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The aims of this study were to characterize drone semen bacterial loads by culture-dependent and independent methods and to describe their variation depending on the method of semen collection, the colony and the apiary. In the first experiment, the bacterial loads of semen collected from the seminal vesicles or from ejaculates was studied using culture-dependent methods. The collection method had a significant influence on the overall bacterial count in semen. Out of the 42 semen samples analyzed, 26 (61.9%) tested positive for bacterial isolation. This encompassed the entirety of samples obtained from the seminal vesicles (21 of 21), whereas only 23.8% of those derived from ejaculates (5 out of 21) showed bacterial isolation. In the second experiment, next-generation sequencing techniques were used to describe the microbiome of ejaculated drone semen for the first time. The most abundant phyla were Proteobacteria, Firmicutes, Bacteroidota and Actinobacteriota, while the most abundant genera were Lactobacillus, Staphylococcus, Prevotella, Alloprevotella and Streptococcus. The results showed that the apiary had a significant effect on the community structure composition and abundance of the seminal microbiota, and significative differences in abundance were observed for the genera Sphingomonas, Methylobacterium-Methylorubrum, Bifidobacterium and Alloprevotella. Significant differences were also observed in the richness of the microbiota between apiaries and colonies.<\/jats:p>","DOI":"10.3390\/insects15060377","type":"journal-article","created":{"date-parts":[[2024,5,22]],"date-time":"2024-05-22T07:56:11Z","timestamp":1716364571000},"page":"377","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Microbiota Analysis of Ejaculated Honey Bee Drone Semen and the Effect of Semen Collection Method on Bacterial Loads"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5316-1703","authenticated-orcid":false,"given":"Jes\u00fas","family":"Y\u00e1niz","sequence":"first","affiliation":[{"name":"BIOFITER Research Group, Environmental Sciences Institute (IUCA), Department of Animal Production and Food Sciences, University of Zaragoza, 22071 Huesca, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3243-3711","authenticated-orcid":false,"given":"Marion","family":"Toquet","sequence":"additional","affiliation":[{"name":"Microbiological Agents Associated with Animal Reproduction (ProVaginBIO) Research Group, Departamento Producci\u00f3n y Sanidad Animal, Salud P\u00fablica Veterinaria y Ciencia y Tecnolog\u00eda de los Alimentos, Facultad de Veterinaria, Universidad Cardenal Herrera-CEU, CEU Universities, Carrer Tirant lo Blanc, 7, Alfara del Patriarca, 46115 Valencia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8991-325X","authenticated-orcid":false,"given":"Pilar","family":"Santolaria","sequence":"additional","affiliation":[{"name":"BIOFITER Research Group, Environmental Sciences Institute (IUCA), Department of Animal Production and Food Sciences, University of Zaragoza, 22071 Huesca, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9260-5792","authenticated-orcid":false,"given":"Miguel Angel","family":"Silvestre","sequence":"additional","affiliation":[{"name":"Department of Cellular Biology, Functional Biology and Physical Anthropology, University of Valencia, 46100 Burjassot, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4268-7846","authenticated-orcid":false,"given":"Raquel","family":"Toledo-Perona","sequence":"additional","affiliation":[{"name":"Microbiological Agents Associated with Animal Reproduction (ProVaginBIO) Research Group, Departamento Producci\u00f3n y Sanidad Animal, Salud P\u00fablica Veterinaria y Ciencia y Tecnolog\u00eda de los Alimentos, Facultad de Veterinaria, Universidad Cardenal Herrera-CEU, CEU Universities, Carrer Tirant lo Blanc, 7, Alfara del Patriarca, 46115 Valencia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4483-3077","authenticated-orcid":false,"given":"\u00c1ngel","family":"G\u00f3mez-Mart\u00edn","sequence":"additional","affiliation":[{"name":"Microbiological Agents Associated with Animal Reproduction (ProVaginBIO) Research Group, Departamento Producci\u00f3n y Sanidad Animal, Salud P\u00fablica Veterinaria y Ciencia y Tecnolog\u00eda de los Alimentos, Facultad de Veterinaria, Universidad Cardenal Herrera-CEU, CEU Universities, Carrer Tirant lo Blanc, 7, Alfara del Patriarca, 46115 Valencia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,5,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3896\/IBRA.1.52.4.09","article-title":"Standard methods for instrumental insemination of Apis mellifera queens","volume":"52","author":"Cobey","year":"2013","journal-title":"J. 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