Response surface model to illustrate the benefits of tryptophan, melatonin, and N,N-dimethylglycine in quail chicks exposed to aflatoxin B₁

Abstract

A dose-response assay in a central composite design platform was conducted to investigate the responses (performance, immunity, and meat quality) of quail chicks to dietary tryptophan (Trp), melatonin (MEL), and N,N-dimethylglycine (DMG) exposed to aflatoxin B₁ (AFB₁). A total of 1,275 quail chicks were randomly allotted to 85-floor pens consisting of 17 treatments with 5 replicates and 15 birds per each pen. Dietary MEL and DMG had a different effect on growth rate and interacted with dietary Trp and AFB₁ during the first 4 wk of age, while their effect disappeared at the last week of the experiment. Dietary Trp and AFB₁ were only significant on the gain of quail chick after d 28 of the assay. During the second and third weeks of age, the reduction in feed intake caused by AFB₁ attenuated by dietary MEL and DMG and dietary Trp profoundly affects feed intake in the last 2 wk of the experiment. Dietary MEL and DMG were effective on feed conversion ratio (FCR) during the second and third weeks of age. AFB₁ decreased breast meat yield (BMY) and thigh meat yield (TMY), but the inclusion of either MEL or DMG removed the adverse effects of AFB₁. Dietary Trp increased BMY, but it did not affect TMY. Increasing dietary Trp linearly increased the Lactobacillus bacteria (LAB) population, and AFB₁ negatively impacts the LAB population. The inclusion of dietary DMG removed that negative effect on LAB. Although AFB₁ decreased the antibody production against SRBC-antigen, increasing dietary Trp in intoxicated quails increased the plasma antibody in SRBC-challenged birds. At low levels of dietary Trp (0.15–0.19%), the addition of DMG increased malondialdehyde (MDA) production while increasing Trp reversed this adverse situation. In conclusion, these supplements may interact with AFB₁ in younger chicks, and dietary Trp and AFB₁ have a significant impact on the growth performance of quail chicks during the fifth and sixth week of age.

INTRODUCTION

Aflatoxin B₁ (AFB₁), the most potent mycotoxin for humans and animals (da Rocha et al., 2014), mainly affects the liver, kidney, immune organs (spleen, bursa of Fabricius, and thymus), and gastrointestinal system of the poultry species (Bagherzadeh-Kasmani and Mehri, 2015; Khanipour et al., 2019; Hou et al., 2022). AFB₁ negatively affects performance production (e.g., weight gain, feed intake, and feed conversion ratio; FCR), induces immunosuppression, and decreases meat quality (Aftabi et al., 2016; Khanipour et al., 2019) mainly by increasing the concentration of malondialdehyde (MDA), a biological marker of oxidative stress (Del Rio et al., 2005). In essence, AFB₁ found in contaminated foods is an inert compound and is activated in the hepatocytes through an epoxidation process by the hepatic cytochromes P450 (CYP450). The complex of CYP450 in phase I of the bioactivation of AFB₁ is responsible to render the inactive AFB₁ into the reactive and electrophilic AFB₁-exo-8,9-epoxide (AFBO) (Monson et al., 2015). In general, the metabolic pathway of AFB₁ in the body consists of 2 phases. In phase I, the inactive molecule of AFB₁ could be transformed into the activated toxic substance, AFBO, which involves the addition of a small polar group, containing both positive and negative charges by any of the reactions including epoxidation, hydration, hydroxylation, O-demethylation, and reduction. In phase II, a conjugation reaction will add endogenous glutathione to the AFBO by glutathione S-transferases (GSTs). This phase is the detoxification process which is required for the excretion of AFB₁ as a water-soluble product by the kidney (Mughal et al., 2017).

Among the physical, chemical, and biological methods to fight aflatoxicosis, the target of the biological approach is the manipulation of the AFB₁ metabolism either in phase I or phase II by the support of the antioxidant capacity of the body and facilitating the excretion of the conjugated molecule of AFB₁, respectively (Deng et al., 2018). It has been postulated that although AFB₁ upregulates the gene expression of cytochrome P450 (Bahari et al., 2014), the positive effects of excess dietary Trp may be related to the Trp-derivatives and Trp-metabolizing enzymes such as tryptophan 2,3-dioxygenase (TDO) (Khanipour et al., 2019). Therefore, the biological approach exhibits the manipulation of phase I of AFB₁ metabolism via controlling the activity of cytochrome P450.

In the present study, it aimed to investigate the concurrent manipulation of 2 metabolic phases of AFB₁ by using dietary Trp and its derivative, melatonin (MEL), and N,N-dimethylglycine (DMG) as a methyl-donor group in quail chicks exposed to AFB₁.

