1. Introduction
Modern poultry breeds selected through genetic breeding have high genetic potential, productivity, and energy requirements [
1]. The high energy concentration of their diets requires high levels of lipids (fats and oils). Lipids (fats and oils) are sensitive to oxidation, and are highly susceptible to oxidative deterioration in the process of feed production, use, and storage [
2]. In poultry feed, the oxidative rancidity of lipids (oil or polyunsaturated fatty acids, etc.) significantly affects the quality of feed and the safety of poultry products [
3].
A large number of studies have found that the addition of antioxidant substances to feed has an important role in improving the oxidative stability of feed, as well as the activity of antioxidant enzymes in the bodies of livestock and poultry, extending the shelf-life of animal products [
4,
5]. Antioxidants, including synthetic antioxidants (for example phenolic antioxidants) and natural antioxidants (for example tocopherols) [
6], block the harmful effects of oxygen production and prevent free radicals from damaging the body by trapping and neutralizing them, The most commonly used synthetic antioxidants in food or in feed are phenolic antioxidants [
6,
7], such as butylated hydroxyanisole (
BHA), butylated hydroxytoluene (
BHT), tertiary butylhydroquinone (
TBHQ) and propyl gallate (
PG), which can be used for the protection of dietary fats and oils.
Antioxidant TBHQ is a highly effective antioxidant for feeds, and can prevent oxidative spoilage caused by air oxidation; protect fats, oils, fat-soluble ingredients and other natural components of feeds; and delay the adverse changes caused by the oxidation of foods [
8,
9]. Numerous studies have shown that the antioxidant TBHQ is essential as a feed preservative in the production of livestock and poultry feeds and can effectively protect feeds against oxidative deterioration [
10,
11]. In vivo, TBHQ can regulate the expression of antioxidant protein genes by activating proteins; it is often used in medicine as an activator of the nuclear transcription factor E2-related factor-2 [
12,
13]. However, antioxidant TBHQ is ingested into the bodies of poultry along with the feed consumed, and its effects on the bodies of poultry have not been adequately studied and evaluated.
In recent years, attention has turned to natural antioxidants due to concerns regarding the safety of synthetic antioxidants. Tea polyphenols (
TP) are a natural antioxidant that can prevent and treat diseases by scavenging free radicals and regulating the activity of different types of oxidases in the body [
14]. After entering the animal body, the mechanism by which TP produces antioxidant effects includes the following processes: the increase in activity of antioxidant enzymes; the inhibition of lipid peroxidation; the scavenging of free radicals [
15]; and a reduction in oxidation via chelation of metal ions [
16]. Complex antioxidants formulated with both synthetic and natural antioxidants, with TBHQ serving as a highly effective feed antioxidant, and TP serving as an important antioxidant for the bodies of poultry, have aroused the interest of this research team. Therefore, the objective of the present study was to evaluate the effects of diets supplemented with an antioxidant complex containing TP and TBHQ on growth performance, meat quality, including regular quality and the chemical composition of breast and leg muscles, serum biochemistry, and the antioxidant capacity of serum, liver, breast meat, jejunum, and ileum, in broilers.
2. Materials and Methods
The Institutional Animal Care and Use Committee (IACUC) of Sichuan Agricultural University approved all procedures used in the study.
2.1. Birds, Experimental Design, Diet, and Management
A total of 600 one-day-old male Arbor Acres broiler chickens with a similar initial body weight were randomly distributed into 3 experimental groups with 10 replicates of 20 birds each using a completely randomized design. The birds were fed a basal diet supplemented with 0, 300, and 500 mg/kg complex antioxidants (
CA), respectively. The trial lasted 42 d, divided into starter (1–21 d) and grower (22–42 d) phases. The main components of the complex antioxidants were ~40%TBHQ, ~20% TP, and ~20% sodium citrate (
SC), and were provided by MuDan Biochech (Xiamen, China). The composition and nutrient levels of the basal diet are shown in
Table 1. The diets were offered in pelleted form. The experimental room temperature and light regimen were maintained at 32 °C to 34 °C and a “23 h on to 1 h off” routine for the first 3 d, gradually decreased to 22 °C at the rate of 2 °C to 3 °C per week, or under “20 h on to 4 h off” for the remainder of the feeding period, respectively. All broilers were housed in cages (2 m length × 0.8 m width, 20 birds/cage) and had free access to water and feed.
