## 2 Materials and Methods
### Experimental Design and Dietary Treatment  
At 20 WOA, birds were weighed individually and randomly allocated to eleven dietary treatments (30 replicates/treatment). Average hen starting weights did not differ significantly between the dietary treatments (\(p>0.05\)). The first three dietary treatments were a standard-protein diet (SP, 17.24% CP); a reduced-protein diet (15.00% CP) with sufficient levels of Lys, Met, and Thr but insufficient levels of other essential AAs (RP); and a reduced-protein diet (15.00% CP) with sufficient levels of all essential AAs (RP-EAA). Eight subsequent dietary treatments were formulated by the deletion method as described by [PERSON], et al. [19], whereby each specific AA was removed from the RP-EAA diet: tryptophan (RP-EAA-Trp), valine (RP-EAA-Val), isoleucine (RP-EAA-Ile), arginine (RP-EAA-Arg), leucine (RP-EAA-Leu), histidine (RP-EAA-His), phenylalanine (RP-EAA-Phe), and glycineequivalent (RP-EAA-Gly) (Table 1). The CP level in the SP diet was per HyperLine Brown nutritional recommendations [35], while the CP levels in the RP diets were selected followed the recommendations of previous studies on RP diets [28; 36]. The levels of essential AAs were selected based on [PERSON] nutritional recommendations [35]. Feed was provided in mash form. The nutritional composition of the major ingredients, including dry matter (DM), gross energy (GE), CP, crude fat, and ash content, was analysed prior to formulating the diets, and these values used for diet formulation (Table 2). \begin{table}
\begin{tabular}{c c c} \hline \hline
**Treatment Number** & **Treatment Code** & **Treatment Description** \\ \hline \hline
1 & SP & A standard-protein diet with sufficient levels of all essential amino acids \\ \hline
2 & RP & A reduced-protein diet with sufficient levels of Lys, Met, and Thr but deficient in Trp, Arg, Ile, Val, Leu, His, Phe, and Gly \\ \hline
3 & RP-EAA & A reduced-protein diet with sufficient levels of all essential amino acids, including Lys, Met, Thr, Trp, Arg, Ile, Val, Leu, His, Phe, and Gly \\ \hline
4 & RP-EAA-Trp & Treatment 3 deficient in Trp \\ \hline
5 & RP-EAA-Val & Treatment 3 deficient in Val \\ \hline
6 & RP-EAA-Ile & Treatment 3 deficient in Ile \\ \hline
7 & RP-EAA-Arg & Treatment 3 deficient in Arg \\ \hline
8 & RP-EAA-Leu & Treatment 3 deficient in Leu \\ \hline
9 & RP-EAA-His & Treatment 3 deficient in His \\ \hline
10 & RP-EAA-Phe & Treatment 3 deficient in Phe \\ \hline
11 & RP-EAA-Glycineequivalent & Treatment 3 deficient in Gly \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Description of dietary treatments. \begin{table}
\begin{tabular}{l c c} \hline \hline
**Ingredients (\%, Otherwise as Indicated)** & **SP** & **RP** \\ \hline Wheat & 38.44 & 49.52 \\ Barley & 4.00 & 4.00 \\ Sorghum & 20.00 & 20.00 \\ Soybean meal & 13.77 & 2.68 \\ Canola meal & 10.00 & 9.66 \\ Canola oil & 2.59 & 0.80 \\ Limestone (fine) & 4.93 & 4.94 \\ Limestone (coarse) & 4.93 & 4.94 \\ Wheat & 38.44 & 49.52 \\ Barley & 4.00 & 4.00 \\ Sorghum & 20.00 & 20.00 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Nutrient composition of the standard- and reduced-protein diets (as-is basis). The compositions of the final dietary nutrients were also analysed and presented in Table 3. Generally, analysed nutrient contents were close to the calculated values, indicating the dietary formulation objectives were achieved. Additionally, the RP diet deficient in all essential AAs except Lys, Met, and Thr and the RP-EAA diet sufficient in all essential AAs but with lower CP levels relative to the SP diet were obtained as expected (Tables 2 and 3).