MATERIALS AND METHODS

Ethics Statement

The Research Animal Ethic Committee of the University of Zabol and the 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

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, Iran and fed a grower diet based on the recommendation of NRC (1994) from hatch to 7 d of age. At d 8, a total of 1,275 quail chicks were randomly allotted to 85-floor pens consisting of 17 treatments with 5 replicates with 15 birds per pen. Body weight and feed intake were recorded weekly to measure body weight gain (BWG) and FCR. The temperature was held at 26°C ± 2.0°C at the third week of age afterward, with a relative humidity of 60% ± 3.5. The lighting program was 23L:1D during the study. The 17 experimental diets were formulated based on central composite design (CCD; Tables 1 and 2) to meet or exceed the nutritional requirements of quail chicks, according to NRC (1994). All protein-containing feed ingredients were analyzed for CP (method 990.03, AOAC, 2006) and amino acid profile (method 982.30, AOAC, 2006) before the beginning of the experiment. As described by Hasanvand et al. (2018), feed samples were prepared using 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.

Table 1.

Arrangement of the input variables based on central composite design codes.

Treatment	Tryptophan	Aflatoxin B1	Melatonin	Di-methylglycine
1	1	−1	1	1
2	0	0	1.68179	0
3	0	0	−1.68179	0
4	−1	−1	1	−1
5	0	0	0	1.68179
6	0	−1.68179	0	0
7	1.68179	0	0	0
8	−1	1	1	1
9	−1	1	−1	1
10	0	0	0	0
11	1	1	−1	−1
12	1	1	1	−1
13	0	1.68179	0	0
14	0	0	0	−1.68179
15	−1	−1	−1	−1
16	1	−1	−1	1
17	−1.68179	0	0	0

Table 2.

Composition of experimental diets.