2.2. Data and Sample Collection
At 21 and 42 days of age, after 12 h of feed withdrawal, birds were weighed, and the feed intake was obtained by cage. Body weight (BW), average daily gain (ADG), average daily feed intake (ADFI), and feed-to-gain ratio (F:G) were calculated for the periods of 1–21 d, 22–42 d, and 1–42 d of age.
On d 42, one bird from each replicate (n = 10) with body weights close to the replicate mean were selected and sampled. The blood samples were collected via jugular vein puncturing and placed into tubes without heparin sodium (an anticoagulant). Then, serum was isolated and stored at −20 °C for serum biochemical determination. The birds from which blood was collected were euthanized through exsanguination. Then, liver, pectoral muscle, jejunum, and ileum were collected and stored at −20 °C for antioxidative capacity assays. Some pectorals and leg muscles were then excised and subsequent meat quality parameters were determined, including the pH value, meat color, drip loss, and cooking loss at 45 min, 24 h, and 48 h. Other pectorals and leg muscles were excised and stored at −20 °C for nutrient composition determination.
2.3. Serum Biochemical Indices
The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as well as the content of serum albumin (ALB), total protein (TP), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), triglyceride (TG), and uric acid (UA), were analyzed using an automatic biochemical analyzer (HATICHI 7180, Tokyo, Japan).
2.4. Determination of Meat Quality
The meat color and pH of the pectoral and leg muscles were determined at 45 min, 24 h and 48 h post mortem, using the method described by [
17,
18,
19]. Briefly, the pH and color parameters, including lightness (L*), redness (a*), and yellowness (b*), were measured at the thickest part of pectoral and leg muscles at 45 min post mortem, and after 24 and 48 h of placement at 4 °C, as previously described. Drip loss was estimated by suspension method: the pectoral and leg muscles were taken and cut into cube-sized (2 cm
3) meat samples; the meat samples were suspended in self-sealing bags with wire and stored at 4 °C for 24 h. The difference between the initial and final weight of the meat sample as a percentage of the initial weight is the drip loss, according to [
20]. Cooking loss was determined by the method described by [
21].
2.5. Antioxidant Capacity Assays
Tissue samples from liver, pectoral muscle, jejunum, and ileum were prepared as 10% tissue homogenates (1:9), according to the experimental instructions and stored at −20 °C for the determination of antioxidant indices. Total antioxidant capacity (T-AOC, A015-1-2), malondialdehyde (MDA, A003-1-1), total superoxide dismutase (T-SOD, A001-1-1), catalase (enzyme) (CAT, A007-1-1), glutathione-s-transferase (GST, A004-1-1), and hydrogen peroxide (H
2O
2) activities were measured in serum and tissue using an enzyme labeling instrument (Tecan Infinite 200 Pro, Männedorf, Switzerland) according to [
22]. These kits were purchased from Nanjing Jian Cheng Bioengineering Institute, and all operations were performed according to the kit instructions.
2.6. Statistical Analysis
The data were analyzed by one-way ANOVA using SPSS software (Version 26.0 for windows; SPSS Inc., Chicago, IL, USA). Each replicate was considered an experimental unit. Differences among treatments were detected using Duncan’s multiple range tests. Probability values ≤ 0.05 were considered significant, while values of 0.05 < p < 0.1 were considered a tendency. The data are expressed as the mean ± standard deviation (SD).
4. Discussion
The present study comprehensively evaluated the effectiveness of CA containing TBHQ and TP on broilers based on growth performance, serum biochemical parameters, and meat quality, including the regular quality and chemical composition of breast and leg muscle, and the antioxidant capacity of serum, liver, breast meat, jejunum, and ileum. We found that broilers fed a diet only supplemented 300 mg/kg CA presented a better growth performance, meat quality, and body antioxidant ability than broilers fed the control diet. The results suggest that the CA containing TBHQ and TP is a good antioxidant product for broilers; however, there is limited information about the effect of TBHQ on growth performance and meat quality in animals or poultry. TBHQ is the active metabolite produced by BHA through a demethylation reaction [
23]. In vivo studies have shown that BHA is mainly metabolized to TBHQ in humans, dogs, rats and mouse, and this process is mediated by cytochrome P450 [
24]. Imbabi et al. [
25] found that supplementing growing rabbits with 50–100 mg/kg BHA may promote growth performance and meat quality via maintaining the redox balance.
