INTRODUCTION
Reducing the crude protein (CP) content in diets for broilers has created a mechanism capable of decreasing feed costs and nitrogen emissions from the production of meat products [
1,
2]. However, low-protein diets tend to impair performance by decreasing weight gain, worsening feed conversion, and increasing carcass fat content [
3]. The protein requirement in the diet of birds involves both the need for essential amino acids (AAs) and the supply of non-essential AAs and nitrogen. Meeting the nutritional requirements of essential AAs makes it possible to carefully reduce the CP in the diet without harming the performance of the birds [
4]. The concern with meeting only the needs of essential AAs increase the chance of a non-essential AAs becoming limiting in the diet, due to the amount of precursors for a given AA being insufficient or due to the delay of metabolic processes [
5]. Dietary supplementation of glycine (Gly), which is a non-essential AA, was shown to be able to promote improvement in the productive parameters of broilers during the starter and grower phases [
4,
6,
7]. Even with the ability of birds to synthesize Gly endogenously, this synthesis may not be metabolically efficient to meet the AA requirements of broilers in the initial phase [
8]. Thus, the diet must be formulated to supply at least 40% of the total Gly requirement to enable the growing birds to meet the necessary metabolic demand for protein deposition and production of biomolecules, such as uric acid (UA), creatine, glutathione and collagen [
9]. Since Gly can be reversibly metabolized to serine (Ser) and, on a molar basis, Ser plays the same functions as Gly in chicken, it becomes necessary that the requirements of these AAs be considered jointly as Gly equivalents (Gly
equi) in diet formulations [
4].
Moreover, threonine (Thr) serves as a possible metabolic precursor of Gly and occurs as an active process through a series of enzymatic reactions [
9], which is potentially necessary in fulfilling the Gly requirement for poultry [
4,
10,
11]. When Thr is degraded by the Thr aldolase enzyme, Thr generates Gly and acetaldehyde as by-products or via Thr dehydrogenase with 2-amino-3-ketobutyrate as an intermediate step generating acetyl-CoA, which can be directly converted into Gly [
12]. Thr being considered as the third limiting AA in corn-soybean meal-based diets provided to broilers, in addition to playing a fundamental role in the synthesis of proteins and mucin, also allows the lowest concentration of Gly
equi to be contained in the diets [
13,
14]. Studies have shown that diets with marginal Gly
equi levels, which is commonly observed in low-CP diets formulated with plant-based ingredients, can be viable by having enough Thr for broilers [
10,
13,
15]. If the premise that increasing dietary Thr in low-protein diets increases performance is true, this suggests that Thr concentrations may be acting equivalently with Gly, demonstrating that these AAs may be crucial for diets formulated with reduced CP content. Thus, the present study was conducted to evaluate the Gly
equi requirements of broilers from 21 to 42 days of age, by offering reduced CP diets with varying standardized ileal digestible threonine (SID Thr) levels.
DISCUSSION
Lowering the dietary CP content offers several significant advantages for sustainable chicken meat production, including reduced dietary costs, lower emissions of nitrogen and ammonia, improved litter quality, and enhanced bird welfare due to less indigestible protein entering the hindgut to support the growth of potential pathogens [
2,
23,
24]. A substantially reduced-CP diet for broiler chickens raised during the growing period contains between 2% and 3% less CP compared to a conventional diet [
10,
25]. Thus, the present study examined a CP concentration of 16.5%, which is lower than the Cobb-Vantress [
26] recommended requirements for corn-soybean based diets. Since low-CP diets are gaining popularity in poultry nutrition, it is essential to meet the Gly
equi and threonine requirements of broiler chickens for optimal growth performance because these AAs are considered essential in low-CP corn-soybean-meal-based diets [
5,
13,
27]. The present study investigated the interactive effect of a Gly
equi and SID Thr levels in low CP diets on the performance of growing broilers (22 to 42 d of age). The results obtained for weight gain revealed that in low-protein diets, Thr supplementation at levels lower than and/or equal to recommended SID Thr could increase the Gly
equi requirements. However, supplementing Thr to provide excess dietary SID Thr levels reduces the requirement of Gly
equi, thus confirming the result of previous studies [
9,
10,
13]. This shows that the decline in weight gain of birds fed with low-protein diets could be prevented when excess SID Thr is provided with marginal Gly
equi levels. Chrystal et al [
28] observed that, when there is deficient dietary Gly
equi levels, Thr reduces feed consumption, causing losses in the productive response of birds. However, some studies have shown that, excess Thr supplementation reduces the need for Gly
equi, since the enzymes Thr aldolase and Thr dehydrogenase are able to degrade excess Thr into Gly [
8,
13]. Consequently, more energy (9 ATP/mol) needed in the synthesis of Gly is conserved, hence making an increased amount of energy available for optimal growth [
10]. Interactive effects between total Gly
