Optimising Energy-to-Protein Ratio in Practical Reduced-Protein Diets for Laying Hens

Abstract

This study evaluated the optimal energy-to-protein ratio in practical reduced-protein diets to evaluate the production performance, nutrient digestibility, and egg quality parameters of laying hens between 20 and 35 weeks of age. The best feed conversion ratio (FCR) of 2.548 was achieved at 15.5% crude protein (CP) and 100% recommended apparent metabolizable energy (AME) level with a reduced-protein diet, followed by 14% CP and 95% recommended AME levels (2.634) from 20 to 35 weeks of age (WOA) (p < 0.05). The yolk index was reduced only in diets containing 17% CP at 90% AME levels (p < 0.01) at week 35. Reduced dietary protein from 17% to 14% decreased egg weight and body weight gain between 20 and 35 WOA (p < 0.05) as well as decreased hen weight and shell breaking strength at 35 WOA (p < 0.05). However, it also reduced nitrogen excretion by 30% (p < 0.001) and increased protein digestibility by 17% (p < 0.01). Between 20 and 35 weeks of age, reducing dietary energy from 100% to 90% AME increased feed intake (p < 0.001), while excreta moisture, dry matter digestibility, and energy digestibility decreased at week 35 (p < 0.001). At week 27, reducing energy from 100% to 90% AME increased shell weight (p < 0.05), whereas lowering protein from 15.5% to 14% increased shell proportion (p < 0.05). Thus, it can be concluded that reducing dietary protein level from 17% to 15.5% with a 100% recommended AME level is optimal to improve feed efficiency while maintaining egg quality in laying hens from 20 to 35 WOA.

1. Introduction

Reduced dietary crude protein (CP) has gained increased interest in the poultry industry due to increased protein digestibility and feed efficiency, improved gut health and litter quality, as well as decreased water intake, nitrogen, and ammonia emissions [1,2]. Moreover, decreasing the reliance on arable land for cultivating oilseed meals may help stabilize feed costs and promote more sustainable poultry production systems [3]. In previous studies, inclusion of crystalline amino acids (AA) in broiler grower diets reduced dietary soybean meal levels by 50% [4]. Furthermore, supplementing low-protein corn and soybean meal diets (14% CP) with key AA such as methionine, lysine, threonine, tryptophan, isoleucine, and valine can help sustain feed efficiency and egg production in laying hens [5]. Finally, feeding reduced-protein diets may improve gut health and increase the numbers of beneficial microbiota populations in laying hens with subsequent effect on increasing numbers of clean eggs. Little attention has been paid to gut health in laying hens as the conventional cage systems could effectively prevent birds from fecal–oral exposure. However, as egg production moves towards cage-free systems, gut-related problems and incidence of foot pad lesions may increase in laying hen flocks [6].

In recent years, reduced-protein diets have become increasingly popular. Due to differences in digestion dynamics between low- and standard-protein diets [7], a better understanding of AA requirements for laying hens is required. In particular, determining the sequence in which essential AA becomes limiting in laying hen diets is crucial for optimizing AA supplementation. This limiting order was investigated in hens fed diets containing wheat, sorghum, and soybean meal, which are common ingredients in poultry feeds [8]. A study by Jahan et al. [8] recorded that valine was identified as the fourth limiting AA, while tryptophan, isoleucine, arginine, and histidine were each considered the co-fifth limiting AA. In contrast, leucine, phenylalanine, and glycine were found to be non-limiting in terms of their effect on feed conversion ratio (FCR), following lysine, methionine, and threonine [8]. These findings could enable more precise feed formulation and support the wider adoption of reduced-protein diets in laying hen production. They also support the hypothesis that reduced CP diets can be effective when essential AA profiles are balanced appropriately. Beyond performance implications, balanced reduced CP diets contribute to sustainability by reducing nitrogen excretion, reducing land use, and lowering feed costs at laying hen farms. The ideal protein concept, expressing AA ratios relative to lysine, serves as a foundation for precision feeding in such diets [4]. Nevertheless, continuous evaluation of AA requirements remains necessary, as optimal levels are influenced by factors such as dietary protein content, bird genotype, environmental conditions, and specific production goals [9].

Reduced-protein diets offer benefits, yet ongoing adjustments are essential to resolve existing issues and ensure peak performance in laying hens. For instance, Rozenboim et al. [10] reported that laying hens consuming a diet high in energy but low in protein (3000 kcal apparent metabolizable energy (AME) per kg, 13% CP) showed reductions in feed consumption, body weight, egg weight, and egg production compared to those fed a control diet containing 2750 kcal AME/kg and 17.5% CP. Further, high energy and low CP diets increased plasma liver enzyme levels (alkaline phosphatase and aspartate aminotransferase), liver hemorrhagic score, colour score, and fat content, indicating liver dysfunction [10]. This physiological change is strongly associated with fatty liver hemorrhagic syndrome, an important metabolic disorder in layer hens. These results confirm that low-protein, high-energy diets impair performance and pose serious health risks to hens, which is relevant to commercial egg producers concerned with both production and welfare. Similarly, Jiang et al. [11] reported that laying hens fed a low-protein, high-energy diet (3040 kcal AME/kg, 11.3% CP) had lower feed intake and egg production, higher liver fat and abdominal fat pads, and higher levels of serum leptin-like protein, osteocalcin, estrogen, and lower keel osteocalcin than control hens (2735 kcal AME/kg diet, 15.8% CP). These outcomes are directly correlated with insufficient protein supplies for egg synthesis and metabolic control. However, several obvious disadvantages could be seen in these studies. Firstly, reducing dietary protein to very low concentrations (11% to 13% CP) in these studies may result in inadequate levels of essential and non-essential AA (e.g., Thr, Trp, Arg, Val, Ile, Leu, His, Phe, Gly) as well as other dietary elements such as electrolyte balance and potassium, which were not considered during the experiments. Secondly, because of the antagonistic interactions among AA, a deficiency in essential AA like Arg may cause an imbalance in the arginine-to-lysine ratio, potentially impairing laying performance in hens [12,13]. Thirdly, a deficiency in an essential AA may result in the degradation or conversion of other essential AA for nonessential purposes, decreasing protein synthesis and egg production [14,15]. Thus, the reduced laying performance in hens fed high-energy and low-protein diets, compared to control hens in studies conducted by Rozenboim et al. [10] and Jiang et al. [11], might not only be due to the high energy-to-protein ratios but other nutritional factors in these studies. For instance, in the study by Jiang et al. [11], only Lys and Met were supplemented in the reduced-protein diets, while Rozenboim et al. [10] did not report the detailed dietary composition. Other research with less severe differences in dietary energy and CP levels between the treatments showed similar findings. For example, Novak et al. [16] reported that White Leghorn hens consuming a low-protein corn–soybean meal diet with a recommended energy (2871 kcal AME/kg, 14% CP) showed reduced feed efficiency compared to hens given diets with recommended protein and energy (2871 kcal AME/kg, 17% CP) or low protein combined with low energy (2785 kcal AME/kg, 14% CP) during the period of 39 to 50 weeks of age. Likewise, Meluzzi et al. [17] reported that Hy-Line Brown laying hens consuming reduced-protein corn–soybean meal diets with the recommended energy level (2854 kcal AME/kg, 15% CP) experienced reductions in laying rate, egg mass, egg weight, and feed efficiency compared to hens on control diets with 17% CP and equivalent energy levels between 33 and 40 weeks of age.

