3. Results
Assay 1: determination of NMR NRmaxT, and Lys requirement using utilization efficiency.
The results of the nitrogen balance assays are shown in
Table 6. The increase in dietary protein levels influenced all the variables studied (
p < 0.0001). Furthermore, a gradual increase in NI and NEX was observed between the levels of Lys and protein in the diet. However, the ND was gradual up to level five, with a daily difference of only 48.58 mg/BW
kg0.67 between levels five and six, and 35.93 mg/BW
kg0.67 between levels six and seven. The same effect was observed for NMO and NR, with a slight decrease from level five to six (55.22 mg/BW
kg0.67 and 103.80 mg/BW
kg0.67, respectively) and an increase up to level seven (68.12 mg/BW
kg0.67 and 177.94 mg/BW
kg0.67, respectively). The difference in dietary Lys levels provided a range of 78.06% in NMO, which influenced the difference of 68.35% from level one to level seven of Lys in NR.
The exponential function between NI and NEX was used to estimate the NMR (
Figure 1). The NMR value obtained was 425.27 mg/BW
kg0.67 per day for quail in the laying period, where NI was equal to zero. With the adjustment of the non-linear regression between NI and NR, it was possible to estimate the value of 3386.61 mg/BW
kg0.67 of daily NR
maxT (
Figure 2).
Based on the models used to calculate nitrogen utilization efficiency, that is, the dietary protein quality (b = [ln(NRmaxT) − ln(NRmaxT − NR)]/NI), it was possible to estimate the value of b = 0.000486. Using the ratio of b and the concentration of the limiting amino acid in the protein (c), it was possible to estimate the Lys efficiency of utilization Lys bc−1 = 0.000101. Based on the parameters derived from the models and on the response of Japanese quail breeders in this study, the Lys intake was required to reach 80% of the NRmaxT value, which was 961.90 mg/BWkg0.67 per day for a bird weighing 0.16 kg Moreover, the daily intake of Lys is 291 mg/bird/day or 1.164% Lys in the diet, considering a feed intake of 25 g/bird.
Assay 2: Determination of amino acid utilization efficiency, requirements, and optimal amino acid ratio.
The results of the nitrogen balance test for Japanese quail breeders are presented in
Table 7. From these data, the values of
b and
bc−1 were derived, which enabled the determination of the IAAR. The birds that consumed diets with amino acid deletion showed, a reduction in nitrogen retention, ranging from 1.47% to 45.69%, in the nitrogen balance, with the addition of NMO. Within this range glycine + serine was the amino acid treatment with the smallest reduction (18.60 mg/BW
kg0.67) and valine was the treatment with the highest (579.98 mg/BW
kg0.67) when compared with CD. These results were influenced by the NI, which differed for the birds that consumed a diet limited in a valine (
p = 0.0165) and glycine + serine (
p = 0.0022) compared to that of the CD treatment group. All birds showed an increase in NEX after the treatments with individually limited amino acids, with an overall average that was 31.90% higher than that of the CD treatment group. In particular, birds that consumed a leucine-limited diet had an increased NEX of 42.23% when compared with that of the CD group.
Body weight was compromised in birds that received diets limited in threonine and valine, whereas the others showed no difference compared with that in the CD group (p > 0.05). In contrast, the NMO was lower than 376.56 mg/BWkg0.67 (the mean CD) for all treatments with one limiting amino acid, but only diets limited in lysine and valine differed significantly from CD (p < 0.05), with 168.85 and 223.52 mg/BWkg0.67, respectively.
After a more specific evaluation of the responses using the estimation of parameter b, the limiting diets presented lower protein quality when compared with that of CD (
p < 0.0001). An exception to this was the diet limited in glycine + serine (
p = 0.1064). When evaluating the values of
bc−1, the highest use efficiency of an amino acid was for tryptophan (0.000226), and the lowest was for leucine, phenylalanine + tyrosine, arginine, and lysine (
Table 8).
