# The Impact of Grafted Larvae and Collection Day on Royal Jelly's Production and Quality
## 3 Results and Discussion
### Effect of Larval Age at Grafting on Royal Jelly Composition and Yield  
While beekeeping practice conventionally recommends second-day larvae for optimal RJ production, our experimental data reveal significant compositional differences associated with larval age at grafting. Comparative analysis of RJ derived from first-day (Group A2), second-day (Group B2), and third-day (Group C2) larvae demonstrates distinct physicochemical profiles (Table 2). These findings challenge the assumption of uniform RJ quality across the standard grafting window and suggest that larval developmental stage serves as a critical determinant of RJ properties. The observed variations, particularly between RJ produced from first-day versus third-day larvae, may explain anecdotal reports of textural differences in commercial production. Notably, while all samples were harvested at the standard 72 h interval post grafting, the initial larval age at grafting appears to initiate divergent biochemical pathways that persist through RJ secretion. This temporal effect was consistent across all 36 collected samples (12 per experimental group), reinforcing the statistical validity of the observed trends.  
The yield per cell varied significantly among the groups (_p_ < 0.05): B2 gave the highest yield (0.332 \(\pm\) 0.10 g), followed by C2 (0.241 \(\pm\) 0.09 g) and A2 (0.193 \(\pm\) 0.06 g). The observed yield differences between experimental groups likely reflect distinct nutritional requirements during larval development. Group A2 (first-day larvae) demonstrated reduced RJ accumulation, potentially attributable to the extended secretion period required to achieve adequate provision mass. These findings align with established apian physiology, where nurse bees modulate RJ production in response to larval pheromonal cues and nutritional needs [29; 30]. The yield patterns further suggest that current RJ harvesting protocols, typically optimized for second-day larvae [31], may require reevaluation to account for these developmental influences on production efficiency.  
Interestingly, none of the physicochemical parameters (moisture, proteins, sugars, or 10-HDA) were significantly affected by larval age (_p_ > 0.05). This suggests that while larval age influences the quantity of RJ, it does not significantly impact its composition. Moisture content ranged between 60.5% and 71.8%, with values comparable to those reported in the literature [32; 33]. Protein levels ranged from 11.3% to 13.7%, and sugar and 10-HDA content fell within similar levels. The relatively high sucrose content is attributed to supplemental feeding with carbohydrate-rich syrup, a practice known to influence sugar levels in RJ [34; 35]. These findings lead to the rejection of the null hypothesis (H\({}_{2}\)), indicating that larval age does affect RJ characteristics.  
Regarding larval age, conventional beekeeping practices favor second-day larvae for grafting, a recommendation empirically supported by higher RJ yields [31], while the findings of [PERSON] et al. [29] for _A. cerana_ suggested the use of third-day larvae may reduce the quality of RJ. Our results corroborate those of [PERSON] et al. [31], because in group B2, higher production was achieved.