mellifera and Scaptotrigona mexicana. Five incubation times (5, 10, 15, 20, and 30 min per fluorochrome) were tested. The shortest tested intervals allowing consistent fluorescence interpretation across all five microscopic fields in every biological replicate were 20 min per fluorochrome in A. mellifera and 30 min per fluorochrome in S. mexicana. Using these conditions, sperm vitality and acrosomal integrity were assessed by fluorescence microscopy after one week of storage at 22 °C in FEM7, saline solution (SS), or modified Kiev. Sperm vitality was determined by Hoechst 33342/PI live–dead classification, and localized green FITC-PNA fluorescence was the criterion for acrosomal damage. Binomial generalized linear mixed models were used to analyze the observed counts in relation to species, diluent, and their interaction. A significant species × diluent interaction was detected. Modified Kiev yielded the highest estimated sperm vitality in A. mellifera, without a significant difference from FEM7, whereas FEM7 yielded the highest estimated sperm vitality in S. mexicana. SS yielded the lowest estimated sperm vitality in both species. Acrosomal damage was detected exclusively in spermatozoa classified as dead. These results establish operational staining conditions for the combined assay and show that the relative performance of complete diluent formulations differed between species under the collection and storage conditions tested.