## 1 Introduction  
Protein is a pivotal dietary component, considered to be one of the most expensive nutrients in commercial poultry rations [1]. The global increase in urbanisation and human population has reduced the availability of agricultural land, and climate change has negatively impacted sustainable crop production [2]. Consequently, accessibility of feed ingredients for poultry diets has decreased, and the costs of these ingredients have increased substantially, making meeting commercial poultry feed demands challenging. Refinement of dietary amino acid (AA) profiles might allow for reductions in dietary crude protein (CP) levels, reducing dependency on protein-rich feed ingredients such as soybean meal [3], and thus reducing feed costs, the use of arable land to produce oilseed meals, and nitrogen excretion into the environment [4; 5]. Biologically, birds are unable to sufficiently synthesise several AAs (the essential AAs), meaning dietary protein from feed ingredients is still required, while in reduced-protein (RP) diets, the inclusion rates of protein sources such as soybean meal are reduced, and proportions of crystalline AAs and other cereal grains (e.g., wheat) are increased, to ensure that nutrient demands are being met [3; 6]. Thus, supplementing poultry diets with crystalline or synthetic AAs can reduce reliance on soybean meal [7; 8]. In recent years, feed-grade AAs have become more commercialised and available at competitive prices in the market, allowing for their inclusion in poultry feed formulations [9]. A reduction of 45 g/kg dietary CP was found to significantly increase ileal AA digestibility coefficients by 6.18% in broiler chickens raised under tropical conditions [10]. Additionally, reducing dietary CP levels in wheat-soybean meal diets by 30 g/kg and in maize-soybean meal-based diets by 45 g/kg increased ileal digestibility by 9.10% and 5.82%, respectively [11; 12]. In contrast, a high concentration of protein-bound AAs from high-CP corn-based diets increases undigested protein reaching the hindgut, which can result in more nitrogen excretion to the environment and proliferation of gut-specific pathogens, including _Clostridium perfinogens_, _Escherichia coli_, and _Salmonella_ spp. This affects gut health integrity and production performance [13; 14; 15; 16]. Supplementation of AAs could reduce the dietary requirement of soybean meal in wheat-based diets by up to 50% and CP by 20%, without affecting performance, compared to standard-protein diets. Furthermore, nitrogen excretion can be reduced by 3 to 10% per 10 g/kg lower dietary CP [3; 17]. Determining the optimal AA profiles and order of limiting AAs (the critically important AAs) is required when replacing bound CP in soybean meal with crystalline AAs in RP diets, to ensure requirements for growth and production are being met [18]. Numerous studies have presented positive effects when feeding poultry RP corn and soybean meal-based diets supplemented with crystalline AA, but a lack of consensus about which supplemental AA are the most crucial is apparent [19; 20; 21]. The order of limiting AAs varies between the diets, dictated by the AA profiles of the major feed ingredients [22]. Crystalline AAs are absorbed rapidly, and do not need to be digested. In a recent review, [PERSON], et al. [8] indicated that inclusion rates of crystalline AAs could be increased from 7.23 to 38.49 g/kg, and soybean meal inclusion rate could be reduced by 66% (113 versus 334 g/kg) when formulating reduced-CP diets. Further research is warranted into the implications of this in the laying hen industry. There are ten essential AAs required in poultry diets for optimal production performance: lysine (Lys), methionine (Met), tryptophan (Trp), threonine (Thr), arginine (Arg), isoleucine (Ile), leucine (Leu), histidine (His), phenylalanine (Phe), and valine (Val). Lysine, Met, and Thr are the first three limiting essential AAs for both broilers and layers [23; 24; 25]. Glycine (Gly) and serine (Ser) are non-essential limiting AAs [26].