## 4 Discussion  
The precise formulation of RP diets must ensure a balanced AA profile, with the inclusion of crystalline or synthetic AAs being crucial to achieve this [50]. Incorporating synthetic Met, Lys, and Thr is a common practice during the formulation of RP diets for laying hens, to reduce feed cost without compromising laying performance. Determining the next limiting AA for laying hens is necessary to minimise diet costs further and reduce nitrogen excretion. This study aimed to determine the order of limiting AAs after Met, Lys, and Thr in wheat-sorghum soybean meal-based RP diet for laying hens. The study showed that egg weight, egg mass, FCR, protein intake and protein excretion were compromised in hens fed the RP diet; however, the diets deficient in Val and Ile had most adverse effects compared to the SP and RP-EAA diets. In this study, hens fed the RP diets (15.00% CP) with sufficient levels of all essential AAs had similar laying performance compared to hens offered the SP diet (17.24% CP), while hens fed the RP diets deficient in certain AAs or all essential AAs after Met, Lys, and Thr had relatively lower laying performance compared to those fed the SP diet. This suggests that RP diets could affect laying performance in absence of adequate supplemental essential AAs. These results are consistent with previous research findings showing reduced egg weight [36; 51], egg mass [16; 52], and egg production [53; 54], and increased FCR [52; 53; 55] in birds fed RP diets where the levels of essential AAs or other nutrients might be deficient. In this study, a tendency of decreasing hen-day egg production in the RP diet corresponds \begin{table}
\begin{tabular}{l l l l l l l l} \hline \hline
**Treatment** & _Lactobacillus_ & _Ruminococcus_ & _Bacteroides_ & _Bacillus_ & _Bifidobacteria_ & _Enterobacteria_ & **Total Bacteria** \\ \hline SP & 8.98 & 9.45 & 10.88 & 7.84 & 9.65 & 7.27 & 12.59 \\ RP & 8.70 & 9.34 & 11.05 & 7.58 & 9.76 & 7.61 & 12.48 \\ RP-EAA & 8.65 & 9.53 & 10.95 & 7.85 & 9.85 & 7.40 & 12.52 \\ RP-EAA-Trp & 8.67 & 9.41 & 11.01 & 7.84 & 9.80 & 8.07 & 12.55 \\ RP-EAA-Val & 8.86 & 9.42 & 11.04 & 7.68 & 9.79 & 7.18 & 12.51 \\ RP-EAA-Ile & 8.69 & 9.40 & 11.09 & 7.77 & 9.83 & 7.30 & 12.52 \\ RP-EAA-Arg & 8.72 & 9.59 & 10.96 & 7.86 & 9.91 & 7.32 & 12.57 \\ RP-EAA-Leu & 8.71 & 9.50 & 10.94 & 7.77 & 9.79 & 7.14 & 12.59 \\ RP-EAA-His & 8.69 & 9.48 & 10.95 & 7.84 & 9.83 & 7.60 & 12.53 \\ RP-EAA-Phe & 8.68 & 9.52 & 11.10 & 7.93 & 9.78 & 7.52 & 12.56 \\ RP-EAA-Gly & 8.81 & 9.50 & 11.06 & 7.76 & 9.80 & 7.88 & 12.56 \\ SEM & 0.11 & 0.05 & 0.05 & 0.14 & 0.06 & 0.59 & 0.05 \\ \(p\)-value & 0.744 & 0.082 & 0.067 & 0.908 & 0.373 & 0.991 & 0.903 \\ \hline \hline \end{tabular}
\end{table} 
Table 8: Caecal microbial quantification of the hens fed the dietary treatments at 40 weeks of age (log10 copies/g). with those reported in other studies, and might be attributed to AA deficiency and/or an imbalance in AA ratio resulting from the reduction in body protein reserves [53; 56]. In addition, AAs have functional properties, improving feed efficiency by increasing metabolic activity. In this study, hens fed the RP diet exhibited lower feed intake compared to those fed the SP diet, irrespective of AA deficiency, which might be partly due to the deficiency of total sulfur AAs in these diets [57; 58]. Isoleucine appears to be the fourth, and Val the fifth, limiting AA for laying hens fed the wheat-sorghum soybean meal diet, based on egg mass in this study. Also, hens fed the Ile- and Val-deficient RP diets had lower egg weight and egg mass compared with the SP diet in this study. These findings are consistent with [PERSON] and [PERSON] [59], who reported that supplementation of either Ile or Val to corn-soybean meal-based RP diets (13.8% CP) with sufficient levels of Met, Lys, Thr, Trp, and Arg increased egg mass in Hy-Line Brown laying hens from 31 to 38 WOA. Isoleucine, Val, and Leu are branched-chain AAs that act as precursors of other essential AA and proteins.