Ingredients	Diets
	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17
Wheat	74.48	70.28	70.04	70.04	27.01	27.01	27.27	27.27	27.25	27.27	27.27	13.53	13.54	13.54	13.59	38.27	70.05
Soybean meal	0.00	0.00	0.00	0.00	13.72	13.72	13.73	13.73	13.72	13.73	13.73	25.44	25.44	25.44	25.44	24.72	0.00
Corn gluten meal	4.67	14.34	14.34	14.34	15.40	15.40	15.40	15.40	15.40	15.40	15.40	6.50	6.50	6.50	6.50	3.75	14.34
Corn	3.19	1.50	1.50	1.50	30.43	30.43	30.43	30.43	30.43	30.43	30.43	43.65	43.65	43.65	43.65	24.45	1.50
Glutamate	8.21	4.39	4.39	4.39	2.02	2.02	2.02	2.02	2.02	2.02	2.02	2.54	2.54	2.54	2.54	2.27	4.39
Limestone	1.43	1.47	1.47	1.47	1.48	1.48	1.48	1.48	1.48	1.48	1.48	1.50	1.50	1.50	1.45	1.43	1.47
L-lysine hydrochloride	1.28	1.18	1.18	1.18	0.78	0.78	0.78	0.78	0.78	0.78	0.78	0.47	0.47	0.47	0.47	0.47	1.18
Sunflower oil	1.04	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.87	1.00
Di-calcium phosphate	1.03	0.97	0.97	0.97	0.84	0.84	0.84	0.84	0.84	0.84	0.84	0.75	0.75	0.75	0.75	0.75	0.97
Sodium bicarbonate	0.84	0.84	0.84	0.84	0.86	0.86	0.86	0.86	0.86	0.86	0.86	0.51	0.51	0.51	0.51	0.10	0.84
L-arginine	0.73	0.58	0.58	0.58	0.22	0.22	0.22	0.22	0.22	0.22	0.22	0.00	0.00	0.00	0.00	0.00	0.58
L-threonine	0.67	0.51	0.51	0.51	0.31	0.31	0.31	0.31	0.31	0.31	0.31	0.29	0.29	0.29	0.29	0.31	0.51
Corn-AFB1	0.59	0.00	0.95	0.95	0.00	0.59	0.59	0.59	0.59	0.59	0.59	0.24	0.24	0.94	0.94	0.59	0.24
Rice hull	0.00	1.72	0.97	0.97	4.86	4.27	4.30	4.27	4.27	4.27	4.27	2.69	2.69	2.02	2.02	0.00	1.71
DL-methionine	0.45	0.26	0.26	0.26	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.23	0.23	0.23	0.23	0.26	0.26
L-isoleucine	0.44	0.26	0.26	0.26	0.12	0.12	0.12	0.12	0.12	0.12	0.12	0.11	0.11	0.11	0.11	0.08	0.26
L-Valine	0.35	0.14	0.14	0.14	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.14
Mineral premix1	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25
Vitamin premix2	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25
KCl	0.07	0.05	0.05	0.05	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.05
NaCl	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.24	0.00
Di-methylglycine	0.03	0.0122	0.05	0.05	0.30	0.30	0.00	0.03	0.06	0.03	0.03	0.0478	0.0478	0.0122	0.0122	0.03	0.01
Melatonin	0.0015	0.0006	0.0024	0.0006	0.0015	0.00	0.0015	0.0015	0.0015	0.003	0.0015	0.0024	0.0006	0.0024	0.0006	0.0015	0.0024
Nutrient analysis
AME (kcal/kg)	2900	2900	2900	2900	2900	2900	2900	2900	2900	2900	2900	2900	2900	2900	2900	2900	2900
CP (%)	24	24	24	24	24	24	24	24	24	24	24	24	24	24	24	24	24
Ca	0.80	0.80	0.80	0.80	0.80	0.80	0.80	0.80	0.80	0.80	0.80	0.80	0.80	0.80	0.80	0.80	0.80
P available	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30	0.30
Di-methylglycine (mg/kg)	300	122	478.40	478.40	300	300	0.00	300	600	300	300	478.40	478.40	122.00	122.00	300	122
Melatonin (mg/kg)	15.00	6.10	24.00	6.10	15.00	0.00	15.00	15.00	15.00	30.00	15.00	24	6.10	24	6.10	15	24
Total arginine	1.25	1.25	1.25	1.25	1.25	1.25	1.25	1.25	1.25	1.25	1.25	1.25	1.25	1.25	1.25	1.25	1.25
Total isoleucine	0.98	0.98	0.98	0.98	0.98	0.98	0.98	0.98	0.98	0.98	0.98	0.98	0.98	0.98	0.98	0.98	0.98
Total leucine	1.15	1.97	1.97	1.97	2.39	2.39	2.39	2.39	2.39	2.39	2.39	1.98	1.98	1.98	1.98	1.77	1.98
Total lysine	1.30	1.30	1.30	1.30	1.30	1.30	1.30	1.30	1.30	1.30	1.30	1.30	1.30	1.30	1.30	1.30	1.30
Total methionine + cysteine	0.90	0.91	0.91	0.91	0.91	0.91	0.91	0.91	0.91	0.91	0.91	0.91	0.91	0.91	0.91	0.91	0.91
Total methionine	0.65	0.58	0.58	0.58	0.54	0.54	0.54	0.54	0.54	0.54	0.54	0.57	0.57	0.57	0.57	0.57	0.58
Total threonine	1.04	1.04	1.04	1.04	1.04	1.04	1.04	1.04	1.04	1.04	1.04	1.04	1.04	1.04	1.04	1.04	1.04
Total tryptophan	0.15	0.17	0.17	0.17	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.23	0.23	0.23	0.23	0.25	0.17
Total threonine	0.74	0.73	0.73	0.73	0.84	0.84	0.84	0.84	0.84	0.84	0.84	0.71	0.71	0.71	0.71	0.66	0.73
Total valine	0.95	0.95	0.95	0.95	0.95	0.95	0.95	0.95	0.95	0.95	0.95	0.95	0.95	0.95	0.95	0.95	0.95
DEB (mEq/kg)3	240	240	240	240	240	240	240	240	240	240	240	240	240	240	240	240	240

¹Mineral premix provided per kilogram of diet: Mn (from MnSO₄·H₂O), 65 mg; Zn (from ZnO), 55 mg; Fe (from FeSO₄·7H₂O), 50 mg; Cu (from CuSO₄·5H₂O), 8 mg; I [from Ca (IO₃)₂·H₂O], 1.8 mg; Se, 0.30 mg; Co (from Co₂O₃), 0.20 mg; Mo, 0.16 mg.

²Vitamin premix provided per kilogram of diet: vitamin A (from vitamin A acetate), 11,500 U; cholecalciferol, 2,100 U; vitamin E (from dl-α-tocopheryl acetate), 22 U; vitamin B₁₂, 0.60 mg; riboflavin, 4.4 mg; nicotinamide, 40 mg; calcium pantothenate, 35 mg; menadione (from menadione dimethyl-pyrimidinol), 1.50 mg; folic acid, 0.80 mg; thiamine, 3 mg; pyridoxine, 10 mg; biotin, 1 mg; choline chloride, 560 mg; ethoxyquin, 125 mg.

³DEB: dietary electrolyte balance represents dietary Na + K − Cl in mEq/kg of diet.

Preparation of AFB1

AFB₁ was synthesized by the PTCC-5286 strain of Aspergillus parasiticus grown on the corn grain through anaerobic reactions under constant stirring and controlled temperature (Dashkevicz and Feighner, 1989). The concentration of AFB₁ in the samples was measured by an ELISA method (Ridascreen Aflatoxin B₁ Art. No. 1211, R-Biopharm, Darmstadt, Germany) and the contaminated corn was incorporated into the basal diet to provide the desired amounts of AFB₁ according to the CCD platform.

