## 4 Discussion
Branched-chain AAs can regulate fatty acid metabolism in the liver [60], which plays a vital role in hepatic lipoprotein production, an important limiting factor for egg formation [61]. It has been presumed that the Leu requirement can be met by various sources of protein in diets [25], but it appears that supplementation of Ile and Val in RP diets is necessary to maintain egg production, egg weight, and egg mass [28; 62]. Moreover, an antagonism effect between Ile, Val, and Leu may appear at higher concentrations in higher-protein diets compared to the RP diets [63; 64]. The importance of Ile was highlighted in earlier studies. For instance, it has been indicated that the deficiency of Ile in the corn-soybean meal RP diet compromised egg weight and egg mass in Hy-Line Brown laying hens during 35 to 43 WOA [65]. Others have reported that adding Ile to the RP diet reduced CP requirements (2%) without affecting laying hen performance during 20 to 46 WOA [66]. In contrast, [PERSON], et al. [32] found that Ile supplementation from 0.1 to 0.4% to a corn-soybean meal RP diet (14% CP) with sufficient levels of Met, Lys, Thr, Trp, and Val did not affect laying performance in [PERSON] laying hens during 28 to 40 WOA. The lack of response to Ile supplementation in this scenario might be due to the insufficient levels of Arg and Phe in the diet, as Trp, Arg, and Phe were shown to be of equal importance (the sixth co-limiting AA) in the RP diets in the current study. Arginine stimulates luteining hormone secretion, which impacts ovarian follicle development and ovulation [67]. This supports the present findings, in that Arg deficiency negatively impacts egg weight and egg mass, reaffirming the importance of considering the Arg requirement in RP diet formulations. Tryptophan and Phe are aromatic AAs, reducing stress by the synthesis of neurotransmitters such as serotonin and dopamine [68; 69], thus influencing hen behaviour. Adequate supplementation of these AA in RP diets is required to maintain hens' reproductive organs' growth and development [70; 71]. On the other hand, the findings of this study suggested that Leu, His, and Gly may be considered non-essential AA as the egg mass of hens fed these diets was similar to those fed the RP-EAA and SP diets. The main function of His in poultry nutrition is to synthesise carosine, which substantially increases the antioxidant capacity of muscles, having minimal impact on laying performance [72; 73]. The lack of effect of Gly in the present study supports previous studies that supplementation of Gly did not affect egg production and egg mass [74; 75]. However, [PERSON], et al. [76] found some positive effects of Gly supplementation on egg weight and egg mass in Mandara laying hens during 28 to 40 WOA that might be due to breed differences from the current study. In this study, if the AA order is ranked based on FCR, Val may be considered the fourth limiting, and Trp, Ile, Arg and His may be considered as co-fifth limiting AA in wheat-sorghum soybean meal RP diets for laying hens. This ordering is different from [PERSON], et al. [55], who demonstrated Trp as the 4 th, and Val and Ile as co-fifth, limiting AA in com-soybean meal RP diets, based on the laying performance, including FCR. The discrepancy between the studies might be due to the differences in diet composition and study duration. Our research findings support the prediction of [PERSON], et al. [77], who indicated that Val may be the next limiting AA after Met, Lys, Trp, and Thr in a RP diet (14.8% CP) for laying hens, based on the requirements of digestible Val: Lys ratios in commercial corn-soybean meal layer hen diets. The necessity of Val in layer hen diets is evident in other studies showing that increasing Val level significantly decreases FCR [78; 79; 80]. Moreover, [PERSON], et al. [80] stated that Val requirement in laying hens fed a corn-peanut-based diet was highest to optimise egg mass, followed by egg production, and lowest to optimise FCR.