The above studies on reduced-protein diets only consider a limited number of EAA during formulation, usually Lys, Met, Met plus Cys, Thr, and/or Trp. This could explain why performance outcomes were influenced by deficiencies of other EAA rather than the dietary energy-to-protein ratio. Moreover, laying hens fed a high-fat and low-protein diet are more likely to suffer from fatty liver and skeletal problems [18,19]. Thus, ensuring adequate energy and protein intake is critical to promote production efficiency and the birds’ health [16]. The energy-to-protein ratio must be adjusted when protein levels are reduced to maintain laying performance and physiological balance; however, this research topic has not been fully explored yet. A laying hen study conducted by Li et al. [20], where a 4 × 3 factorial design was used with the factors of dietary AME (2400, 2550, 2700 and 2850 kcal/kg diet) and CP levels (14.5, 16.0 and 17.5%), showed promising results. In more detail, it was found that feeding Lohmann Brown hens a diet containing 2400 kcal/kg of AME/kg and 16% CP which contained corn, wheat bran, soybean meal, cottonseed meal, and canola meal enhanced egg production and egg mass, compared with the other diets [20], whereas hens offered diets with low energy and recommended protein levels (2400 kcal AME/kg diet and 17.5% CP) had the lowest egg production, egg mass, eggshell thickness, and highest broken egg proportion compared to the other diets [20]. A study by Li et al. [20] also suggested that an AME intake ranging from 325.7 to 331.7 kcal/day and CP intake between 19.5 and 20.7 g/day are optimal for maximizing egg production, egg mass, and FCR in Lohmann Brown laying hens aged 26 to 38 weeks. As the ideal digestible AA pattern recommended by Lemme [21] was considered in the study conducted by Li et al. [20], the AA deficiency, imbalance, and antagonism might be minimized. Li et al. [20] proposed that a balanced diet may increase laying hen performance with moderate reductions in AME and CP. This information may be useful for egg producers who may want to reduce the feed cost and/or utilize more low-energy feed ingredients such as barley, oat, and wheat millrun when these ingredients become more available. In this study, we investigated energy-to-protein ratios in reduced-protein diets for laying hens, with all essential AA considered when formulating the diet, thereby minimizing the chances of deficiency or imbalance—a known issue with reduced-protein diets. Three dietary energy levels and three protein levels were used to assess their interaction with key performance indicators. This study aimed to match study objectives and outcomes by assessing whether the impact would affect production performance, nutrient utilization, and egg quality. Specifically in regions with limited soybean production, this research addresses a current gap in the literature regarding developing sustainable, low-CP diets to maintain laying hen performance while reducing environmental impact and import dependency. We hypothesize that a moderate reduction in dietary protein together with balanced essential AA and appropriate dietary energy levels can improve feed efficiency without compromising egg production and egg quality.

2. Materials and Methods

2.1. Experimental Design and Housing Conditions

The study was conducted in the Laureldale Layer Cage Research Facility Center, Cluny Road, University of New England (UNE), NSW, Australia, in 2024. The UNE Animal Ethics Committee approved the study (Approval number: ARA24-002) and it met the Australian code of practice for use and care of animals for scientific purposes [22].

The study was conducted in a layer cage facility over a period of 16 weeks from 20 to 35 weeks of age using a 3 × 3 factorial design with dietary crude protein levels (17, 15.5, and 14% CP), as well as dietary energy levels (90, 95, or 100% of dietary AME levels based on breed recommendations). Therefore, there were nine dietary treatments with 13 replicate cages of two hens per cage per treatment (n = 234). Each experimental unit consisted of an individual cage housing two hens. The energy levels in the 90%, 95%, and 100% recommended AME diets were 2453, 2589, and 2725 kcal/kg, respectively. The optimal energy-to-protein ratio from 20 to 35 weeks of age was selected based on FCR and egg quality. The Hy-Line Brown laying hens were evenly distributed between the dietary treatments at the start of the experiment. Birds were housed in individual cages (30 cm wide, 50 cm deep, 45 cm high) with a nipple drinker and feed trough per cage. Lights were kept on for 16 h and dark for 8 h throughout the study. Lights are turned on at 4 am and off at 8 pm according to the Hy-Line Brown laying hen management guide [23]. Ambient temperature and relative humidity were measured twice daily throughout the experimental period, but environmental conditions were not regulated. Mashed feed was provided to the hens. Feed and water were available to the birds ad libitum for the duration of the study. Figure 1a,b presents the weekly averages of temperature and relative humidity recorded in the hen house. Upon completion of the study, the hens were rehomed.

2.2. Experimental Diets

The experimental diets were formulated using wheat, sorghum, soybean meal, barley, and canola meal as base ingredients. The sequence of limiting essential AA identified by Jahan et al. [8] was applied to guide the formulation of the reduced-protein diets used in this study. The standard-protein diets had sufficient CP levels according to the breed nutritional recommendations [24], whereas the reduced-protein diets had 1.5 to 3.0 percentage points (15 to 30 g/kg) lower CP level compared to the standard-protein diets. Primary feed ingredients like wheat, sorghum, soybean meal, barley, and canola meal were analyzed for key nutritional components, including metabolizable energy, CP, AA, crude fat, crude fibre, minerals, and ash content using a near-infrared reflectance (NIR) spectroscopy system (Foss NIR 6500, Hillerød, Denmark) [25]. The results were standardized using Adisseo calibration parameters prior to diet formulation. All diets were formulated using commercial feed formulation software (Concept 5, CFC Tech Services, Inc., Pierz, MN, USA), ensuring that nutrient specifications met the recommended nutritional requirements for Hy-Line Brown laying hens, as outlined by Hy-Line International [24]. A vitamin–mineral premix was incorporated into all experimental diets in accordance with the manufacturer’s recommendations (Rabar Pty Ltd., Beaudesert, Queensland, Australia). The mixed diets were analyzed for gross energy, crude protein, dry matter, ash, and mineral content using standard procedures outlined by AOAC [26] to verify their formulated nutrient levels.

Table 1. Ingredient composition of experimental diets (as-fed basis).

