# The Impact of Grafted Larvae and Collection Day on Royal Jelly's Production and Quality
## 3 Results and Discussion
### Effect of Harvesting Day on Physicochemical Characteristics and Yield of Royal Jelly  
Beekeepers commonly observe RJ samples with unusually low moisture content, often associated with suboptimal collection timing. To systematically evaluate how harvest duration influences RJ composition and production yield, we analyzed samples collected at three critical time points (24 h, 48 h, and 72 h post grafting) from colonies grafted with second-day larvae. Our findings (Table 1) demonstrate that the temporal interval between grafting and harvesting significantly affects both the physicochemical profile and total yield of RJ, with pronounced differences observed between the 24 h (Group A1), 48 h (Group B1), and 72 h (Group C1) collection groups. These results provide empirical validation of beekeepers' practical observations while offering new insights into the time-dependent biochemical dynamics of RJ secretion. The mean RJ quantity one day after the grafting was only 0.052 \(\pm\) 0.023 g per cell, insufficient for analysis; thus, multiple cells were collected from two consequent graftings. On the second and third days, RJ deposition increased significantly (\(p\) = 0.001) to 0.138 \(\pm\) 0.095 g and 0.326 \(\pm\) 0.128 g per cell, respectively, indicating a near-exponential \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Parameter** & **Group A1** & **Group B1** & **Group C1** \\  & **(_n_ = 9)** & **(_n_ = 18)** & **(_n_ = 12)** \\ \hline Moisture & 48.3 \(\pm\) 1.05 c & 58.1 \(\pm\) 1.95 b & 67.8 \(\pm\) 1.73 a \\ (\%) & (46.5–49.9) & (54.9–61.1) & (64.8–70.5) \\ \hline Protein Content & 18.6 \(\pm\) 0.56 a & 16.2 \(\pm\) 0.78 b & 12.3 \(\pm\) 0.82 c \\ (\%) & (17.8–19.3) & (15.2–17.7) & (11.3–13.6) \\ \hline Fructose & 2.99 \(\pm\) 0.32 a & 3.32 \(\pm\) 0.70 a & 3.00 \(\pm\) 0.41 a \\ (\%) & (2.28–3.31) & (2.24–5.54) & (2.24–3.64) \\ \hline Glucose & 2.92 \(\pm\) 0.43 a & 3.13 \(\pm\) 0.29 a & 3.07 \(\pm\) 0.37 a \\ (\%) & (2.26–3.44) & (2.66–3.61) & (2.26–3.51) \\ \hline Sucrose & 1.24 \(\pm\) 0.36 b & 1.69 \(\pm\) 0.49 a & 1.88 \(\pm\) 0.44 a \\ (\%) & (0.75–1.92) & (1.03–2.55) & (1.12–2.89) \\ \hline
10-HDA & 2.45 \(\pm\) 0.27 a & 2.33 \(\pm\) 0.42 a & 2.29 \(\pm\) 0.39 a \\ (\%) & (2.01–2.83) & (1.77–3.02) & (1.61–2.80) \\ \hline \hline \end{tabular} Where different latin letters are used, statistically significant differences were found (a = 0.05). The values in parentheses refer to the range of values for each parameter. Where _n_: the number of samples in each group. A1 Group: 24 h after grafting; B1 Group: 48 h after grafting; C1 Group: 72 h after grafting.  
\end{table} 
Table 1: Physicochemical parameters of RJ samples collected in different days after the grafting (average \(\pm\) standard deviation). pattern of secretion. This pattern supports the hypothesis that worker bees modulate RJ production in response to larval age and nutritional demand. Moisture content showed a statistically significant increase across the three groups: 48.3 \(\pm\) 1.05% in Group A1, 58.1 \(\pm\) 1.95% in B1, and 67.8 \(\pm\) 1.73% in C1 (\(p\) = 0.002). These findings suggest that water is added progressively into RJ, as the jelly matures in the queen cell. Notably, Group A1 exhibited moisture levels that were lower than any previously reported values in the literature [24]. Conversely, the total protein content decreased significantly over time (\(p\) < 0.001), dropping from 18.6 \(\pm\) 0.56% (A1) to 16.2 \(\pm\) 0.78% (B1) and finally to 12.3 \(\pm\) 0.82% (C1). This trend could be attributed either to dilution by added moisture or to the enzymatic transformation and binding of proteins with other constituents. Nevertheless, the measured range (11.3-19.3%) aligns with reported range of RJ protein content in previous research [25; 26]. In contrast, the concentrations of glucose and fructose remained statistically unaltered across groups (\(p\) > 0.05), indicating that early or late harvesting does not significantly affect these sugars.