equi and SID Thr intake have been frequently reported in the literature [
14,
15,
28]. Ospina-Rojas et al [
10] providing diets with 0.84% to 0.92% Thr and 1.44% to 1.76% Gly
equi observed an interaction between Gly levels and Thr levels feed:gain, but not in weight gain in broilers aged 21 to 35 days. On the other hand, Corzo et al [
13], working with grower broilers fed with diets containing 0.72% to 0.81% Thr and 1.64% to 1.76% Gly
equi, obtained an interaction for weight gain, but not for feed:gain. According to Bernardino et al [
29], Gly supplementation decreases the activity of enzymes that catabolize Thr, thus saving Thr from being degraded to Gly. Moreover, Ospina-Rojas et al [
10] demonstrated that Gly supplementation would be necessary to maintain dietary Thr levels rather than reducing it, because the use of Thr for physiological roles such as production of gastrointestinal mucin, among other functions, would be reduced via Gly addition, thus resulting in improved growth rates in broilers. Therefore, increased Thr supplementation is unnecessary in diets containing adequate or high Gly
equi concentrations; because excess Thr compromises the productive performance of birds, increasing the elimination of UA, as this process requires high consumption of metabolic energy [
10]. Also, our results are in accordance with earlier reports that showed Thr supplementation at levels higher than its recommendation could serve as a source of dietary Gly
equi with marginal AA concentrations, promoting the use of low-protein diets for broilers without any negative effects on growth performance [
10,
15]. Thus, this outcome shows evidence to support the idea that the needs for Gly
equi are reduced when dietary SID Thr is provided in excess of its requirement. The feed:gain in this present study tended to be lower when adequate SID Thr concentration in the diet was offered to the birds compared to when SID Thr levels exceeded its requirement. This was in line with the results of previous studies, which have shown that the feed:gain of birds improved when they received the appropriate concentration of the Thr amino acid [
8,
10,
30]. The feed:gain of birds that received the adequate level (0.68%) of SID Thr may have improved due to the stabilization and maintenance function of the intestinal tract performed by Thr, favoring the digestion of nutrients [
10,
31]. These same authors point out that adequate levels of Thr allow the digestive system to benefit from increased amylase secretion and mucin production, improving the digestibility of nutrients present in the diet.
An increase in the requirement of Gly
equi resulted in increased pectoral muscular creatinein of birds fed with varying dietary levels of SID Thr with reduced CP concentration. Since the need for Gly
equi in the synthesis of creatine is considered essential at the starting period of the broiler’s growth, increasing levels of Gly
equi resulted in a positive response with respect to pectoral muscular creatine during grower stage (22 to 42 d) when low-protein plant-based diets are provided. The present study suggests that Thr supplementation to provide dietary SID Thr at levels lower or greater than the required (0.68%) showed an increment in the Gly
equi requirement for muscle creatine, thus confirming the report of Ospina-Rojas et al [
8] and Aguihe et al [
15]. Creatine, in addition to having considerable importance in increasing growth performance, can also be considered an important source of energy for muscle tissue, since it allows the immediate resynthesis of ATP [
32]. Furthermore, when the amount of pectoral muscular creatineis increased, there is a better use of nutrients for muscle growth in this tissue [
33]. However, plant-based diets do not provide a sufficient amount of creatine as required by birds, as there is inadequate dietary creatine supplied via plant ingredients. Therefore, to supply this deficit, greater amounts of Gly are required to synthesize creatine [
34]. Therefore, increasing total Gly
equi up to 1.56% in the diet through Gly supplementation may be favorable as a source of creatine, thus, promoting improved utilization of nutrients and energy for proper muscle growth in this tissue [
33]. SID Thr levels influenced the pectoral muscular creatine content, which was higher in broilers that received adequate (0.68%) and excessive (0.78%) SID Thr diets compared to those offered deficient SID Thr (0.58%). This result suggests that increasing dietary SID Thr at or above recommended level in broilers fed with low-protein diets is essential for promoting the synthesis of creatine in muscle tissues. One possible explanation for the positive response to increasing Thr levels on pectoral muscular creatine is that it acts as a readily available precursor to Gly, thus invariably increasing the amount of Gly
equi needed for the creatine synthesis pathway [
8]. This observation is supported by a previous report that demonstrated that the catabolism of Thr yields Gly to support metabolic processes such as the conversion of guanidino acetic acid to creatine [
35]. Therefore, the synthesis of creatine depends on increased availability of Gly
equi for methylation of guanidino acetic acid, which is the only metabolic intermediary product and precursor of creatine in muscles [
36]. Creatine levels are good indicators of protein and AA metabolism, which stimulates protein synthesis in muscles and are involved in energy metabolism and storage [
37].
