# The Impact of Grafted Larvae and Collection Day on Royal Jelly's Production and Quality
## 1 Introduction  
Royal jelly (RJ) is a complex and highly nutritious secretion synthesized by the hypopharyngeal and mandibular glands of young worker bees (_Apis mellifera_ L.), primarily to nourish developing larvae and the queen bee. Its critical role in determining bee caste development is well-established: all larvae are initially fed with RJ, but only those continuously fed beyond the third day develop into queens. This exclusive diet drives profound phenotypic differences between queens and workers, despite their identical genetic background [1]. The nutritional and epigenetic mechanisms triggered by RJ include enhanced reproductive capacity, increased longevity, and anatomical distinctions [2], positioning RJ not only as a vital factor in hive development but also as a product of interest in the functional food, pharmaceutical, and cosmetic industries. Chemically, RJ comprises water (50-70%), proteins (9-18%), carbohydrates (7-18%), lipids (3-8%), vitamins, minerals, and a variety of bioactive components, including major royal jelly proteins (MRJPs) and 10-hydroxy-2-decenoic acid (10-HDA), a fatty acid uniqueto RJ with recognized bioactivity [3; 4]. These constituents are responsible for RJ's diverse biological properties, including antioxidant, antimicrobial, immunomodulatory, and anti-aging effects [5; 6]. The chemical profile of RJ is, however, highly sensitive to a range of intrinsic and extrinsic factors. While seasonal variation, botanical environment, and geographic origin are known to influence RJ composition [7; 8], procedural aspects related to its production--such as the age of grafted larvae and the day of collection--are critical, yet underexplored, determinants of quality and consistency. Pollen is the primary source of proteins, lipids, vitamins, and minerals in the honeybee diet and plays a pivotal role in the development and functioning of the hypopharyngeal glands, which are responsible for RJ secretion [9]. A diet rich in high-quality pollen has been shown to enhance both the quantity and nutritional value of RJ, primarily by upregulating glandular protein synthesis and metabolic activity in nurse bees [10; 11]. Conversely, pollen scarcity or low-quality pollen sources can lead to reduced RJ production and altered biochemical composition, particularly with respect to protein content and 10-HDA concentration [12]. Therefore, the availability, diversity and nutritional adequacy of pollen sources must be carefully considered in any evaluation of RJ yield and quality [8]. In commercial RJ production, techniques involve grafting young worker larvae into queen cups, prompting nurse bees to produce large quantities of RJ. Although it is standard practice to use larvae less than 24 h old for optimal results, the exact age at grafting can significantly impact both the quantity and quality of produced RJ. Larval age affects not only the biochemical signaling within the hive but also the intensity and composition of the glandular secretions from nurse bees [13; 14]. As the age of the grafted larvae increases, the nurse bees' responsiveness may decline, resulting in changes in the nutritional profile of the jelly. These compositional changes may include fluctuations in MRJP concentrations, total protein content, and key sugars such as glucose and fructose. Furthermore, larval age might influence the lipid profile, especially bioactive fatty acids like 10-HDA, which serve as essential markers for RJ quality and authenticity [3; 15; 16]. The biological activity and commercial value of RJ are thus closely tied to these factors, yet standard guidelines for larval age are often based on anecdotal or empirical practices rather than systematic study. The timing of RJ harvest after larval grafting--typically after 48, 72, or 96 h--plays a pivotal role in determining its final physicochemical properties. While a 72 h collection period is generally adopted in commercial settings, variations in this interval can significantly affect the yield of RJ and the stability of bioactive compounds.