Humoral Immunity Responses

Four birds in each replicate were challenged with sheep red blood cell (SRBC) antigens at d 18 and 25 and after the second challenge, antibody production against SRBC-antigen was measured by hemagglutination inhibition test in the serum samples according to Cheema et al. (2003).

Malondialdehyde Measurement

Four birds of each replicate were randomly selected and sacrificed by cervical dislocation and deboned meat of thigh portions were grounded with a blender and stored at −20°C for 30 d to determine the oxidation stability in meat samples. As described previously (Mehri et al., 2015), 1 g of grounded meat sample was weighed and homogenized (Polytron homogenizer, PCU, Malters, Switzerland) with 4 mL of 5% aqueous trichloroacetic acid (TCA) and 2.5 mL of 0.8% butylated hydroxytoluene, and then centrifuged at 3,000 × g for 3 min. The top hexane layer was discarded, and the bottom layer was filtered and made to 5 mL volume with 5% TCA and then added to a screw-capped tube containing 3 mL of 0.8% aqueous 2-thiobarbituric acid (TBA). Finally, tubes were placed in a 70°C water bath for 30 min, then immediately cooled under 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 the calculation of the MDA concentration in the samples. Tetraethoxypropane (1, 1, 3, 3-tetraethoxy propane, T9889, 97%, Sigma, St. Louis, Missouri) was used as the MDA precursor in the standard curve. The concentration of MDA was expressed as milligrams per kilogram of meat sample.

Bacterial Populations of Ileal Content

The ileal contents of 4 birds in each replicate were separately collected into the sterile tubes for serial dilution as described by Ghazaghi et al. (2014). In brief, 1 g of ileal digesta was added into the test tube containing 9 mL of sterilized phosphate-buffered saline, and buffered solutions were transferred to the microbial laboratory. Microbial populations were determined by serial dilution (10⁻⁴–10⁻⁶) of ileal samples before inoculation onto Petri dishes. Plates for lactic acid bacteria (LAB; grown in deMan, Rogosa and Sharpe, MRS agar) colonies were counted between 24 and 48 h after inoculation. Colony-forming units were defined as distinct colonies measuring at least 1 mm in diameter.

Design of Experiment

In this experiment, a 4-factor, 5-level CCD was used. The CCD is one of the most commonly used response surface designs for fitting second-order models. The CCD consists of F factorial points, 2 × k star points, which are located at a distance [±α] equal to 1.682 from the center point, and nc (number of center points). The factorial points are used to fit linear and interaction terms. The star points provide additional levels of the factor which are useful for the estimation of the quadratic term (Montgomery, 2008). According to the CCD platform, the levels of independent factors were as follows:

Tryptophan (%): 0.15, 0.17, 0.20, 0.23, 0.25

AFB₁ (ppm): 0.00, 0.40, 1.00, 1.60, 2.00

Melatonin (mg/kg): 0.00, 6.10, 15.0, 24.0, 30.0

N,N-dimethylglycine (mg/kg): 0.00, 122, 300, 478.4, 600

Statistical Analysis

The pen was used as the experimental unit in this study. The linear and quadratic terms of the independent variables as well as their interactions were evaluated. A polynomial regression equation was generated in the following form:

$$y=\beta 0+\sum _{i=1}^k{\beta }_0{x}_i+\sum \sum {\beta }_{ij}{x}_i{x}_j+\sum _{i=1}^k{\beta }_{ii}{x}_i^2$$

where y is response of interest, β₀ is the intercept, and β_i, β_ii, and β_ij are the coefficients estimated by the model. All data were analyzed by Design-Expert (Stat-Ease Inc., Minneapolis, MN, Version 12) to estimate the optimal levels of independent variables using desirability function (Mehri, 2014). In some cases, nonsignificant variables are included to the models to support the hierarchy of the models.

RESULTS

G_7–14: The linear effects of Trp and DMG and the interaction of Trp × AFB₁, Trp × MEL, and AFB₁ × DMG along with the quadratic effects of MEL were significant on the G_7–14. The quadratic effect of Trp also showed a decreasing trend on G_7–14 (P = 0.06). Increasing AFB₁ decreased G_7–14 at higher dietary levels of Trp while the maximum gain was achieved at the highest level of dietary Trp (Figure 1A). The interaction effect of Trp × MEL was significant on G_7–14, where the maximum gain was achieved at highest levels of Trp and MEL. In fact, dietary MEL showed a synergistic effect with Trp to increase the G during the second week of age (Figure 1B). Dietary DMG also resulted in synergistic effect with Trp on quail gain, where the maximum G_7–14 was obtained at the highest levels of Trp and DMG (Figure 1C). Increasing DMG in toxin-free diets decreased quail growth while decreasing effects of AFB₁ on quail G_7–14 was attenuated with increasing DMG (Figure 1D).