In addition, TP are natural antioxidants typical in flavonoids. Studies have shown that broilers fed diets containing 0.5% to 2% tea polyphenols can improve growth performance, muscle antioxidant capacity and meat quality, and the effect is better than diets supplemented with 0.1% oxytetracycline calcium [
26]. Jelveh et al. [
27] showed that feeding green tea extract and green tea phytosomes compensated the negative effects of
Eimeria infection on growth performance in broilers. Chen et al. [
28] observed that supplementing 1% green tea powder to the diet could improve the quality of breast meat. Water-holding capacity (WHC) is considered as a key meat quality characteristic of chickens, which is mainly expressed through dripping loss and cooking loss [
29]. Consistent with previous findings, we observed that the addition of 300 mg/kg CA to the diet led to a reduction in both dripping loss and cooking loss of the muscle and resulted in a decrease in post-mortem yellowness of the muscle, along with an increase in the postmortem pH value. The postmortem pH value of the muscle increased, indicating improved quality. This may be due to the strong antioxidant activity of the CA. Indeed, Xi et al. [
10] found that TBHQ played an important role in attenuating PM2.5-induced pyroptosis and necroptosis in chicken primary alveolar epithelial cells by inhibiting the generation of reactive oxygen species (
ROS). Liu et al. [
30] also found that TBHQ attenuates podocyte injury in diabetic nephropathy by inhibiting NADPH oxidase-derived ROS generation via the nuclear factor E2-related factor-2 /hemeoxygenase-1 signaling pathway. Studies have shown that TBHQ can disrupt the metabolic processes of exogenous toxic compounds and reduce the synthesis of toxic metabolites in the body, thereby reducing oxidative stress damage [
9]. Yan et al. [
14] conducted a review about antioxidant mechanism of TP and its impact on health benefits. In vivo experiments shoed that TP can increase levels of rat serum catalase, glutathione peroxidase and SOD, and can reduce the production of MDA as well as the content of TC in serum. Consistent with the previous studies, the results of our study demonstrated that dietary supplementation of CA increased the activities of antioxidant enzymes in the serum, liver and jejunum of broilers and decreased the liver MDA level as well as serum TC content. Therefore, we suggest that the CA containing TBHQ and TP improved the growth performance and meat quality by increasing the antioxidant capacity of broilers.
It is worth noting that broilers fed a diet with 500 mg/kg CA did not present further improvement in growth performance and meat quality in comparison to diets with 300 mg/kg CA. Meanwhile, our study observed a significant increase in serum AST and UA levels in the 500 mg/kg dose group compared to the control group. Serum biochemical indicators are frequently used to reflect the changes in material metabolism and tissues and organs function in broilers [
31]. When the body’s liver is damaged or has metabolic disorders, ALT and AST enter the blood circulation from liver cells. Therefore, a high AST level in the serum could be an important indicator of liver damage [
32]. These findings suggest that the high dose of CA may have a detrimental effect on the liver of broiler chickens on d 42. Furthermore, AST’s involvement in protein or amino acid metabolism and the urea cycle could explain the high uric acid levels observed in 42 d birds with the same 500 mg/kg CA dose [
33]. Synthetic phenolic antioxidants are widely used in feed; considering their potential risks, it is very important to monitor the content of these in feed. The European Food Safety Authority [
34] established that BHA and TBHQ, individually or in combination, in the same foods, can be used at a maximum permitted level of 200 mg/kg. Limits to the quantity of BHT, BHA and TBHQ, individually, or in combination in the same feed, are all 150 mg/kg in feed according to Chinese Feed Hygiene Standards. However, TBHQ alone, or in combination only with BHT and/or BHA, can be used with a maximum 200 mg/kg by weight of the fat and oil content and not by the weight of the commercial complete feed, according to the Code of Federal Regulations [
35]. Therefore, attention should be paid to the appropriate supplemented amount of TBHQ in broiler commercial feed.