	Dietary Treatments
Energy level (AME, kcal/kg)	2725	2725	2725	2589	2589	2589	2453	2453	2453
Crude protein level (%)	17	15.5	14	17	15.5	14	17	15.5	14
Ingredient (%)
Sorghum	27.1	27.0	27.0	27.1	27.0	27.0	27.0	27.0	27.0
Wheat	22.9	27.7	32.8	22.2	25.4	28.8	15.7	18.9	22.3
Soybean meal	19.3	14.7	9.30	14.6	9.50	4.30	15.3	11.0	6.00
Barley	15.0	15.0	15.0	15.0	15.0	15.0	15.0	15.0	15.0
Canola meal	2.00	2.00	2.00	8.80	10.0	10.0	10.0	10.0	10.0
Lime coarse	6.80	6.80	6.80	6.70	6.80	6.80	6.70	6.70	6.70
Limestone fine	3.00	3.00	3.00	3.00	3.00	3.00	3.00	3.00	3.00
Canola oil	2.30	1.90	1.40	1.00	0.90	0.90	1.20	1.20	1.20
Arbocel RC fine	0.70	0.70	0.70	0.70	0.70	0.70	0.70	0.70	0.70
Monocalcium phosphate	0.20	0.20	0.20	0.20	0.30	0.30	0.30	0.30	0.30
Salt	0.20	0.15	0.10	0.19	0.15	0.10	0.20	0.16	0.12
Sodium bicarbonate	0.17	0.24	0.32	0.16	0.22	0.29	0.15	0.21	0.28
Celite	0.00	0.00	0.00	0.00	0.40	1.40	4.40	5.20	6.10
Potassium carbonate	0.000	0.000	0.140	0.000	0.010	0.160	0.000	0.005	0.150
D,L-methionine	0.175	0.215	0.265	0.155	0.190	0.245	0.155	0.195	0.245
L-lysine HCl	0.040	0.175	0.335	0.070	0.205	0.365	0.045	0.175	0.335
Vitamin-mineral premix 1	0.100	0.100	0.100	0.100	0.100	0.100	0.100	0.100	0.100
Choline chloride	0.045	0.065	0.090	0.010	0.010	0.010	0.010	0.010	0.010
Phytase Axtra PHY Gold	0.003	0.003	0.003	0.003	0.003	0.003	0.003	0.003	0.003
Xylanase Axtra XB TPT 201	0.010	0.010	0.010	0.010	0.010	0.010	0.010	0.010	0.010
Pigment Jabiru red	0.004	0.004	0.004	0.004	0.004	0.004	0.004	0.004	0.004
Pigment Jabiru yellow	0.003	0.003	0.003	0.003	0.003	0.003	0.003	0.003	0.003
L-arginine	0.000	0.000	0.090	0.000	0.000	0.130	0.000	0.000	0.110
L-valine	0.000	0.020	0.105	0.000	0.020	0.110	0.000	0.015	0.100
L-isoleucine	0.000	0.020	0.110	0.000	0.045	0.135	0.000	0.035	0.125
L-threonine	0.000	0.035	0.115	0.000	0.035	0.115	0.000	0.030	0.105
Total ingredient	100	100	100	100	100	100	100	100	100

¹ Vitamin–mineral premix (Rabar Pty Ltd., Beaudesert, Queensland, Australia) provided the following per kilogram of vitamin–mineral premix: vitamin A, 10 MIU; vitamin D, 3 MIU; vitamin E, 20 g; vitamin K, 3 g; nicotinic acid, 35 g; pantothenic acid, 12 g; folic acid, 1 g; riboflavin, 6 g; cyanocobalamin, 0.02 g; biotin, 0.1 g; pyridoxine, 5 g; thiamine, 2 g; copper, 8 g as copper sulphate pentahydrate; cobalt, 0.2 g as cobalt sulphate 21%; molybdenum, 0.5 g as sodium molybdate; iodine, 1 g as potassium iodide 68%; selenium, 0.3 g as selenium 2%; iron, 60 g as iron sulphate 30%; zinc, 60 g as zinc sulphate 35%; manganese, 90 g as manganous oxide 60%; antioxidant, 20 g.

,

Table 2. Calculated nutrient content of experimental diets.

	Dietary Treatments
Energy level (kcal/kg ME)	2725	2725	2725	2589	2589	2589	2453	2453	2453
Crude protein level	17	15.5	14	17	15.5	14	17	15.5	14
Calculated nutrients (%, otherwise as indicated)
AMEn 1, kcal/kg	2725	2725	2725	2589	2589	2589	2453	2453	2453
Crude protein	17.0	15.5	14.0	17.0	15.5	14.0	17.0	15.5	14.0
Crude fat	4.1	3.7	3.2	2.9	2.9	2.8	3.1	3.0	3.0
Crude fibre	3.5	3.4	3.3	3.9	3.9	3.8	3.9	3.8	3.7
Ash content	12.9	12.7	12.6	13.0	13.2	14.0	17.3	17.9	18.7
Dig 2. lysine	0.74	0.74	0.74	0.74	0.74	0.74	0.74	0.74	0.74
Dig. methionine	0.42	0.43	0.46	0.41	0.42	0.45	0.41	0.43	0.45
Dig. methionine + cysteine	0.66	0.66	0.66	0.66	0.66	0.66	0.66	0.66	0.66
Dig. threonine	0.54	0.52	0.51	0.54	0.52	0.51	0.55	0.52	0.52
Dig. isoleucine	0.63	0.57	0.57	0.61	0.57	0.57	0.61	0.57	0.57
Dig. leucine	1.28	1.15	1.00	1.25	1.12	0.97	1.25	1.13	0.98
Dig. tryptophan	0.21	0.19	0.16	0.21	0.18	0.16	0.21	0.18	0.16
Dig. arginine	0.95	0.83	0.77	0.92	0.79	0.77	0.93	0.81	0.77
Dig. histidine	0.37	0.33	0.28	0.36	0.32	0.27	0.37	0.33	0.28
Dig. valine	0.70	0.65	0.65	0.70	0.65	0.65	0.70	0.65	0.65
Dig. phenylalanine	0.74	0.66	0.56	0.71	0.62	0.53	0.71	0.63	0.53
Dig. glycine	0.52	0.47	0.40	0.53	0.48	0.41	0.53	0.48	0.41
Calcium	4.00	4.00	4.00	4.00	4.01	4.01	4.01	4.01	4.01
Available phosphate	0.32	0.32	0.32	0.32	0.32	0.32	0.32	0.32	0.32
Sodium	0.18	0.18	0.18	0.18	0.18	0.18	0.18	0.18	0.18
Chloride	0.18	0.18	0.18	0.18	0.18	0.18	0.18	0.18	0.18
Potassium	0.65	0.58	0.58	0.64	0.58	0.58	0.65	0.58	0.58
Linoleic acid	1.4	1.3	1.2	1.1	1.1	1.1	1.1	1.1	1.1
Choline, mg/kg	1450	1450	1450	1611	1570	1450	1657	1561	1450
Dietary electrolyte balance, mEq/kg	194	176	176	192	176	176	192	176	176

¹ AMEn: apparent metabolizable energy corrected to zero N retention. ² Dig: standard ileal digestible amino acid coefficients as determined by near-infrared spectroscopy (Foss NIR 6500, Hillerød, Denmark) standardized with Adisseo calibration.

and

Table 3. Analyzed nutrient content of experimental diets (as-fed basis).