Figure 1.

Gain_7–14 = 32.72 + 6.45*A−0.10*B + 0.65*C − 4.15*D − 10.86*AB + 5.66*AC + 4.44*AD + 8.26*BD − 3.53*A² − 4.41*C². A: Tryptophan; B: AFB₁; C: Melatonin; D: N,N-dimethylglycine; Significant parameters are bold (P < 0.05). Underlined parameter showed trend (P = 0.06).

G_7–21: The linear effect of Trp was only significant on G_7–21 while the quadratic effect of Trp (P = 0.08), and cross product of Trp × DMG (P = 0.06), and AFB₁ × MEL (P = 0.11) showed nonsignificant trend on G_7–21. Increasing dietary DMG in low-Trp diets decreased the growth whereas the increasing DMG at higher levels of dietary Trp increased the quail growth and maximum G_7–21 was achieved at maximum levels of Trp and DMG (Figure 2A). Although the simple effect of MEL was not significant on G_7–21, the increasing levels of MEL in AFB₁-contaminated diets removed the negative impact of AFB₁ on growth (Figure 2B).

Figure 2.

Gain_7–21 = 74.34 + 8.94*A+1.75*B + 2.62*C − 1.70*D +12.48*AD + 6.80*BC − 5.93*A². A: Tryptophan; B: AFB₁; C: Melatonin; D: N,N-dimethylglycine; Significant parameters are bold (P < 0.05); Underlined parameters showed trend (P = 0.06, 0.11, 0.08, respectively).

G_7–28: None of independent variables have significant effects on G_7–28, while the cross product of Trp × MEL (P = 0.06) and Trp × DMG (P = 0.07) showed decreasing and increasing trend on G_7–28, respectively. Increasing dietary MEL in low-Trp diet showed an increasing trend on quail growth and the interactive effect of Trp × MEL revealed a nonsignificant trend on G_7–28 (Figure 3A). Increasing dietary DMG in low-Trp diet showed a decreasing trend on G_7–28 but the maximum growth rate during 7 to 28 d of age was achieved at maximum levels of Trp and DMG, indicating a positive synergism on G_7–28 (Figure 3B).

Figure 3.

Gain_7–28=106.98+7.03*A + 0.45*B + 3.51*C +  2.27*D −9.36*AC + 14.09*AD. A: Tryptophan; B: AFB₁; C: Melatonin; D: N,N-dimethylglycine; Significant parameters are bold (P < 0.05); Underlined parameters showed trend (P = 0.13, 0.06, 0.07, respectively).

G_7–32: The linear effects of Trp and AFB₁ were significant on G_7–32. As shown in Figure 4, the overall assessment of predictor variables showed that the significant variables on G_7–32 were dietary Trp and AFB₁ and the remaining variables did not impact the gain of growing quails (Figure 4).

Figure 4.

Gain_7–32 = 124.96 + 5.95*A − 5.38*B. A: Tryptophan; B: AFB₁; Significant parameters are bold (P < 0.05).

F_7–14: The linear effects of Trp and MEL, quadratic effect of AFB₁, and cross product of AFB₁ × DMG were significant on feed intake during the second week of age. At the highest levels of AFB₁ and DMG, the feed intake was maximized (95.3 g/b) while in toxin-free diets, increasing DMG decreased the feed intake. Inclusion of DMG in toxin-free diets decreased feed intake during the second week of age while in AFB₁-contaminated diets increasing DMG increased feed intake linearly, where the maximum feed intake was achieved in quail fed on AFB₁-diet supplemented with 600 mg/kg DMG. The cross product of AFB₁ × DMG was significant in polynomial equation of feed intake during the second week of age (Figure 5A). The quadratic effect of AFB₁ was significant on feed intake in quail chicks from 7 to 14 d of age, while dietary Trp linearly increased feed intake. At all level of AFB₁, the maximum feed intake was obtained at the top level of dietary Trp. In fact, increasing dietary Trp removed the adverse effect of AFB₁ on feed intake during the second week of age (Figure 5B). The linear effects of Trp and MEL were significant on feed intake and the maximum feed intake was achieved at maximum levels of Trp and MEL, suggesting the synergistic effect of MEL and Trp to increase feed intake in quail chicks during the second week of age (Figure 5C).

Figure 5.

F_7–14 = 77.83 + 9.46*A − 0.88*B + 3.64*C + 0.17*D +13.04*BD + 6.19*B². A: Tryptophan; B: AFB₁; C: Melatonin; D: N,N-dimethylglycine; Significant parameters are bold (P < 0.05).

