1. Introduction
The use of a low-crude-protein diet fortified with essential amino acids is a part of a precision feeding and has gained commercial acceptance to improve protein utilization, minimize environmental impact and thus increase the sustainability of broiler production, because it reduces the cost of production [
1,
2,
3]. Protein accounts for the second most expensive nutrient in poultry diets, following metabolizable energy [
4,
5]. In the typical corn–soybean meal diet, approximately 80 and 60% of dietary protein is digested and utilized by broilers and layers, respectively [
6,
7]. The undigested protein is excreted into the environment, causing ammonia accumulation in poultry houses. The nitrogen run off from poultry litter used as a fertilizer causes the overstimulation of the growth of aquatic plants and algae in groundwater [
8,
9]. In addition to the excessive N excreted leading to environmental issues, undigested dietary protein has been reported to induce necrotic-enteritis-related symptoms by hindering gut fermentation [
10,
11]. Thus, feeding a low-crude-protein diet is a feasible strategy to address the environmental and economic concerns related to nitrogen excretion [
12,
13,
14,
15,
16]. Moreover, the recent commercial availability of the essential amino acids, including arginine, threonine, tryptophan and valine, made it possible to further reduce the crude protein levels in broiler diets [
13,
17,
18,
19,
20,
21]. In addition, the 2021 global supply chain crisis due to the COVID-19 pandemic created a shortage and uncertainty for the supply of available ingredients, particularly of crystalline amino acids, on the market for broiler production, and the conditions became even worse following the Russia–Ukraine conflict [
21,
22,
23,
24].
Therefore, it is crucial to test the effect of single essential amino acids and their combination on broiler performance and meat yield. However, to date there is limited research focusing on single-amino-acid supplementation in a low-protein diet, since most of the research used two or more amino acids [
25,
26,
27].
Broilers fed a low crude protein meeting essential amino acids requirements have been reported to yield an equivalent performance and carcass protein compared to a conventional protein diet [
28,
29,
30]. However, Dean et al. [
31] reported that broiler performance deteriorates due to a limited availability of amino acids when a low-protein diet with essential amino acids is fed. These inconsistent results were identified because of a potential glycine shortage in the low-protein diet [
27,
32].
Thus, the main objective of the present study was to investigate the effect of satisfying the essential amino acids requirements for methionine+lysine, arginine, threonine, valine, isoleucine or tryptophan individually or in combinations on the sustainability of broiler growth performance, carcass yield, meat quality and nitrogen excretion in the early growth period. The present work consists of two experiments: the first aiming to investigate the effect of essential amino acid supplementation on a low-protein diet (18.3 vs. 22.5% CP) during 1–28 days of age. As there was a lack of a positive effect from amino acids supplementation in a low-protein diet on growth performance in experiment 1, a second experiment was conducted, in which there were the same differences in protein level (18.3 vs. 22.5%) but with the low-protein diets were formulated to contain proteins from only vegetable or vegetable plus animal sources. This tested if a higher amino acids availability from protein animal sources could restore the growth of broilers during the early growing phase (1–21 days of age).
3. Results
The growth performance results of experiment 1 are presented in
Table 3. The diet with 18.3% of CP supplemented with Lys and Met (NC) compromised body weight gain, protein intake and protein conversion ratio compared to the PC group during 1–28 days of age (
p < 0.05). Supplementing Arg in the NC diet increased bodyweight gain compared to the NC group; however, the Arg group is still lower than the PC group (
p < 0.05). The supplementation of Val or amino acid combinations increased body weight gain compared to the NC group (
p < 0.05), reaching a similar level as the PC group. Additionally, supplementation with the essential amino acid combination increased the intact protein intake compared to the NC group (
p < 0.05); however, it was still lower than the PC group (
p < 0.05). Initial body weight, feed intake, FCR and SR were not significantly affected by different dietary protein and amino acids supplementations.
The growth performance and N excretion results of experiment 2 are presented in
Table 4. Broilers fed the low-crude-protein diet at 18.3% with Lys and Met supplementation showed a lower body weight gain for both vegetable–animal and only-vegetable protein diets compared to the PC group with 22.5% CP. Supplementation with Val or amino acids combinations in a low-protein diet with meat and fish meals improved the body weight gain compared to the NC group and it reached the same level as the PC group (
p < 0.05). Similarly, supplementation with either single amino acids or a combination in the only-vegetable diet increased the body weight gain compared to the low-protein-diet NC group; however, it was still lower than in the PC group (
p < 0.05). Protein intake significantly decreased in low-protein-amino-acids-supplemented diets, while PCR was improved.