	Dietary Treatments
Energy level (kcal/kg ME)	2725	2725	2725	2589	2589	2589	2453	2453	2453
Crude protein level	17	15.5	14	17	15.5	14	17	15.5	14
Analyzed nutrients (%, otherwise as indicated)
Dry matter	91.5	91.5	91.0	91.3	91.1	91.3	91.9	91.8	91.7
Gross energy, kcal/kg	3681	3672	3605	3604	3594	3533	3483	3432	3386
Crude protein	16.4	15.4	13.7	16.3	15.0	13.6	16.6	14.9	13.6
Ash content	12.6	11.7	12.3	12.9	12.1	13.6	16.7	17.2	17.1
Calcium	3.74	3.63	3.77	3.85	3.62	3.68	3.55	3.76	3.39
Phosphorus	0.39	0.38	0.37	0.46	0.48	0.43	0.51	0.50	0.45
Aspartic acid	1.56	1.36	1.02	1.44	1.10	0.90	1.43	1.16	0.83
Serine	0.83	0.74	0.63	0.82	0.68	0.58	0.74	0.68	0.52
Glutamic acid	3.73	3.52	3.10	3.67	3.13	2.99	3.50	3.10	2.59
Glycine	0.71	0.65	0.57	0.76	0.64	0.56	0.68	0.69	0.52
Histidine	0.40	0.36	0.30	0.41	0.35	0.29	0.37	0.35	0.26
Arginine	1.04	0.94	0.83	1.03	0.81	0.80	0.96	0.87	0.69
Threonine	0.60	0.57	0.57	0.63	0.59	0.56	0.58	0.58	0.48
Alanine	0.81	0.73	0.64	0.81	0.69	0.63	0.78	0.69	0.57
Proline	1.20	1.14	1.07	1.30	1.18	1.08	1.16	1.17	0.98
Tyrosine	0.53	0.47	0.39	0.53	0.46	0.36	0.46	0.43	0.34
Valine	0.79	0.72	0.69	0.81	0.65	0.69	0.76	0.69	0.61
Methionine	0.43	0.42	0.46	0.41	0.44	0.42	0.35	0.41	0.38
Lysine	0.86	0.84	0.83	0.89	0.94	0.81	0.80	0.79	0.70
Isoleucine	0.74	0.67	0.67	0.75	0.64	0.66	0.68	0.64	0.60
Leucine	1.43	1.28	1.14	1.45	1.22	1.07	1.31	1.20	1.00
Phenylalanine	0.91	0.84	0.70	0.95	0.79	0.63	0.79	0.79	0.61

present the ingredient composition, calculated nutrient profiles, and laboratory-analyzed nutrient values of the experimental diets, respectively.

2.3. Data Collection and Laboratory Analysis

Egg production was determined by collecting, counting, and weighing all eggs laid per cage on a daily basis, while feed intake was recorded weekly. Egg mass and feed conversion ratio (FCR) were calculated using data on egg production, egg weight, and feed consumption. The FCR was determined as kilograms of feed consumed per kilogram of eggs produced. Hens were weighed at 20 and 35 weeks of age. Egg quality was measured for 13 eggs per treatment, and for each cage one egg was selected for quality analysis (117 eggs in total) at 27 and 35 weeks of age following the procedures described by Dao et al. [27]. All eggs used for quality assessment were tested within 3 h of collection on the same morning without storage time. Eggshell reflectivity was assessed using the TSS QCE-QCM device (Technical Services and Supplies, Dunnington, York, UK) on a scale from 0 to 100, where 0 corresponds to black and 100 corresponds to white. Thus, lower eggshell reflectivity values indicate darker eggshell colour. Egg dimensions, including length and width, were determined with a digital calliper, and the egg shape index was calculated as the ratio of width to length. Additional egg quality parameters such as eggshell breaking strength, shell thickness, albumen height, Haugh unit, yolk colour, yolk height, yolk diameter, and yolk index were evaluated using a digital egg tester (DET6500, Nabel Co., Ltd., Kyoto, Japan). Egg yolks were carefully collected using filter paper (CAT No. 1541-090, Whatman, Buckinghamshire HP7 9NA, UK) and then weighed. Eggshells were rinsed, completely dried, and weighed. The albumen weight was determined by subtracting the weights of the yolk and shell from the total egg weight. Subsequently, the proportion of each egg component was calculated by dividing its weight by the total intact egg weight.

A total excreta collection method was used to evaluate excreta moisture, nitrogenous waste, and apparent digestibility of dry matter, energy, and protein from dietary treatments. This assessment was carried out over a consecutive 3-day period (72 h) at 35 weeks of age, involving seven cages per treatment and a total of 63 cages. Excreta samples were collected from each cage twice daily, at 8:00 and 16:00, following the removal of feathers and feed residues, and subsequently stored at 4 °C. The dry matter, gross energy, and crude protein content of the excreta were analyzed to determine dry matter, energy, and protein retention. The dry matter content of the feed and the total feed intake from each individual cage within each treatment over the 3-day excreta collection period were measured to determine the intake of dry matter, gross energy, and crude protein. Apparent digestibility of dry matter, protein, and energy was then calculated using the equations outlined by Dao et al. [27]. Specifically, apparent protein digestibility was determined by dividing the average amount of protein retained by the average protein intake over the 3-day excreta collection period, then multiplying by 100. Protein intake was calculated by multiplying the average feed intake during this period (3-day) by the crude protein content of the feed. Protein retained was calculated by subtracting the average protein excreted in the excreta during the 3-day collection period from the total protein intake. The amount of protein excreted was determined by multiplying the average excreta volume collected over the 3 days by the crude protein content of the excreta. A similar approach was applied to calculate the digestibility of dry matter and energy for dietary treatments. All measurements and calculations were expressed on a dry matter basis.

Apparent energy and protein digestibility were calculated using equations described by Dao et al. [27].

Apparent protein digestibility (%) = (CP_retained/CP_intake) × 100

Apparent energy digestibility (%) = (GE_retained/GE_intake) × 100

CP_intake (g/day) = CP_feed (%) × FI (g/day/hen)

GE_intake (kcal/day) = GE_feed (kcal/g) × FI (g/day/hen)

CP_retained (g/day) = CP_intake − CP_excreta (%) × excreta volume (g/day/hen)

GE_retained (kcal/day) = GE_intake − GE_excreta (kcal/g) × excreta volume (g/day/hen)

where CP, GE, and FI are crude protein, gross energy, and feed intake, respectively.

2.4. Statistical Analysis

Data analysis was conducted using R Commander (version 3.3.1, R Foundation for Statistical Computing, Vienna, Austria). Prior to analysis, all datasets were assessed for normality and homogeneity of variance. A multifactorial ANOVA was performed to examine the interaction effects between energy levels (90%, 95%, and 100% of the recommended dietary AME) and protein levels (standard versus reduced protein). When significant effects were detected, Tukey’s post hoc test was applied for pairwise comparisons among treatments. Statistical significance was set at p < 0.05.

3. Results

3.1. Laying Performance

Table 4. Laying performance of hens fed the dietary treatments from weeks 20 to 35.

Energy (AME) Level (%)	Protein Level (%)	Egg Weight (g)	Hen Day Egg Production (%)	Egg Mass (g)	Feed Intake (g)	FCR (kg Feed/kg Egg)
100	17	59.8	84.4	53.1	126	2.994 bc
	15.5	60.7	84.9	55.8	125	2.548 a
	14	57.7	84.4	51.8	121	2.715 ab
95	17	58.7	87.5	54.5	129	3.282 cd
	15.5	61.1	85.0	55.6	127	3.235 cd
	14	57.5	85.3	53.3	126	2.634 a
90	17	59.9	86.2	55.8	136	2.772 ab
	15.5	58.4	86.1	53.3	130	3.335 d
	14	58.6	84.3	53.1	130	3.117 cd
Main effect
Energy level	100	59.4	84.6	53.6	124 a	2.753
	95	59.1	86.0	54.5	127 ab	3.068
	90	58.9	85.5	54.0	132 b	3.091
Protein level	17	59.4 ab	86.1	54.4	130	3.030
	15.5	60.0 b	85.4	54.9	127	3.047
	14	58.0 a	84.6	52.8	126	2.839
Pooled SEM		0.30	0.69	0.45	0.86	0.069
p-values	Energy	0.781	0.707	0.747	<0.001	0.065
	Protein	0.018	0.721	0.136	0.055	0.341
	Energy × protein	0.111	0.940	0.369	0.804	0.031

^a,b,c,d Means within columns not sharing a common suffix are significantly different at the 5% level of probability. Energy levels of diets containing 100%, 95%, and 90% recommended AME levels were 2725, 2589, and 2453 Kcal/kg diet, respectively.

shows the performance of laying hens from 20 to 35 weeks of age.

