Reproductive Performance and Sperm Quality in the Spermatheca of Apis mellifera Queens as a Function of Fertilization Method and Floral Availability

ABSTRACT The reproductive performance of honey bee queens ( Apis mellifera ) depends on the interaction between mating strategy and seasonal fluctuations in floral resources. This study evaluated the effects of floral season (floral scarcity vs. beginning of flowering) and fertilization method natural mating (NM) vs. instrumental insemination (II) on queen rearing success, oviposition performance and the quality of sperm stored in the spermatheca. A total of 75 queens were reared per season using the Doolittle method. From these, ten queens per season (5 NM, 5 II) were established in nucleus colonies and monitored for oviposition over six consecutive weeks. Spermathecae were subsequently dissected from NM queens ( n = 5 per season) and from a subset of II queens ( n = 2 per season/control) to evaluate seminal parameters: volume, sperm concentration, motility (phase‐contrast microscopy), viability, acrosome integrity and mitochondrial activity (fluorescence microscopy). Season significantly affected queen‐rearing outcomes. Larval acceptance and queen‐cell size did not differ, but queen emergence success and weight were lower during floral scarcity (40.7%; 180 mg) than at the beginning of flowering (77.9%; 195 mg). Oviposition showed a season × fertilization interaction. Under floral scarcity, II queens produced more eggs than NM queens (626 ± 15 vs. 521 ± 21), suggesting that controlled insemination can buffer resource limitation. During the beginning of flowering, NM queens achieved higher brood production (2232 ± 30 vs. 1756 ± 19), reflecting enhanced reproductive capacity under floral abundance conditions. Sperm quality was primarily influenced by season. Sperm quality within the spermatheca was primarily driven by season. Floral abundance significantly increased sperm concentration (7.10 vs. 4.60–6.74 × 10 6 /μL), motility (scale 0–4: 3.0 vs. 2.0), viability (80% vs. 55%), acrosome integrity (64% vs. 60%–61%), mitochondrial activity (75% vs. 62%) and seminal volume (3.0 vs. 2.2–2.5 μL). Fertilization method had a limited effect with NM queens showing higher sperm motility only under floral abundance conditions. Overall, II supports higher oviposition under resource scarcity, whereas NM maximises brood production and sperm performance when floral availability is abundant. These results provide further practical guidance for optimising queen‐rearing schedules and selecting mating strategies in apicultural management.