The ideal ratio derived from the efficiency of the individual amino acids (
bc−1) in this study, using the Goettingen approach, is presented in
Table 8. A 40% reduction was sufficient to estimate the ideal ratio for all essential amino acids tested, except for glycine + serine, owing to the efficiency of use (
bc−1).
4. Discussion
To the best of our knowledge, this study is the first to determine the Lys requirement for Japanese quail breeders. We used the nitrogen balance and estimated the NR
maxT, NMR,
b, and
bc−1 [
11,
13,
14]. Moreover, using dietary protein utilization efficiency, we determined the ideal ratio of essential amino acids based on amino acid deletion.
Birds had lower feed intake amounts at lower concentrations of limiting amino acids (N1 and N2,
Table 3), an effect related to the Lys content [
18]. Dietary Lys deficiency renders the rate of protein synthesis unfeasible for cell cycle proliferation, leading to cell apoptosis [
19]. This can lead to lower ovary and oviduct weight and a consequent decrease in broiler breeders’ production [
9]. The reduction in egg production decreases energy requirements, which induces intake regulation [
20]. Considering that the energy content at all levels of Lys were the same, the amount of energy required for production would become smaller, and, consequently, the feed intake would be lower. Thus, the lowest concentration of Lys (N1 = 3.4 g/kg and N2 = 5.0 g/kg) made it impossible to maintain production [
20]. In this study, a significant increase in egg output was observed up to N5.
NMR is the minimum nitrogen retention, which was estimated considering the intercept of the exponential function when NI = 0 [
21]. According to Liebert [
21], the NMR value does not consider nitrogen loss from feathers and skin desquamation. Therefore, the NMR results were reported as the approximate average amount of nitrogen [
14]. The daily NMR of 425 mg/BW
kg0.67 for Japanese quail breeders was 1.7 times higher than that found for broiler breeders [
22]. Using the comparative slaughter technique, Silva et al. [
22], found that the NMR is 760 mg/BW
kg0.67 per day for commercial Japanese quails, a value 1.8 times higher than that found in this study for adult birds.
Another important factor causing the difference between the NMR values is the methodology used. NEX determined using the comparative slaughter technique accounts for the nitrogen lost via feathers, which is not included in the nitrogen balance technique [
23]. The food restriction imposed in the study conducted by Silva et al. [
22], i.e., a lower feed supply (80, 60, and 40%) as the form of limitation, makes homeostasis unfeasible and modifies the anabolic and catabolic responses of the animal. Body proteins are targeted for oxidation and are converted into glucose or ketone bodies for energy generation [
24]. Due to the lack of scientific studies determining NMR for Japanese quail breeders, the value found is a reference for other studies (425.27 mg/BW
kg0.67 per day).
The value of 3386.61 mg/BW
kg0.67 is the maximum daily retained nitrogen, under non-limiting conditions, for Japanese quail breeders. This value is expressed as the theoretical limit of the exponential function [
13,
21]. One application of this constant for nutritional programs is to determine the maximum potential of the strain and allow the estimation of demand according to the production objective. In addition, it is fundamental for practical modelling applications [
21].
The estimated NR
maxT values for laying breeders were 1639.9 mg/BW
kg0.67 and 1554.2 mg/BW
kg0.67 for 31–35 and 46–50 weeks, respectively [
14], and 1883 mg/BW
kg0.67 for commercial laying hens [
15]; both studies used the nitrogen balance methodology. The values approached a 51% difference between the values found for Japanese quail breeders and that for heavy breeders, and the difference decreased slightly to 55% when compared with commercial layers. Comparing NR
maxT results is impossible due to variations in strains, age, feed consumption, and diet characteristics [
15,
21].
Parameter
b was estimated as 0.000486, representing the function’s growth rate. The interpretation of b depends on protein quality and is independent of nitrogen intake [
21]; the amino acid variation in the protein reduces or increases the amount of nitrogen to its maximum potential [
23]. Likewise, calculating the daily requirement of Lys depends only on the efficiency of the dietary amino acid; thus, the parameter
bc−1 was established [
25].