The results indicated that reducing dietary protein level from 17% to 15.5% did not affect egg weight in laying hens but reducing it from 15.5% to 14% significantly decreased egg weight (p < 0.05, Table 4). There was an increase in feed consumption when the dietary AME level decreased from 100% to 90% of the recommended AME level (p < 0.001, Table 4). In contrast, feed intake tended to decrease as the dietary protein level reduced from 17% to 14% (p = 0.055, Table 4). A significant interaction between energy and protein was observed for the FCR result (p < 0.05, Table 4). The reduction in dietary protein level from 17% to 15.5% significantly reduced FCR (14.9%) in 100% AME diets, but it had no effect on FCR in 95% AME diets and increased FCR in 90% AME diets (p < 0.05). Further reductions in dietary protein level from 15.5% to 14% did not affect FCR in 100% and 90% AME diets, but lowered FCR was recorded in 95% AME diets (p < 0.05, Table 4). The best FCR results were obtained in laying hens fed the reduced-protein diet with 15.5% CP and 100% AME levels and the reduced-protein diet with 14% CP and 95% AME levels (p < 0.05, Table 4). Hen day egg production and egg mass did not differ significantly among the dietary treatments throughout the study period from 20 to 35 weeks of age. No mortalities were recorded in this study.

3.2. Hen Weight

The hen weight of the dietary treatments over the experimental period is given in

Table 5. Hen weight of the dietary treatments during the experimental period.

Energy (AME) Level (%)	Protein Level (%)	Hen Weight Week 20 (g)	Hen Weight Week 35 (g)	Weight Gain Weeks 20–35 (g)
100	17	1675	2255	580
	15.5	1654	2272	618
	14	1656	2157	501
95	17	1639	2199	560
	15.5	1649	2262	613
	14	1645	2196	551
90	17	1661	2218	557
	15.5	1621	2166	545
	14	1603	2107	504
Main effect
Energy level	100	1662	2228	567
	95	1644	2219	575
	90	1628	2164	536
Protein level	17	1658	2224 ab	566 ab
	15.5	1641	2233 b	592 b
	14	1635	2154 a	519 a
Pooled SEM		8.11	13.54	11.56
p-values	Energy	0.255	0.100	0.335
	Protein	0.481	0.028	0.033
	Energy × protein	0.744	0.508	0.691

^a,b Means within columns not sharing a common suffix are significantly different at the 5% level of probability. Energy levels of diets containing 100%, 95%, and 90% recommended AME levels were 2725, 2589, and 2453 Kcal/kg diet, respectively.

.

Initial average hen weight at 20 weeks of age did not differ significantly among the treatments. Furthermore, no significant interaction between dietary energy and protein levels was observed for hen weight at 35 weeks or for weight gain between weeks 20 and 35. A reduction in dietary protein level from 15.5% to 14% significantly decreased hen body weight at 35 weeks and overall weight gain from weeks 20 to 35, as indicated by the main effect of protein level (p < 0.05; Table 5).

3.3. Egg Quality

The external egg quality, internal egg quality, and egg proportions of the dietary treatments at week 27 are shown in

Table 6. External egg quality of hens fed dietary treatments at week 27.

Energy (AME) Level (%)	Protein Level (%)	Shell Breaking Strength (kgf)	Shell Thickness (mm)	Egg Length (mm)	Egg Width (mm)	Egg Shape Index	Reflectivity (%)
100	17	5.24	0.453	55.6	44.3	0.797	21.3
	15.5	4.72	0.453	56.7	44.3	0.781	22.9
	14	5.30	0.463	54.8	43.5	0.794	22.9
95	17	5.38	0.466	55.3	43.8	0.793	22.8
	15.5	5.14	0.447	55.8	44.2	0.793	20.8
	14	4.99	0.457	55.7	44.3	0.796	23.2
90	17	5.18	0.461	55.7	44.4	0.798	22.2
	15.5	5.27	0.463	55.1	44.5	0.807	21.9
	14	5.39	0.472	55.2	44.2	0.800	22.3
Main effect
Energy level	100	5.10	0.456	55.7	44.0	0.791 a	22.4
	95	5.16	0.457	55.6	44.1	0.794 ab	22.3
	90	5.28	0.465	55.3	44.4	0.802 b	22.2
Protein level	17	5.26	0.460	55.5	44.2	0.796	22.1
	15.5	5.05	0.454	55.9	44.3	0.794	21.9
	14	5.22	0.464	55.3	44.0	0.796	22.8
Pooled SEM		0.07	0.002	0.15	0.11	0.002	0.26
p-values	Energy	0.564	0.141	0.565	0.477	0.050	0.941
	Protein	0.430	0.144	0.228	0.474	0.815	0.329
	Energy × protein	0.305	0.330	0.101	0.389	0.287	0.202

^a,b Means within columns not sharing a common suffix are significantly different at the 5% level of probability. Energy levels of diets containing 100%, 95%, and 90% recommended AME levels were 2725, 2589, and 2453 Kcal/kg diet, respectively.

,

Table 7. Internal egg quality of hens fed dietary treatments at week 27.

Energy (AME) Level (%)	Protein Level (%)	Albumen Height (mm)	Yolk Colour	Haugh Unit	Yolk Height (mm)	Yolk Diameter (mm)	Yolk Index
100	17	9.10	11.8	91.3	22.6	40.9	0.556
	15.5	9.86	11.6	96.5	22.8	40.0	0.569
	14	7.50	11.0	84.1	22.5	39.5	0.570
95	17	9.40	12.3	95.5	22.4	40.5	0.556
	15.5	8.82	11.0	90.0	22.8	40.0	0.572
	14	9.35	13.2	93.4	22.5	40.0	0.562
90	17	8.95	12.2	92.4	22.6	40.4	0.561
	15.5	7.50	12.2	84.1	22.8	39.7	0.574
	14	8.31	11.8	87.4	22.7	40.5	0.562
Main effect
Energy level	100	8.82	11.5	90.6	22.6	40.1	0.565
	95	9.19	12.2	92.9	22.6	40.2	0.563
	90	8.25	12.1	88.0	22.7	40.2	0.566
Protein level	17	9.15	12.1	93.1	22.5	40.6	0.557
	15.5	8.73	11.6	90.2	22.8	39.9	0.572
	14	8.39	12.1	88.3	22.5	40.0	0.565
Pooled SEM		0.25	0.20	1.55	0.07	0.21	0.003
p-values	Energy	0.303	0.295	0.424	0.818	0.993	0.942
	Protein	0.454	0.460	0.445	0.327	0.343	0.166
	Energy × protein	0.219	0.124	0.366	0.917	0.837	0.959

Energy levels of diets containing 100%, 95%, and 90% recommended AME levels were 2725, 2589, and 2453 Kcal/kg diet, respectively.

and

Table 8. Egg proportions of hens fed dietary treatments at week 27.

