Esteso, Cristina MilagrosToledano Díaz, AdolfoCastaño, CristinaHiges, MarianoMartín Hernández, RaquelLópez Goya, AgustinDe la Rúa, PilarMartínez Madrid, Carmen BelénSantiago Moreno, Julián2025-03-282025-03-282025Esteso, M. C., Toledano-Díaz, A., Castaño, C., Higes, M., Martín-Hernández, R., López-Goya, A., De la Rúa, P., Martínez-Madrid, B., & Santiago-Moreno, J. (2025). Storage Conditions of Sperm Samples and Gametic Characterization by Sperm Head Morphometry in Drones (Apis mellifera). Animals, 15(5), 672. https://doi.org/10.3390/ani1505067210.3390/ani15050672https://hdl.handle.net/20.500.14352/119028Author Contributions: Conceptualization, M.C.E., J.S.-M., M.H., R.M.-H. and A.L.-G.; methodology, A.T.-D., C.C. and P.D.l.R.; software, A.T.-D.; validation, J.S.-M.; formal analysis, M.C.E.; investi gation, M.C.E., J.S.-M., M.H., R.M.-H., A.L.-G. and P.D.l.R.; resources, M.C.E.; data curation, C.C.; writing—original draft preparation, M.C.E. and B.M.-M.; writing—review and editing, J.S.-M., A.T.-D. and A.L.-G.; visualization, A.T.-D.; supervision, M.C.E.; project administration, M.C.E.; funding acquisition, M.C.E. All authors have read and agreed to the published version of the manuscript.The present study aimed to evaluate an optimal method to transport and store drone sperm samples, as well as to characterize drone spermatozoa through sperm head morphometry. A total of 291 mature drones were used. We performed three experiments. In a first experiment, sperm variables were assessed under different incubation conditions (5 °C, 15 °C, and 37 °C with 5% CO2). Results showed that sperm viability was optimally maintained at 15 °C (p ˂ 0.05). In the second experiment, the supplementation of extender with catalase (200 UI) improved (p ˂ 0.05) the sperm viability and motility during liquid storage at different incubation times. Finally, a morphometric analysis of sperm head was made: length 5.13 µm, width 0.85 µm, area 3.78 µm2, perimeter 15.01 µm, acrosome length 3.50 µm. The variability in sperm head morphometry was calculated by coefficients of variation (CV) within- and between-drones. The CV within-drone was higher than the CV between-drones for all morphometric parameters regardless of hive origin, indicating a high degree of sperm pleomorphismSimple Summary: Environmental factors may produce morphological and morphometric alterations in the honey bee’s sperm. These changes in drone spermatozoa can produce variations in their physical characteristics, which could cause the loss of ability to remain viable within the queen bee’s spermatheca. The storage and conservation of sperm is an effective strategy to protect the genetic diversity of honey bees and contribute to selective breeding programs. The extender choice greatly impacts sperm quality. The use of antioxidants in extenders for preservation could improve the functional semen characteristics. Indeed, the supplementation of extender with catalase (200 UI) improved the sperm viability and motility during liquid storage. The accurately measured dimensions of sperm head allow improving the understanding of reproductive biology and might even be used as indicators of environmental influences. The variability of sperm head morphometry was calculated, revealing a high degree of sperm pleomorphism in drones, suggesting the existence of sub-populations that should be investigated in the future.engAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/journal article2076-2615https://doi.org/10.3390/ani15050672open access636.082.4Short-term storageSeminal qualityCatalaseSperm morphometryHoney beesProducción animal3104 Producción Animal