The daily value of Lys intake for a bird of 0.16 kg (291 mg/bird) was defined by the efficiency of Lys utilization for the studied diet. This estimated value for the daily intake of Lys reached 80% of the NR
maxT, which was observed in birds of treatment 7 (N7 = 16.8 g/kg of feed). These results characterize the maximum genetic potential of the animal [
23,
26].
Lys has physiological functions in all cells and tissues during the synthesis of various indispensable compounds [
19]. In peak-laying birds, in addition to vital functions, the Lys metabolic pathway, in addition to vital functions, is directed towards yolk and albumen formation, where 87% and 67% of the Lys in the yolk and albumen at peak production, respectively, are from dietary sources [
6]. Therefore, Lys intake in the diet is directly related to egg production [
9], and insufficient Lys intake makes egg production unfeasible. Establishing Lys requirements is extremely important, as it is considered the reference amino acid to establish the proportions of other essential amino acids [
6].
Such proportions were determined from the IAAR based on the deletion of amino acids by the use efficiency of use of dietary protein from the nitrogen balance compared to a CD. Among all the amino acids studied, reduction the of valine in the diet had the biggest influence, reducing feed consumption (30.51%), body weight (12.50%), and nitrogen excreted in the egg output (57.60%), compared with those in birds from the CD group. However, some studies have shown that attention must be paid to the levels of leucine and isoleucine when considering the dietary levels of valine [
27,
28]. This is because they have a branched chain (BCAA), and their excesses or deficiencies can result in antagonism [
29]. Proportional differences were observed in the CD for Leu:Val and Ile:Val at 100:50 and 100:115, respectively, as well in the valine-limited diet where the ratio for Leu:Val and Ile:Val was 100:30 and 100:69, respectively. Therefore, leucine and isoleucine were proportionately higher in the limiting diet. Metabolically, excess leucine induces branched-chain aminotransferase activity, leading to the catabolism of other BCAAs [
30]. Valine is the amino acid most susceptible to antagonism and enzymatic degradation [
31]. Furthermore, excess leucine stimulates the synthesis of protein and inhibits protein degradation. However, with a deficiency of this amino acid, the stimulation of synthesis further exacerbates the amino acid imbalance in the plasma pool [
32]. This corroborates the weight loss observed among the birds, which may be justified by the breakdown of muscle proteins to maintain the plasma balance of amino acids. The detection of an amino acid deficiency in the anterior piriform cortex induces the animal to reduced feed intake [
33] as a preventive mechanism. In other studies, the animal reduced the intake of a limiting diet [
34,
35], which corroborates with the results of this study.
The requirements for the use of an amino acid were divided into the need for maintenance and the efficiency of protein retention, which for birds in production is related to egg output. This was achieved using a factorial approach. This is affected by the deletion of lysine and valine in the diet, via nitrogen deposition, and the logical approach that the 40% limitation made amino acids unavailable for protein synthesis in egg production. In a study by Azzam et al. [
36], the non-addition and lower level (1 g/kg) of L-val in the diet significantly affected the serum albumin level. Notably, hepatic production of yolk lipoproteins is regulated by BCAAs [
37]. The lysine-related NMO reduction is directly related to egg weight and can be explained by the reduction in egg protein when birds are fed low-lysine diets [
38,
39]. In lysine metabolism, muscle tissue is manipulated by the rate of egg [
6]. In addition, broiler breeders subjected to a dietary lysine deficiency of 44.75% showed a significant difference in egg and chick weights [
39]. Kim et al. [
9] demonstrated broiler breeder hens fed a 30% lysine reduced diet (0.55% Lys in the diet) had lower oviduct and ovary weights and follicular recruitment. This was accompanied by a delay in ovulation due to apoptosis and necrosis the ovarian follicles, which culminated in a drop in egg production. Therefore, lysine deficiency in the diet makes optimal production impossible because excess lysine is destined for producing eggs only after meeting the muscular requirement [
6].