Initial body weight, feed intake, FCR, EN and SR were not significantly affected by different dietary protein and amino acids supplementations.
Results of the carcass yield of experiment 1 are shown in
Table 5. There was no difference for dressing, breast and wings, whole legs, liver, or abdominal fat among treatments.
The carcass yield and meat composition results of experiment 2 are shown in
Table 6. Supplementation of essential amino acid combination in a low-protein diet with fish and meat meals increased the dressing percentage. Moreover, broilers fed an all-vegetable NC diet or with the supplementation of Thr, Arg, Val or amino acid combination showed a higher relative spleen weight compared to the PC group (
p < 0.05).
The survival rate in the two experiments was not significantly affected by different amino acids supplementations in low-protein diets based on maize–soybean meal diets or maize–soybean meal diets with animal protein. This shows the safe effects of low-protein diets on the survivability of broilers up to 28 days of age.
4. Discussion
Our findings agree with Soares et al. [
4] and Abreu et al. [
5], who observed that 19% CP based on commercially available feedstuffs impaired the FCR of broilers during the growing period. However, when the negative control diets were supplemented with Met+Lys, the results constituted smallest body weight gains compared to the other groups, suggesting that Met+Lys were not the only limiting amino acids during 1–28 or 1–21 days of age in experiments 1 and 2, respectively. Thus, Val or AA mix with Gly addition to the NC diet improved growth to a level like a PC diet containing animal protein supplements. The FCR was not statistically different among treatments although PC groups showed a numerically best FCR. Abreu et al. [
5] reported that broiler diets containing 19% CP formulated with unusual ingredients of starch, corn gluten bran and soybean hulls and supplemented with Thr, Arg, Trp, Val and Ile displaying a similar growth performance and production factor as the PC.
In partial agreement with the present results, Jensen and Mendonca [
37] reported that the addition of 0.1% or 0.2% of L-valine to the 16% CP diet did not increase weight gains or feed efficiency while reducing abdominal fat deposition: however, the addition of 0.2% L-valine improved feed efficiency. Likewise, Cuca and Jensen [
38] found that addition of arginine to 20, 21 or 22% CP diets significantly increased body weight and feed efficiency to the same level as the 22.5% CP diet. The differing effects of Val and Arg increased BWG when supplemented in diets containing animal proteins such as fish and meat meals compared to solely vegetable sources may be related to the metabolic availability of the Val and Arg. It has been reported that fish meal or meat and bone meal have a higher AA availability than soybean meal except for Val [
39,
40]. Thus, supplementation of Val in the NC group with an animal protein source supported the BWG equivalent to the PC diet. However, wide variation in the AA digestibility of animal protein ingredients has been documented, and AA availability is highly related to the protein quality [
41]. Smith [
42] reported that Val and Arg availabilities in fish meal were 67.3 and 62.6%, respectively. Along the same line, Miller and Kifer [
43] found that Arg addition to poor quality fish meal improved the performance of chicks. Furthermore, Baker [
12] and Boorman and Burgess [
44] indicated that synthetic Arg had higher availability than natural Arg. Similarly, supplementing a low-protein diet with Lys, Met, Thr and Trp was found to improve the growth of broilers [
13]. The present results revealed that Val supplementation in a low-protein diet containing fish and meat meals increased growth and EPEV to the level of the positive control diet when compared to a diet containing soybean meal as the sole protein source. It is worth noting that the growth and EPEV of broilers fed diets containing corn–soybean meal and fish meat meal were better than those fed a single protein source based on soybean meal only; however, the FCR was similar between animal protein and corn–soybean containing diets. The better performance and EPEV observed in broilers fed an animal protein source is also associated with its higher AA availability.
Supplementation with Trp increased growth in experiment 2; however, compared to Val it was found to be less effective in the animal-protein-containing diet; however in the plant-protein-containing diet, it showed an effect comparable to other amino acids for increasing growth performance and EPEV. Griminger et al. [
45] reported that dietary protein did not linearly increase the requirements by the chick for Trp; however, Rogers and Pesti [
46] showed that the Trp requirements increased linearly with the increases in CP % in the diet. Maynard et al. [
47] reported that reducing Trp in a diet did not reduce body weight gain during 15 to 22 d of age.