Energy (AME) Level (%)	Protein Level (%)	Yolk Weight (g)	Albumen Weight (g)	Shell Weight (g)	Yolk (%)	Albumen (%)	Shell (%)
100	17	13.9	41.6	6.24	22.7	67.2	10.1
	15.5	13.4	42.9	6.18	21.6	68.5	9.88
	14	13.3	39.6	6.13	22.6	67.0	10.4
95	17	13.6	40.2	6.20	22.7	66.9	10.4
	15.5	13.4	41.9	6.11	21.9	68.1	10.0
	14	13.6	41.3	6.23	22.2	67.6	10.2
90	17	13.6	42.1	6.31	22.0	67.8	10.2
	15.5	13.7	42.5	6.47	22.0	67.7	10.4
	14	13.3	41.5	6.40	21.7	67.8	10.5
Main effect
Energy level	100	13.6	41.4	6.19 a	22.3	67.6	10.1
	95	13.5	41.1	6.18 a	22.3	67.5	10.2
	90	13.5	42.0	6.40 b	21.9	67.7	10.3
Protein level	17	13.7	41.3	6.25	22.5	67.3	10.2 ab
	15.5	13.5	42.4	6.26	21.8	68.1	10.1 a
	14	13.4	40.8	6.25	22.2	67.4	10.4 b
Pooled SEM		0.08	0.36	0.04	0.16	0.18	0.05
p-values	Energy	0.995	0.587	0.020	0.566	0.885	0.180
	Protein	0.182	0.180	0.997	0.277	0.174	0.036
	Energy × protein	0.490	0.567	0.663	0.722	0.506	0.202

^a,b Means within columns not sharing a common suffix are significantly different at the 5% level of probability. Energy levels of diets containing 100%, 95%, and 90% recommended AME levels were 2725, 2589, and 2453 Kcal/kg diet, respectively.

, respectively.

No significant energy × protein interactions were obtained for egg quality parameters at week 27. However, reducing dietary energy level from 100% to 90% recommended AME level resulted in increased egg shape index (p = 0.050, Table 6) and shell weight (p = 0.020, Table 8) at week 27 as shown by the main effect of energy level. Meanwhile, reducing dietary protein level from 15.5% to 14% increased shell proportion at week 27 as shown by the main effect of protein level (p < 0.05, Table 8). The external egg quality, internal egg quality, and egg proportions of the dietary treatments at week 35 are shown in

Table 9. External egg quality of hens fed dietary treatments at week 35.

Energy (AME) Level (%)	Protein Level (%)	Shell Breaking Strength (kgf)	Shell Thickness (mm)	Egg Length (mm)	Egg Width (mm)	Egg Shape Index	Reflectivity (%)
100	17	5.13	0.434	57.2	45.9	0.802	24.3
	15.5	4.90	0.431	57.3	46.3	0.809	24.3
	14	4.68	0.434	56.1	45.6	0.813	24.7
95	17	4.96	0.436	56.8	46.1	0.811	24.0
	15.5	4.92	0.433	57.1	46.2	0.809	23.01
	14	4.84	0.435	56.7	45.5	0.803	25.3
90	17	5.02	0.439	57.1	46.5	0.816	24.0
	15.5	5.10	0.437	56.6	45.5	0.804	25.4
	14	4.93	0.442	57.2	46.5	0.813	24.7
Main effect
Energy level	100	4.99	0.432	56.7	46.0	0.812	24.3
	95	5.03	0.440	57.2	46.1	0.806	24.2
	90	4.80	0.435	56.9	46.0	0.809	24.8
Protein level	17	5.08 b	0.436	56.6	45.9	0.810	25.3
	15.5	5.08 b	0.436	56.9	46.0	0.808	24.1
	14	4.66 a	0.435	57.1	46.2	0.809	23.9
Pooled SEM		0.07	0.002	0.15	0.12	0.002	0.25
p-values	Energy	0.379	0.246	0.406	0.919	0.457	0.597
	Protein	0.021	0.989	0.423	0.417	0.855	0.055
	Energy × protein	0.365	0.411	0.698	0.391	0.127	0.978

^a,b Means within columns not sharing a common suffix are significantly different at the 5% level of probability. Energy levels of diets containing 100%, 95%, and 90% recommended AME levels were 2725, 2589, and 2453 Kcal/kg diet, respectively.

,

Table 10. Internal egg quality of hens fed dietary treatments at week 35.

Energy (AME) Level (%)	Protein Level (%)	Albumen Height (mm)	Yolk Colour	Haugh Unit	Yolk Height (mm)	Yolk Diameter (mm)	Yolk Index
100	17	8.38	11.9	89.2	22.4	41.5	0.542 ab
	15.5	9.61	11.7	95.0	23.3	43.0	0.547 ab
	14	7.91	11.4	84.9	22.6	40.9	0.555 b
95	17	8.76	10.1	90.7	22.5	40.2	0.563 b
	15.5	9.36	11.7	93.4	22.9	41.5	0.554 b
	14	9.02	12.5	93.3	22.3	43.1	0.520 ab
90	17	7.37	10.9	81.0	22.4	45.0	0.499 a
	15.5	7.93	13.0	86.1	22.0	41.3	0.532 ab
	14	8.70	11.8	88.5	22.5	41.2	0.549 ab
Main effect
Energy level	100	8.48	11.5	88.5	22.6	42.1	0.540
	95	8.58	11.8	89.6	22.6	41.8	0.547
	90	8.62	11.7	89.3	22.3	42.0	0.534
Protein level	17	8.32	11.7	88.5	22.4	41.4	0.544
	15.5	8.09	11.6	86.2	22.5	42.5	0.532
	14	9.27	11.7	92.6	22.7	42.0	0.544
Pooled SEM		0.22	0.20	1.41	0.09	0.29	0.004
p-values	Energy	0.957	0.878	0.943	0.352	0.907	0.470
	Protein	0.068	0.994	0.176	0.416	0.284	0.393
	Energy × protein	0.119	0.757	0.236	0.215	0.064	0.007

^a,b Means within columns not sharing a common suffix are significantly different at the 5% level of probability. Energy levels of diets containing 100%, 95%, and 90% recommended AME level were 2725, 2589, and 2453 Kcal/kg diet, respectively.

, and

Table 11. Egg proportions of hens fed dietary treatments at week 35.

Energy (AME) Level (%)	Protein Level (%)	Yolk Weight (g)	Albumen Weight (g)	Shell Weight (g)	Yolk (%)	Albumen (%)	Shell (%)
100	17	16.0	42.8	6.42	24.6	65.5	10.0
	15.5	16.3	43.9	6.30	24.6	65.9	9.60
	14	15.1	41.5	6.08	24.2	65.9	9.92
95	17	15.2	43.3	6.31	23.4	66.7	9.81
	15.5	16.0	43.1	6.30	24.4	66.0	9.63
	14	15.7	41.5	6.20	24.8	65.4	9.78
90	17	15.8	43.1	6.34	24.3	66.0	9.73
	15.5	14.6	42.4	6.21	23.2	67.0	9.84
	14	15.1	44.7	6.48	22.8	67.4	9.78
Main effect
Energy level	100	15.3	42.9	6.23	23.8	66.5	9.77
	95	15.9	42.8	6.36	24.4	65.8	9.81
	90	15.5	43.0	6.30	23.9	66.3	9.78
Protein level	17	15.6	42.1	6.24	24.4	65.8	9.84
	15.5	15.3	42.9	6.32	23.8	66.4	9.79
	14	15.7	43.8	6.33	23.9	66.4	9.73
Pooled SEM		0.13	0.35	0.04	0.18	0.19	0.05
p-values	Energy	0.168	0.987	0.435	0.318	0.263	0.950
	Protein	0.441	0.155	0.567	0.341	0.326	0.626
	Energy × protein	0.878	0.606	0.277	0.726	0.831	0.646

Energy levels of diets containing 100%, 95%, and 90% recommended AME levels were 2725, 2589, and 2453 Kcal/kg diet, respectively.