To determine the IAAR, the statistical difference between the individual amino acids and CD must be confirmed in the evaluation of parameter
b [
11]. However, the Gly + Ser-limiting diet did not produce a significant decrease in protein quality when compared to that of CD, indicating an excess of this amino acid in the CD.
The other limiting diets, in terms of individual amino acids, reduced protein quality, which made it possible to estimate the IAA value. Among the studies that recommended amino acid requirements in the literature, only the study by Hanafy and Attia [
40] used Japanese quail breeders. However, the recommendation was not estimated (0.2% in the diet) but based on the treatment that resulted in better productivity and reproductive performance. Thus, one can observe the fragility of amino acid nutrition for hens that are fed diets formulated according to recommendations set for commercial laying hens [
4,
5]. We expect that the dissimilarity in the amino acid requirements is likely due to the difference in genetic potential. When we analysed the recommendation by Rostagno et al. [
5], the IAA for all amino acids, except for Val, which was above the recommended value in this study, ranged from 4 (Thr = 0.70%) to 19% (His = 0.48%) among all amino acids studied, except Gly + Ser. According to the NRC [
4], the recommended value was transformed into a digestible amino acid considering 89% [
16]; therefore, the values for Lys, Met + Cys, Thr, Trp, Arg, Val, Leu, His, and Phe + Try were below those determined in this study (0.79, 0.55, 0.59, 0.15, 0.93, 0.73, 1.12, 0.33, and 1.11% in the diet), with differences reaching 56, 38, and 32% for Met + Cys, Trp, and Val, respectively. Only the IAA for Ile was higher than that for commercial quail (1.12% in the diet). Thus, according to the tables currently used as a basis for formulating rations for breeders, no amino acids presented IAA values close to the results of this study, thereby confirming the modification of the requirement by the genotype.
Furthermore, Lima et al. [
41] and Sarcinelli et al. [
42] recommended 0.78% and 0.70% of Thr in the diet of Japanese quail, respectively, which corresponds to values 14% and 5% higher than that found in this study (0.67%). Moreover, AAI values were determined in a study by Lima et al. [
43], where the estimate for Arg was 17% higher (1.14%) than that found in this study (1.17%). For Trp, they estimated 0.22% in their diet [
42], which was 6% higher than the determined value (0.21%). However, the amount of Val in the diet was 62% lower (0.59%) in a study by Martinez et al. [
44], in which the authors considered an 11 g/day of egg output and 0.17 kg of body weight. Lower values were also observed for Met + Cys, with a difference of 12% from the estimate by Sarcinelli et al. [
42] (0.76% Met + Cys in the diet). No studies have been found in the literature on the IAA for other amino acids.
However, based on the results found in the IAAR of essential amino acids proportional to Lys, breeders need to intake higher proportion of the amino acid intake of Met + Cys, Thr, Trp, Arg, Val, and Ile. For Leu, His, and Phe + Tyr, the recommendations for commercial quails are proportionately higher than those of Rostagno et al. [
5]. Thus, when we used the Lys requirement estimated in this study for a 0.16 kg bird (291 mg/bird/day) to determine the IAAR intake of Met + Cys, Thr, Trp, Arg, Val, Ile, Leu, His, and Phe + Tyr as 252, 195, 60, 340, 279, 192, 413, 113, and 387 mg/bird/day, respectively, all amino acids except for His had a higher intake requirement for breeders when compared to that recommended by Rostagno et al. [
5] for commercial quails.
Several factors may explain these observations. First, with genetic improvements made in these birds through crosses and specific selections, more efficient commercial birds that need a lower amino acid intake were developed. According to differences related to the methods used to derive the IAAR, studies by Rostagno et al. [
5], compiling previous studies, used different diets, ages, and methods to estimate the IAAR with different response criteria. Most of the studies cited in this discussion for IAA are dose-response studies [
43,
44], unlike the present study, which used a nitrogen utilization model to determine individual efficiency with only an experimental diet.