Ethics Statement. The Research Animal Ethic Committee of the University of Zabol and Iranian Council of Animal Care approved this experimental protocol. Experiments comply with the “Animal Research: Reporting of In Vivo Experiments” (ARRIVE) guidelines (https://arriveguidelines.org) and with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Bird management. The experimental procedures for animal trials were approved by the Animal Ethics Committee of the University of Zabol. One-day old straight-run quail chicks (Coturnix coturnix Japonica) were provided from the meat-type Quail Genetic Stock Centre at the Research Center of the Research Institute of Zabol (RCRIZ, Sistan, Iran) and fed on grower diet based on the recommendation of NRC 16 from hatch to 20 d of age. At d 21, a total of 420 quail chicks were randomly allotted to 28 floor pens consisted of 7 treatments with 4 replicates with 15 birds per pen (460 cm2). The temperature of experimental house was set at 26⁰C ± 2.0 in the third weeks of age afterward with relative humidity of 60% ± 3.5. The lighting program was 23L:1D during the study.
Bird management and experimental diets. As described by Mehri, et al. 13, the basal diet consisting of wheat, soybean meal, corn, and corn gluten meal was mixed and formulated to provide an adequate concentration of all nutrients for growing Japanese quails, except Trp (Table 1), which was supplemented with 7 concentrations of L-Trp at the expense of cornstarch providing a range of dietary Trp from 2.12 to 2.90 g/kg of diet with 0.13 g/kg increments. Quail chicks were fed ad libitum throughout the trial and had free access to the water from 21 to 35 d of age. As usual, all protein-containing feed ingredients of basal diet and mixed experimental diets were analyzed for CP method 990.03, 37 and amino acid profile method 982.30, 37 before beginning the experiment. As described by Hasanvand, et al. 7, feed samples were prepared using a 24-h hydrolysis in 6 N hydrochloric acid at 110°C under an atmosphere of nitrogen. For Met and Cys, performic acid oxidation was done before acid hydrolysis. Samples for Trp analysis were hydrolyzed using barium hydroxide. Chromatographic separations of amino acids were performed with a Waters HPLC system (Waters, Milford, MA).
Serum biochemical analysis. At d 35, four birds per replicate were euthanized by cervical dislocation and blood samples were taken from the jugular into 10 mL heparin tubes. The blood variables including uric acid (UA), albumin (ALB), triglycerides (TG), alkaline phosphatase (ALP), total protein (TP), and aspartate transaminase (AST) were measured by the spectrophotometric method using commercially available kits (Parsazmun, Tehran, Iran). A portion of collected blood collection was centrifuged under refrigeration (1,500 × g, 5 min, 8◦C) to obtain the serum, which was stored at − 80◦C until analysis (d 30 after collection).
Humoral immune response and antibody analysis. Four birds from each replicate were wing banned and 0.1 mL of 5% SRBC in PBS was injected into the breast muscle at 18 and 28 d of age. Blood sampling was performed at 25 (for primary response to SRBC) and 35 d (for secondary response to SRBC) of age and antibody production against SRBC antigen was assessed by a hemagglutination inhibition test in the serum samples according to Cheema, et al. 38. The serial dilution technique was used to measure the IgG using the 2-mercaptoethanol (2-ME) as described by Bartlett and Smith (2003). In brief, the collected serum was inactivated by heat in a 56○C water bath for 30 min. Then after, 50 µL of PBS was placed in the row of wells in a 96-well V-bottom microtitration plate. To the same wells, 50 µL of serum was added, and plates were sealed and incubated at 37○C for 30 min. Serial dilution of the samples was made on successive rows, 50 µL of a 2-ME solution was added to each well, and plates were again sealed and incubated for 30 min. The agglutination in each well was read by holding plates over a lighted mirror. The antibody titer of IgG was reported as log2 of the reciprocal of the last dilution in which agglutination was observed.
Malondialdehyde assay. At d 35, four birds per replicate were randomly selected and euthanized by cervical dislocation and deboned meat of thigh sections were grounded with a blender and stored in -20◦C for 30 d to assess the oxidation stability through the measure of malondialdehyde (MDA) formation. The third-order derivative spectrophotometric method developed by Botsoglou, et al. 39 with minor modifications was implemented. In brief, one gram of grounded meat sample was picked up and homogenized (Polytron homogenizer, PCU, Switzerland) with 4 ml of 5% aqueous trichloroacetic acid (TCA) and 2.5 ml of 0.8% butylated hydroxytoluene, and then centrifuged at 3000 × g for 3 min. The top layer, hexane, was discarded and the bottom layer was filtered and made to 5 mL volume with 5% TCA, then placed into a screw-capped tube containing 3 mL of 0.8% aqueous 2-thiobarbituric acid (TBA). At last step, falcon tubes were heated in 70°C water bath for 30 min, then immediately cooled by tap water and submitted to spectrophotometry (UNIKON 933, Kontron Co. Ltd., Milan, Italy). The height of the third-order derivative peak that appeared at 521.5 nm was used for calculation of the MDA concentration as the secondary product of oxidation in the samples. The precursor of MDA in the standard curve was the tetraethoxypropane (1, 1, 3, 3- tetraethoxy propane, T9889, 97%, Sigma, USA.). The concentration of MDA was expressed as milligrams per kilogram of meat samples.
Water holding capacity. The meat sample parts were centrifuged at 1000 × g at 4°C for 15 min to measure water holding capacity (WHC), which was calculated as follow 36:
WHC (%) = (weight before centrifugation/weight after centrifugation) ×100
Drip loss. The meat sample (20 g) was picked up 24 h post mortem, placed in a plastic bag, and kept at 4°C. After 24 h, the thawed samples were removed dried on absorbent paper, and reweighed. Drip loss was determined at 48 h post mortem 36:
Drip loss (%) = [(initial weight-final weight)/initial weight] × 100
Statistical analysis. All data analyses including one-way ANOVA, one-sample t-test, spline regression analysis 6, and principal components analysis (PCA) were employed by GraphPad Prism software, version 9.3.1. The mean differences were analyzed by Tukey's multiple comparison test at P < 0.05. The appropriate model for each set of data was determined by the model accuracy index (δ):
$$\delta = {R}^{2}+ \left(\frac{1}{RMSE}+\frac{1}{AICc}\right)$$
1
$$RMSE= \sqrt[2]{\frac{\sum _{i=1}^{N}{({x}_{i}-\widehat{{x}_{i}})}^{2}}{N}}$$
2
$$AICc=AIC+ \frac{2{k}^{2}+2k}{n-k-1}$$
3
$$AIC=2k-2\text{l}\text{n}\left(\widehat{L}\right)$$
4
where RMSE is the root mean square error and AICc is the Akaike information criterion corrected for small sample size, AIC is the Akaike information criterion, k is the number of estimated parameters in the model, \(\widehat{L}\) is the maximum value of the likelihood function for the model.