F_7–21: The linear effects of Trp and MEL, the quadratic effects of AFB₁ along with the cross product of AFB₁ × DMG and MEL × DMG were significant on feed intake from 7 to 21 d of age. However, the interactive effect of Trp × DMG showed an increasing trend (P = 0.06). Dietary Trp linearly increased feed intake in quail from 7 to 21 d of age. Although the linear effect of DMG was nonsignificant, the interaction of Trp × DMG revealed a nonsignificant trend (P = 0.06) on feed intake and the maximum feed intake was achieved at top levels of Trp and DMG in a synergistic manner (Figure 6A). Dietary AFB₁ quadratically decreased feed intake while supplementation of DMG interactively (P < 0.05) increased feed intake in quail received AFB₁ and the maximum feed intake was obtained in quail chicks fed on the AFB₁-contaminated diet with the highest level of DMG (Figure 6B). Dietary MEL linearly increased feed intake but increasing levels of DMG interactively decreased feed consumption in quails fed on the diet containing the highest level of MEL. In this case, maximum feed intake was observed in quails received the highest level of MEL without DMG (Figure 6C).

Figure 6.

F_7–21=195.59 + 20.64*A + 2.91*B + 9.99*C − 2.53*D +20.03*AD + 20.85*BD − 14.47*CD + 11.0*B². A: Tryptophan; B: AFB₁; C: Melatonin; D: N,N-dimethylglycine; Significant parameters are bold (P < 0.05); Underlined parameter showed trend (P = 0.06).

F_7–28 and F_7–32: The linear effects of Trp and MEL, the quadratic effect of Trp and cross product of Trp × DMG were significant on feed intake from 7 to 28 and 7 to 32 d of age, where the maximum feed intake was observed at the top levels of DMG and Trp. In addition, the quadratic effect of Trp was had the highest impact on feed intake, followed by the interaction effect of Trp × DMG (Figure 7A). The same pattern was also observed for feed intake from 7 to 32 d of age, except the most impact on feed intake was attributed to the interaction of Trp × DMG followed by the quadratic effect of Trp (Figure 7B).

Figure 7.

(A) F_7–28 = 350.30 + 19.93*A + 16.91*C − 3.38*D +22.78*AD − 25.08*A². (B) F_7–32 = 410.86 + 18.04*A + 17.26*C −4.15*D+27.21*AD − 19.36*A². A: Tryptophan; C: Melatonin; D: N,N-dimethylglycine; Significant parameters are bold (P < 0.05).

FCR_7–14: The interaction effects of Trp × AFB₁ and Trp × MEL were significant on FCR during the second weeks of age, where the minimum FCR values were achieved by either the highest dietary Trp without AFB₁ or the highest levels of Trp and MEL. The linear effects of dietary Trp and DMG, and the quadratic effect of MEL showed the nonsignificant trend on FCR_7–14 (Figure 8).

Figure 8.

FCR_7–14=2.62−0.177*A+0.081*B + 0.053*C + 0.283*D +0.832*AB − 0.585*AC + 0.267*C². A: Tryptophan; B: AFB₁; C: Melatonin; D: N,N-dimethylglycine; Significant parameters are bold (P < 0.05); Underlined parameters showed trend (P = 0.07, 0.07, 0.11, respectively).

FCR_7–21: Only the linear effect of Trp was significant on FCR from 7 to 21 d of age and the cross product of AFB₁ × MEL showed a nonsignificant trend on this response (P = 0.09), where the minimum FCR_7–21 was achieved in quails fed on toxin-free diet without MEL. Although FCR_7–21 was impaired by dietary AFB₁, the addition of MEL into the diet removed the detrimental effects of AFB₁ on FCR (Figure 9).

Figure 9.

FCR_7–21 = 2.77 − 0.162*A + 0.045*B + 0.028*C −0.225*BC. A: Tryptophan; B: AFB₁; C: Melatonin; Significant parameter is bold (P < 0.05); Underlined parameter showed trend (P = 0.09).

FCR_{7–28 and 7–32}: None of predictive variables were significant on FCR from 7 to 28 and 7 to 32 d of age, however, the interactive effects of Trp × MEL showed a nonsignificant trend.

BMY and TMY: The linear effect of Trp and the cross product of AFB₁ × DMG were significant on BMY. The increasing AFB₁ decreased BMY in quails fed on a diet without DMG while the inclusion of DMG in AFB₁-contaminated diet increased BMY linearly (Figure 10A). The interaction of MEL × DMG was only significant on TMY and the quadratic effect of DMG (P = 0.06) and the cross product of AFB₁ × MEL (P = 0.06) showed nonsignificant trend on TMY. AFB₁ decreased TMY in quails fed a diet without MEL but the inclusion of MEL (P = 0.11) into the AFB₁-contaminated diet increased TMY and partly removed the negative effect of AFB₁ (Figure 10B). Although the simple effects of either MEL or DMG were not significant on TMY, the concurrent use of MEL and DMG significantly decreased TMY. The quadratic effect of DMG showed a decreasing trend (P = 0.08) on TMY (Figure 10C).