Supplementation with Ile did not increase growth or improve the FCR of low-protein diets, indicating that intact Ile may be adequate and/or the responses to Ile may be governed by antagonism with Leu due to the fact that Ile and Leu are both branched-chain amino acid [
12,
37,
48]. Combining the four supplemented amino acids with Gly has no additive effect on growth performance over that observed with Arg or Val alone. This provided evidence that the negative control diet supplemented with Met+Lys was not limited in amino nitrogen. There were no marked variations in feed intake among the experimental groups. The results of Lipstein et al. [
49] and Baker [
12] indicated that broilers eat in an attempt to satisfy their protein or amino acid needs.
The protein intake was constantly higher in the PC than NC group. Even though, there were no substantial variations in protein utilization among the experimental treatments in experiment 1; however, in experiment 2, the protein conversion ratio was improved by feeding the Arg- or Val-supplemented diet. The percentage of nitrogen in the excreta was lower by 15% in the low-protein-amino-acid-supplemented diets; however, the variation within and among samples was very high (±0.62), likely accounting for the lack of significance, resulting in the absence of significance. Moran et al. [
50] and Jacob et al. [
51] indicated that a low-protein-amino-acid-supplemented diet decreased nitrogen excretion by 27.5%. Teekel et al. [
52] found that, as dietary protein was reduced, there was less uric acid and ammonia excretion, but amino acid excretion remained constant. Jirjis et al. [
53] indicated that protein level did not affect amino acids in the urine significantly, but urinary nitrogen was higher with the higher protein diet. A meta-analysis by Alfonso-Avila et al. [
54] reported that nitrogen intake and retention linearly decreased as the dietary protein level was reduced, particularly in days 0–21 of age. In addition, N efficiency increased by 2.3% for each 1% reduction in diet CP content regardless of bird age. A reduced crude-protein diet has also been reported to improve litter quality by reducing the litter moisture content [
55]. All of these considerations can explain the increasing interest in reducing the CP level in the diets as a tool to improve the environmental sustainability of poultry production [
56,
57].
There were several cases of mortality in both experiments, but there were not significant differences among the groups, and, in addition, they were apparently not related to dietary treatments, as indicated by post-mortem investigations.
Carcass yield from the different experimental groups was essentially constant, and the absence of a negative effect from a low-protein-amino-acid-supplemented diet in dressing percentage, breast plus wings and thigh plus legs. It was observed that the percentages of abdominal fat and liver were not different among groups fed either the positive control or the negative control supplemented with any of the amino acids. These results agree with the conclusion of Fisher [
58] and Attia et al. [
16], who reported that abdominal fat could be reduced by increasing the total sulfur amino acids (TSAA) content in the broilers’ diet. Furthermore, the addition of Met to high (24%)- or low (17%)-protein diets improved the growth and FCR and reduced fat in the liver [
59].
There were marked variations among the tested groups in spleen %, in which the negative control diet or diets supplemented with Thr, Arg, Val or Arg+Val+Trp+Ile had a significantly higher spleen percentage compared to the positive control diet. On the other hand, reducing CP while satisfying amino acids requirements had no negative effect on the survivability of broilers during early life.
Feeding an 18.3% CP diet supplemented with Met+Lys and Arg or Val can support normal carcass composition, as shown by the lack of significance in the crude protein, ether extract or moisture contents of the breast meat of the broilers. According to Baker [
14], there was no effect of the dietary protein level when amino acids are adequate for normal tissue growth. The current findings are partially in line with those of Lipstein et al. [
49], Leclercq et al. [
60] and Leclercq [
13], who concluded that lowering protein content while insuring adequate EAA (Arg, Ile, Val and Trp) permitted normal protein growth but increased lipid gain in both lean and fat lines of broilers, showing that lipid and protein deposition can be controlled independently and that there is no antagonism between these two meat components. Bunchasak et al. [
61] observed that carcass protein and abdominal fat content were not affected by reducing intact protein from 21 to 17% when supplemented with Met and Lys to stratify these essential amino acid requirements. Van Harn et al. [
55] also reported that a low-protein diet did not compromise the carcass yield. This is supported by the current results, with a lack of any significant effects of a low-protein-amino-acid-supplemented diet on dressing and carcass composition.