## 1 Introduction
The use of synthetic Lys, Met, and Thr in commercial poultry diets is a common practice worldwide. In most cases, other essential AAs will not be deficient, due to the high levels in soybean meal in the diet. However, as the level of soybean meal is reduced in RP diets, these other AAs may become limiting [3; 27]. There is evidence that synthetic AA supplementation can be used as a tool to overcome this. For example, [PERSON], et al. [3] observed that the inclusion of crystalline Met, Lys, Thr, Val, Ile, and Arg into broiler chicken diets permitted the dietary levels ofsoybean meal and CP to be reduced by up to 50% and 20%, respectively. Moreover, [PERSON], et al. [28] found that egg production and feed efficiency were similar in laying hens fed a corn-soybean meal RP diet (140 g/kg CP) supplemented with Lys, Met, Thr, Trp, Ile, and Val compared to those fed a standard-protein diet with 160 g/kg CP. After Met, Lys, and Thr, the next limiting AAs in broiler chickens are Val, Ile, and Arg, in wheat- and sorghum-based soybean meal and canola meal diets [8; 23; 29; 30]. However, information on the order of limiting AAs in laying hens is lacking, particularly in wheat-sorghum soybean meal-based diets, and is likely different from broilers. Early work by [PERSON] [31] suggested that Ile could be the fourth limiting AA in low-protein corn-soybean meal-based diets for laying hens. In contrast, [PERSON], et al. [32] found that supplementation of Ile in RP diets did not affect egg production, egg quality, or immunological parameters in laying hens. This may reflect the greater egg production and different body development rates in modern-strain layers compared to breeds used decades ago. [PERSON], et al. [28] indicated that other AAs, such as Trp, Val and Ile, are just as important as Met, Lys, and Thr for laying hen performance. Thus, priority AAs in formulating RP diets for laying hens still need to be explored. It is evident that the dietary AA requirements of laying hens should be revised, as current recommendations by NRC [33] do not reflect the needs of modern birds. Increasing commercial availability of limiting AA other than Met, Lys, and Thr might allow for more pronounced reductions in dietary CP levels and reduce reliance on soybean meal inclusion. Therefore, this study was focused on how hen laying performance and egg quality were compromised by the individual deficiency of eight essential AAs other than Met, Lys, and Thr in the RP diet compared to the conventional standard-protein diet to determine the limiting order of these essential AAs in the practical Australian wheat-sorghum-based RP diet. Moreover, nutrient digestibility, bone parameters, and the caecal microbiome were further taken into account to determine if deficiency of specific AAs may have a negative effect during the production cycle. It is hypothesised that the limiting orders of essential AAs in the RP wheat-based diets may be different, and deficiency of essential AAs in these diets would reduce laying hen performance, nutrient digestibility, and health conditions. The findings of this study may help to facilitate precise feed formulations of reduced-protein diets for laying hens that may consequently promote sustainable layer hen production.