Figure 10.

(A) BMY = 49.8 + 6.18*A + 0.148*B − 0.876*D +15.8*BD. (B, C) TMY = 27.7 + 1.48*A − 0.428*B − 0.531*C +0.233*D + 3.41*BC − 4.29*CD + 3.04*D². A: Tryptophan; B: AFB₁; C: Melatonin, D: N,N-dimethylglycine; Significant parameters are bold (P < 0.05); Underlined parameter showed trend (P = 0.11, 0.08, respectively).

LAB:Figure 11 shows that the only significant variable on LAB population was dietary Trp and increasing dietary Trp linearly increased the LAB community. However, the interaction of AFB₁ × DMG resulted in nonsignificant trend on LAB, so as to increasing dietary DMG in toxicated birds removed the negative impact of AFB₁.

Figure 11.

LAB = 9.20 + 0.64*A + 0.27*B + 0.16*D + 1.26*BD. LAB: Lactobacilli; A: Tryptophan; AFB₁: Melatonin; D: N,N-dimethylglycine; Significant parameter is bold (P < 0.05); Underlined parameter showed trend (P = 0.13).

SRBC and MDA: Feeding AFB₁ decreased SRBC-antigen production in quail chick but increasing dietary Trp increased the immunity response in toxicated birds. Figure 12A implied that increasing dietary Trp in normal feeding condition had no effect on SRBC-antigen production. Increasing dietary Trp linearly decreased the MDA production. At low levels of Trp, inclusion of DMG into the diet increased the MDA production while the increasing dietary Trp reversed that negative situation (Figure 12B).

Figure 12.

(A) SRBC=8.62−0.02*A+0.93*B+0.36*C−0.75*D −  2.71*AB. (B): MDA = 0.83 − 0.22*A + 0.12*B + 0.05*C + 0.02*D− 0.69*AD − 0.26*CD. A: Tryptophan; B: AFB₁; C: Melatonin; D: N,N-dimethylglycine; Significant parameters are bold (P < 0.05); Underlined parameter showed trend (P = 0.13).

DISCUSSION

In order to understand the possible mechanisms of the feed additives to combat aflatoxicosis, we should know that the AFB₁ found in the contaminated feeds is not toxic and until it the passes 2 metabolic pathways including phase I and phase II in the body then, it becomes toxic for living organisms. Phase I involves the rendering inactive molecule of AFB₁ to the active toxin, AFBO-exo-8,9-epoxide, by the addition of a polar group via epoxidation process. Phase II involves the conjugation of the product of phase I by the addition of endogenous glutathione (GSH) to the AFBO-exo-8,9-epoxide, producing a water-soluble product that can be excreted out by the kidney through a detoxification process (Mughal et al., 2017).

The effects of DMG and MEL supplements on quail performance were age-dependent and the positive consequences of these additives were observed in the early life of quail chicks. These supplements also interacted with AFB₁ during the second and third weeks of age when the younger chicks are more vulnerable to AFB₁ than older quails. Apparently, 2 mechanisms might involve be the beneficial effects of DMG on performance of the young chicks. First, the molecule of DMG is a methyl donor. Sulfur-amino acids, methionine and cysteine, are not only the constitutes of GSH, but also increase the concentration of hepatic GSH (Wang et al., 1997). GSH is involved in the conjugation of active aflatoxin in the body through the phase II of the AFB₁ metabolism. Therefore, the use of methionine for the synthesis of GSH may deplete the pool of liable methyl group and the addition of DMG may compensate for the partial deficiency of the labile methyl group in the toxicity condition. Second, DMG can be a source of glycine through transmethylation process. Glycine (Gly) is usually considered a nonessential amino acid but its importance in the interconversion to serine (Ser) and subsequently to cysteine led to be considered as a semiessential or essential amino acid in poultry nutrition (Siegert and Rodehutscord, 2019). More recently, it was shown that Gly supplementation could alleviate heat stress-induced dysfunction of antioxidant status and intestinal barrier in broilers. The injured tissue of the intestinal border caused by AFB₁ could also be repaired by Gly derived from the DMG.