, respectively. Reducing dietary protein level from 17% to 14% decreased shell breaking strength (p < 0.05, Table 9) and increase albumen height (p = 0.068, Table 10) as shown by the main effect of protein level at week 35. A significant energy × protein interaction was obtained for yolk index at week 35 (p < 0.01, Table 10), where feeding 17% CP diet did not affect yolk index in the diets with 100% and 95% AME levels but decreased yolk index in the diet with 90% AME level. Dietary treatments did not affect egg proportions or egg quality measures at 35 weeks of age (Table 9, Table 10 and Table 11).

3.4. Excreta Moisture, Nitrogen Excretion, and Nutrient Digestibility

Table 12. Excreta moisture, nitrogen excretion, and apparent nutrient digestibility of hens fed the dietary treatments at week 35.

Energy (AME) Level (%)	Protein Level (%)	Excreta Moisture (%)	Nitrogen Excretion (g/day)	Dry Matter Digestibility (%)	Energy Digestibility (%)	Protein Digestibility (%)
100	17	78.3	1.651	71.7	76.9	45.7
	15.5	78.7	1.589	72.7	77.7	47.4
	14	78.0	1.103	75.5	79.2	56.2
95	17	77.5	1.813	69.3	75.3	44.0
	15.5	77.4	1.696	70.5	75.9	46.6
	14	77.4	1.306	72.0	76.8	50.5
90	17	76.4	1.960	67.8	75.7	42.3
	15.5	74.8	1.643	66.7	75.1	45.2
	14	76.1	1.391	66.7	75.8	48.0
Main effect
Energy level	100	78.3 b	1.448	73.3 c	77.8 b	49.8
	95	77.4 b	1.605	70.6 b	76.0 a	47.2
	90	75.7 a	1.665	67.1 a	75.5 a	45.2
Protein level	17	77.4	1.808 b	69.6	76.0	44.0 a
	15.5	76.9	1.642 b	70.0	76.2	46.4 a
	14	77.2	1.267 a	71.4	77.1	51.6 b
Pooled SEM		0.28	0.042	0.46	0.26	0.94
p-values	Energy	<0.001	0.088	<0.001	<0.001	0.086
	Protein	0.752	<0.001	0.240	0.077	0.002
	Energy × protein	0.671	0.580	0.103	0.605	0.766

^a,b,c Means within columns not sharing a common suffix are significantly different at the 5% level of probability. Energy levels of diets containing 100%, 95%, and 90% recommended AME levels were 2725, 2589, and 2453 Kcal/kg diet, respectively.

presents the effects of dietary treatments on excreta moisture, nitrogen excretion, and apparent nutrient digestibility at 35 weeks of age. No significant energy × protein interactions were obtained for excreta moisture content, nitrogen excretion, and apparent nutrient digestibility. However, reducing dietary energy level from 100% to 90% recommended AME level decreased excreta moisture content (p < 0.001), dry matter digestibility (p < 0.001), and energy digestibility (p < 0.001) as shown by the main effect of energy level at week 35. Meanwhile, reducing dietary energy level from 100% to 95% recommended AME level did not affect excreta moisture content but decreased dry matter digestibility (p < 0.001) and energy digestibility (p < 0.001) as shown by the main effect of energy level at week 35. Consistent with expectations, a reduction in dietary protein from 17% to 14% resulted in a 30% decrease in nitrogen excretion (p < 0.001) and a 17% increase in protein digestibility (p < 0.01), as indicated by the main protein effect at week 35.

4. Discussion

4.1. Laying Performance

This study demonstrated that a moderate reduction in dietary CP from 17% to 15.5%, combined with crystalline AA supplementation and the maintenance of 100% of the recommended AME level, improved feed efficiency in laying hens during 20 to 35 weeks of age. This study highlights the importance of AA balance in reduced-protein diets, which is unique from previous studies that mainly focused on dietary CP reduction. Furthermore, the current findings demonstrate that decreasing dietary AME level from 100% to 95% is necessary to improve feed efficiency when the dietary protein level decreases from 15.5% to 14%. Previous studies have shown that laying hens fed a corn–soybean meal-based diet with reduced protein level containing 14% CP and the recommended energy level (2871 kcal AME/kg) had reduced feed efficiency, compared to hens receiving either a diet with both recommended protein and energy levels (17% CP, 2871 kcal AME/kg) or a diet with both low protein and energy levels (14% CP, 2785 kcal AME/kg) between 39 and 50 weeks of age [16]. Similarly, Meluzzi et al. [17] observed lower laying rate, egg mass, egg weight, and feed efficiency in laying hens fed reduced-protein corn–soybean meal-based diets with 15% CP and recommended energy level (2854 kcal AME/kg diet) compared to those fed the control diets with 17% CP and similar energy level from 33 to 40 weeks of age. Nonetheless, the reduced-protein diets in the studies conducted by Novak et al. [16] and Meluzzi et al. [17] were formulated based only on the requirements for Lys, Met, Cys, Thr, and/or Trp. As a result, the performance outcomes observed may have been confounded by suboptimal levels of other essential AA such as Arg, Leu, Val, and Ile rather than by the energy-to-protein ratio of the diets. A deficiency in essential AA such as Arg can lead to an imbalance in the Arg-to-Lys ratio, which may negatively affect laying performance in hens [12,13].

Additionally, when a particular essential AA is deficient, other essential AA may be catabolized or converted for nonessential metabolic functions, resulting in reduced protein synthesis and consequently lower egg production [14,15]. This study minimized potential negative impacts of AA deficiencies by considering both the levels and limiting order of all essential AA when formulating the reduced-protein diets. It is compelling that feeding reduced-protein diets with 15.5% CP and 100% recommended AME level resulted in the best FCR (14.9%), followed by the second best FCR with 14% CP and 95% recommended AME level in the current study. Keshavarz and Austic [28] also indicated that laying hens receiving a corn–soybean meal-based diet supplemented with essential AA, formulated at 13% CP and 2902 kcal AME/kg, exhibited enhanced feed efficiency compared to those fed a diet containing 16% CP with a similar energy level during the period from 36 to 48 weeks of age. However, the reduced-protein diets containing 14% CP in the present study resulted in lower egg weight, indicating a possible deficiency in non-essential AA. It has been indicated that feeding hens reduced-protein diets lower the nitrogen pool, resulting in the deficiency of nonessential AA such as glutamic acid in birds [29]. Previous studies have shown that glutamic acid is the dominant AA in egg protein and plays important roles in intestinal function and development and eggshell calcification [30,31,32].