In the current experiment, carcass yield was influenced by the interaction of Gly
equi and SID Thr levels in the diet. The results obtained for carcass yield suggest that dietary Thr had a compensatory effect under the lowest level of Gly
equi in the diet of birds aged 22 to 42 d. This implies that since the enzymes Thr aldolase and Thr dehydrogenase can catabolize excess Thr into Gly, Thr would be able to decrease Gly requirements. However, in situations of marginal levels of Gly in a diet based on low protein plant-based products, Thr can be considered a relevant source of Gly [
10,
14]. Similar results were found by Corzo et al [
13] for which lower Gly
equi levels resulted in lower carcass yield values that only improved when dietary Thr was increased. Still, Baker et al [
9] mentioned that when Thr was shown to be in moderate excess, the Gly-sparing effect of dietary Thr was found. The influence of Gly
equi levels on breast yield may be associated with the increase in creatine content in the pectoral muscles in response to Gly supplementation, as creatine is considered an important energy source in animal feed [
32]. Unlike by-products of animal origin, ingredients of plant origin do not meet the nutritional requirement for the performance of birds, thus increasing the needs of Gly, an amino acid used for the endogenous synthesis of creatine [
33]. Dietary Gly
equi levels linearly decreased (p<0.05) the abdominal fat content. A previous study illustrated the challenge of providing diets with low CP, as these compromise feed conversion and led to greater fat deposition [
38]. According to Ospina-Rojas et al [
10], digestion of dietary fat is favored by Gly supplementation, considering that this AA is a component of bile salts, constituting about 90% of the total AA present in the bile.
The result of the present study reveal that higher Gly
equi is necessary in low-protein diets with a lower concentration of SID Thr to achieve a lower SUA, while Gly
equi requirements decreased in diets with adequate SID Thr for growing broilers, which may be a reflection of greater efficiency in the use of AAs. The synthesis of UA as the final product of AA catabolism in chickens, is a Glyequi-dissipating process to eliminate excess nitrogen [
13]. SUA is used as an influential parameter to reflect the extent of amino acid utilization in broiler chickens. In SID Thr-deficient low-protein diets, the linear decrease in SUA concentration with increasing Gly
equi levels is an indication of the reduced rate of AA oxidation to spare more protein for improved nitrogen utilization [
39,
40]. The increase in SUA concentration in diets with 0.78% Thr in response to Gly
equi supplementation may be due to an AA imbalance. Dietary AAs when in excess, are deaminated into carbon and ammonia skeleton which, due to their high toxicity to animal tissues, are eliminated as UA by birds [
38]. In poultry, the premise is that UA is the main product of nitrogen excretion; and for each UA molecule formed, a Gly molecule is lost, an indication of a higher metabolic requirement for Gly in broilers [
4]. This could explain the importance of supplementing diets low in CP with adequate levels of essential and non-essential AAs so that the rate of AA oxidation decreases to save more protein for improved performance in broiler chickens [
41]. Increasing dietary Gly
equi levels showed a quadratic response on total protein at an optimum level of 1.40% in the current study. This is an indication that the adequate levels of Gly
equi in dietare necessary to optimize cellular protein synthesis, since total proteins can be used as markers of protein nutritional status [
1].
The findings of the present study show that increased TBARS concentrations associated with higher lipid peroxidation in birds fed with imbalance SID Thr diets were mitigated by the increased supply of dietary Gly
equi provided through Gly supplementation. The main function of Gly is to favor the excretion of excess nitrogen by donating carbon and nitrogen to the synthesis of UA, this AA is also involved in the process of synthesizing glutathione, which plays an antioxidant role in the body of animals [
42]. Glutathione is an important bio-compound that neutralizes and protect cells against the damaging effects of free radicals by enhancing lipid peroxidation, a process leading to the oxidation of polyunsaturated fatty acids to lipid peroxides [
42]. The existence of glutathionine as an antioxidant within cells hinders the generation of free radicals and advancement of peroxidation [
43,
44]. This could explain the observed positive influence of higher dietary total Gly
equi levels on lipid oxidation by preventing intracellular degradation of glutathionine via elevated glutathione peroxidase and glutathione reductase activity [
45]. These enzymes convert radicals and peroxides in innocuous reduced forms, often with the concomitant oxidation of reduced glutathione to its oxidized form [
43,
45]. The current study corroborates the findings of Gao et al [
45] and Deng et al [
46] who demonstrated that Gly supplementation led to a significant increase in glutathione levels and upregulation of antioxidant-related genes, including glutathione peroxidase, catalase, and superoxide dismutase; as well as the decrease in lipid peroxidation caused by reactive oxygen species and a reduction in MDA levels. This positive effect was attributed to the stimulation of lipid metabolism via increased functionality of mitochondria, lipid droplets, and factors involved in lipid metabolism, such as peroxisome proliferator-activated receptors, sterol regulatory element-binding factor, and ATP [
46]. Thus, this present result demonstrates the potential efficacy of sufficient dietary Gly
equi levels in protecting tissues from peroxidative damage.