In the present study, it was shown that increasing dietary Trp significantly decreased the peroxidation process which was indicated by reducing MDA production in meat samples. This result was in agreement with Khanipour et al. (2019) who speculated that Trp-derivatives and involved enzymes in its metabolism possess antioxidant properties. Although DMG reacted as an antioxidant reagent, inclusion of DMG in low-Trp diets increased MDA production. When Trp supply is not sufficient for metabolic needs, the activity of IDO could be increased and the concentration of kynurenine will be elevated. On the other hand, the activity of TDO will be impaired and Trp-derivatives such as 3-hydroxykynurenine will be declined. Kynurenine is an oxidant agent and a set of high kynurenine and low concentration of 3-hydroxykynurenine could attenuate the peroxidation reaction. In addition, the deficiency of Trp will decrease the cellular synthesis of GSH. In contrast, the addition of DMG in high-Trp diets resulted in synergistic effect on oxidative stability of the cell by increasing the synthesis of SOD and GPx. Dietary DMG also reversed the negative impact of AFB₁ on the feed intake in young chicks, possibly by increasing GSH production and improving the conjugation process of AFBO-exo-8,9-epoxide in phase II. The same effect was also observed for the LAB population and BMY, which the negative effect of AFB₁ on the LAB population in small intestine and BMY was removed by the addition of DMG.

Tryptophan is a precursor of the MEL. Melatonin also has the antioxidant properties that stimulate humoral system by increasing T and B lymphocytes. It also scavenge the ROS at cellular level (Calislar et al., 2018). In some cases, MEL could remove the negative effects of AFB₁ on the growth rate and FCR during the first 2 wk of age. Interestingly, among the studied traits, DMG and MEL had no effects on SRBC-antigen production. AFB₁ directly decreased SRBC while Trp increased the blood levels of SRBC-antigen. Since AFB₁ directly affects liver and lymphoid organs, the impairment of the humoral system by aflatoxicosis could be expected. This effect was in agreement with Khanipour et al. (2019) who showed that SRBC-antigen production was declined by AFB₁ without interaction with Trp. In the present study, there was no interaction between AFB₁ and Trp but the simple effect of Trp resulted in elevation of SRBC-antigen production. In fact, the reduced production of antibodies in the body resulted from dysfunctionality of the immune organs such as thymus and bursa, which are involved in the production of T and B cells, respectively. It has been reported that impairment in the production of antibodies in toxin-treated birds caused by AFB₁ could be due to histopathological lesions in lymphoid organs (Figure 13) induced by ROS (Wang et al., 2013; Chen et al., 2014; Peng et al., 2014). In the present study, the improvement of the SRCB-antigen status in birds fed on high-Trp diets might be due to reduction in ROS by Trp-derivatives such as 5-hydroxytryptophan and melatonin or increase in the involved enzymes with antioxidant properties such as TDO (Khanipour et al., 2019). The later factor could be more possible than the former agents because we could not observe any significant effect for MEL, which is a Trp-derivative. The TDO is a rate-limiting enzyme that catalyze the oxygenation reaction of Trp. This reaction consume the superoxide anion as an oxidative cofactor, supporting the oxidation stability as an antioxidant agent (Dairam et al., 2006). This proposed mechanism for increasing dietary Trp as an antioxidant nutrient could be confirmed by decreasing MDA production in meat samples of the birds received high-Trp diets. MDA, as a marker of oxidative stress, is one of the final products of polyunsaturated fatty acids peroxidation in the cells, which could be increased by increasing ROS in the birds fed on AFB₁ (Bagherzadeh-Kasmani and Mehri, 2015; Mohammadi et al., 2015; Aftabi et al., 2016; Khanipour et al., 2019). In the present study, AFB₁ had no significant effect on the production of MDA in meat samples which was in contrast with our previous studies in our lab. Perhaps, dietary supplements of DMG and MEL exerted the permanent inhibitory effects on the microsomal cytochromes at early life so as to we did not observe any significant effect of AFB₁ on the MDA production in the meat samples of mature birds.

Figure 13.

Mode of action of aflatoxin B₁ on splenic macrophage cells to induce apoptosis.

One of the important outcomes of the present study was the interaction of DMG and MEL with AFB₁ and Trp, respectively. Overall, DMG mainly interacted with AFB₁ while MEL showed the interaction with dietary Trp in most cases. DMG is a precursor of GSH and the production of AFBO-exo-8,9-epoxide in microsomal cytochromes calls the peroxidation process, which could be inhibited by an assessable cellular antioxidant such as GSH derived from supplemental DMG. Trp is a precursor of MEL with antioxidant properties, but de novo synthesis of this hormone from Trp needs the sequential action of several enzymes and could not be considered as an assessable antioxidant. Therefore, MEL supplement could be delivered readily as an available melatonin source in the cell, decreasing the rate of conversion of Trp to MEL in the body and supporting the growth rate in the birds fed on low-Trp diets.

In conclusion, this study revealed that the beneficial effects of DMG and MEL supplements in toxin-treated growing quails could be disclosed during the first 3 wk of age and their effects will be disappeared from the 4 wk onward. These supplements could interact with AFB₁ plays the main role in the growth performance during the fifth and sixth week of age.