## 4 Discussion
This study provides new information that Trp may be less important than Val, and that Val requirements for FCR may be higher than the requirement for egg mass in wheat-sorghum soybean meal-based RP diets for laying hens. Leucine, Phe, and Gly may be considered as non-essential AAs for laying hens due to not having effects on FCR compared to the SP and RP-EAA diets, as shown by the results of this study. The differences in the effects of Ile and Val on egg mass and FCR in the present study might be due to the moderate differences between the RP and SP diets. However, determining the orders of essential AAs based on FCR may be more meaningful than the egg mass, as FCR reflects the economic efficiency to the industry [25]. The present study demonstrated that deficiency of essential AA did not affect internal and external egg quality or egg components, which is supported by earlier studies [28; 32; 81]. Moreover, the findings of this study revealed that RP diets with 20 g/kg lower CP level compared to SP diets can be achieved without substantial negative effects on egg quality of the hens, which is consistent with previous findings [28; 53; 56]. This study revealed that protein intake and excretion in hens fed the RP diets were lower than the SP diet, thus improving environmental benefits. This is in agreement with other studies presenting that lowering levels of intact protein in the diet substantially reduced N-excretion [16; 51; 82]. The lower protein intake in hens fed the RP diets in this study might be due to the similar feed intake but lower CP levels in the RP diets compared to the SP diet. Additionally, the effect of Arg, Leu, and Gly deficiency in the RP diets was more pronounced in lowering protein excretion, which might be attributed to the lower protein intake but similar protein retainment in hens offered the respective diets compared to those offered the SP diet in this study. However, AA deficiency may not affect apparent protein and energy digestibility, as shown by the results of this study and others; [PERSON], et al. [51]. No significant difference in serum uric acid level was observed between the dietary treatments in this study. Uric acid is the metabolic end product of protein metabolism, and the relative values of serum uric acid levels and dietary CP are thought to be inversely correlated, as documented in previous broiler studies [5; 83; 84]. In the current study, similar serum uric acid levels were observed in hens fed the SP and RP diets, corresponding to the findings of other laying hen studies [51; 82]. As the RP diets in the current and aforementioned studies had adequate levels of Met, Lys, and Thr supplementation, the findings may indicate the critical roles of Met, Lys, and Thr in protein utilisation in laying hens [84; 85]. Tibia morphology, breaking strength, and ash/mineral content are indicators of bone quality. In the current study, neither dietary protein level nor deficiency of essential AA exerted any effect on tibial characteristics, which supports earlier studies [82; 86; 87]. Therefore, it can be postulated that lowering CP levels might not affect bone quality in laying hens. Proteins are one of the important gut-active nutrients that can be modulated by gut microbiota in the intestinal tract [88]. Reducing dietary CP levels is thought to increase gut microbial utilisation of AAs, as the presence of undigested protein will be decreased in the hindgut [83; 89]. Moreover, numerous studies in laying hens and broiler chickens have shown that when birds are fed RP diets supplemented with crystalline AAs, the populations of beneficial gut bacteria increase while the numbers of pathogenic bacteria decrease [85; 90; 91; 92]. The present study partially supported these previous studies, in that hens fed the RP diet had relatively higher _Bacteroides_ spp. counts compared to those fed the SP diet. Moreover, the RP diets deficient in Val, Ile, Phe, and Gly showed a trend of increasing _Bacteroides_ spp.