The results of the present study demonstrated that lowering the dietary AME level from 100% to 90% led to an increase in feed intake. These results are understandable as birds consume feed to satisfy their energy requirements [33]. Similar findings were observed by Harms et al. [34], who stated that hens offered a low-energy diet (2519 kcal/kg) consumed more feed than those offered a diet with the recommended energy level (2798 kcal/kg) during 36 to 44 weeks of age. Kim and Kang [35] also reported that elevating dietary AME level from 2650 to 2750 kcal/kg resulted in reduced feed intake in laying hens. The increased feed consumption observed in hens fed the 90% AME diet compared to those fed the 100% AME diet in the current study is consistent with previous studies [33,36] and reconfirms that laying hens regulate their feed intake based on energy requirements.

4.2. Hen Weight

Previous studies have shown that feeding low-protein diets reduced weight gain and body weight in laying hens [37,38], which is consistent with the results of the present study that reducing the dietary protein level from 15.5% to 14% reduced hen weight and weight gain over the study duration. Mousavi et al. [39] demonstrated a linear decrease in body weight in laying hens during 25 to 33 weeks of age as dietary protein level decreased gradually from 18.5% to 15.5%. Also, Novak et al. [16] observed that reducing dietary protein level led to decreased body weight gain in laying hens from 18 to 60 weeks of age, signifying that dietary protein contributes to optimal weight gain during the laying phase. The decrease in body weight gain caused by low-protein diets becomes more significant when hens are at their peak egg-laying stage [16]. This effect may be attributed to the redistribution of AA, whereby nutrients are preferentially allocated toward supporting egg production at the expense of body weight gain and maintenance. However, as the average hen weight in all treatment groups at week 35 in the present study exceeded the Hy-Line Brown standards (1.94 to 2.08 kg) [40], the relatively lower hen weight in this case is more favourable.

4.3. Egg Quality

The present study demonstrated that decreasing dietary protein level from 17% to 14% decreased shell breaking strength and tended to increase albumen height at week 35. Kang et al. [41] discovered that hens fed ad libitum diets (12% or 16% CP) had lower shell strength than those fed on a restricted basis (85% of ad libitum feed consumption from the previous period). It is possible that shell strength might be reduced by increasing the shell curvature [42]. Proteins play a key role in the integration and structural support of calcium crystals within the organic matrix, thereby enhancing eggshell strength [43,44]. A reduction in dietary protein may limit the availability of AA such as glycine, proline, and methionine, which are essential for collagen and shell membrane synthesis [45,46]. This may result in thinner or more brittle shells, ultimately reducing shell strength. Additionally, it has been indicated that AA requirements for eggshell formation and internal egg quality may differ considerably [47]. This variability may help explain the contradictory effects of reducing dietary protein level on shell breaking strength and albumen height observed in the current and previous studies [48,49].

The yolk index, calculated as the ratio of yolk height to yolk diameter, has been used as an indicator to assess the freshness of the egg, with higher values reflecting a fresher egg with a more compact yolk [50]. In more detail, eggs with yolk indexes of above 0.38, 0.28–0.38, and below 0.28 are considered extra fresh, fresh, and regular, respectively [50]. The outcomes of this research indicated that a 17% CP diet had no significant impact on yolk index when combined with 100% (2725 kcal/kg) and 95% (2589 kcal/kg) AME levels; however, a reduction in yolk index was observed at the 90% AME level (2453 kcal/kg). These findings are partially supported by Alderey and Dorgham [51], who reported no changes in yolk index in laying hens offered diets containing 14%, 16% or 18% CP level combined with energy levels of either 2700 or 2850 kcal/kg from 25 to 40 weeks of age. In this study, the lowest yolk index of 0.499 was observed in hens fed the diet with 17% CP and 90% recommended AME levels, which was higher than the standard for extra-fresh eggs (yolk index > 0.38) [50]. However, as the yolk flattens and egg freshness decreases with the storage time [50], possible effects on the yolk index should be considered when the dietary energy level decreases from 100% to 90% of the recommended AME level in normal protein diets.

4.4. Excreta Moisture and Nutrient Utilization

Findings from the present study indicated a decline in excreta moisture content, dry matter digestibility, and energy digestibility when reducing the dietary energy level from 100% to 90% recommended AME level at week 35, whereas lowering dietary energy level from 100% to 95% of the recommended AME level did not affect excreta moisture content at week 35 but decreased dry matter and energy digestibility. These findings align with those reported by Li et al. [52], who observed reduced apparent energy digestibility in Taihe Silky Fowl (Gallus gallus domesticus Brisson) during the peak laying period when dietary AME levels were decreased from 2800 to 2600 kcal/kg. Similarly, Kang et al. [34] observed a linear increase in energy digestibility with increasing dietary AME levels. Furthermore, the current study revealed that reducing dietary protein level from 17% to 14% decreased nitrogen excretion by 30% and enhanced protein digestibility by 17% at week 35. These results are in line with those of Zeweil et al. [53], who reported that decreasing dietary protein level in laying hen diets from 16% to 14% and 12% significantly reduced nitrogen excretion by 33.82 and 45.45%, respectively, from 28 to 48 weeks of age. In the same study, apparent protein digestibility increased significantly with reductions in dietary protein level [53]. The improved protein digestibility and reduced nitrogen excretion observed in hens fed the 14% CP diets may be attributed to the greater inclusion of crystalline AA, particularly Arg (0.090–0.130%), Val (0.100–0.110%), Ile (0.110–0.135%), and Thr (0.105–0.115%), which are more readily digestible and bioavailable than the intact protein sources present in the 17% CP diets [54,55]. Similar results were reported by the other investigators [53,56,57]. Consistent with the present study, Aletor et al. [58] found improved energy and protein utilization in diets with lowered protein content compared to standard-protein diets. Additionally, other research has suggested that chickens can use nutrients effectively under limiting AA conditions [59].

5. Conclusions

Reducing dietary protein from 17% to 15.5% while maintaining 100% of the recommended AME level and balanced AA profiles resulted in a 14.9% improvement in feed efficiency without compromising egg quality parameters, compared to the standard 17% CP diet at the same energy level. Further reducing protein from 15.5% to 14% caused declines in egg weight and shell strength. However, when the dietary protein level decreased from 15.5% to 14%, decreasing dietary AME level from 100% to 95% is necessary to improve feed efficiency. Thus, strategic adjustment of both dietary protein and energy levels while ensuring AA sufficiency is essential for optimizing laying hen performance. By developing an optimally reduced-protein with a balanced AA diet for laying hens, this study may help to increase production efficiency while reducing carbon footprint and industry reliance on imported expensive soybean meals, leading to more efficient and sustainable laying hen production. Nevertheless, as this study was conducted on young hens under specific environmental and management conditions, the results may not fully represent performance under commercial farm settings, where variability in climate, feed quality, and health status may influence outcomes. Moreover, the relatively short experimental duration and use of fixed dietary formulations may limit broader applicability of the findings. Therefore, while this study provides valuable insights into reduced-protein feeding strategies for laying hens, further validation under commercial field conditions across longer production periods and diverse production systems is recommended. Additionally, the economic benefits of feeding reduced-protein diets will depend not only on the relative costs of crystalline AA versus conventional feed ingredients, such as soybean meals, but also on its effects on overall production performance and egg quality. Careful evaluation of both economic and practical outcomes is therefore essential to ensure that sustainability and efficiency gains are achieved without compromising the profitability of commercial operations.