1. Introduction
Poultry production is one of the most quickly developing protein sources worldwide, which has become very important to worldwide food sources. Recent poultry feeds are manufactured to satisfy their nutritional needs. Still, more research is required to determine whether these feeds sufficiently maintain the birds’ immunity system or if additional components are needed to enhance their immune function [
1]. Furthermore, the growth performance improvements in broiler chickens are associated with intestinal health [
2,
3,
4,
5]. Using antibiotics as growth promoters are prohibited because of the rise of multiple and cross-resistance antibiotics used in treating human and animal infections [
6]. Earlier studies demonstrated that phytobiotics with antiviral, antimicrobial, fungicidal, anticoccidial, and antioxidant properties could be used as antibiotic alternatives in poultry diets [
7,
8].
Hibiscus sabdariffa L. (Roselle) is traditionally used in herbal beverages as a food, in the food industry as a flavoring agent, and as an herbal medication. Its extracts exhibited antioxidant, antibacterial, hepato- and nephroprotective, diuretic effect, hypolipidemic, antihypertensive, antidiabetic effects. These effects may be related to intense antioxidant activities, inhibition of α-glucosidase and α-amylase, direct vasorelaxant effect, inhibition of angiotensin-converting enzymes (ACE), and modulation of calcium channel. Roselle has an excellent record of safety and tolerability [
9]. The pharmacological actions of roselle are related to its component such as flavonoids, anthocyanins, organic acids, and polysaccharides [
10]. Roselle extracts contain a high proportion of organic acids as hibiscus acid, hydroxycitric acid, citric acid, tartaric and malic acids as leading composites, and ascorbic and oxalic acid as small compounds [
11].
Anthocyanins are glycosidic forms of anthocyanidins that give fruits and flowers bright and attractive colors, and they form a subgroup of flavonoids [
12]. Anthocyanins have several properties in humans and animals, such as anti-inflammatory, antimicrobial, antioxidant activities. They prevent oxidative liver damage and hyperglycemia, prevent cardiovascular diseases and diabetes, improve vision and obesity control [
13,
14,
15]. Moreover, anthocyanins prevent atherosclerosis, reduce free radical activity, and decrease inflammation and aging [
16]. Therefore, anthocyanin can be used as a suitable feed additive. Many studies demonstrated the anthocyanin effects on different animal species [
17,
18,
19,
20]. Dietary anthocyanins may enhance intestinal barrier function, improve host/bacteria bonding, and boost the multiplication of beneficial bacteria such as
Lactobacillus spp. and
Bifidobacterium spp. [
21]. These bacteria benefit host health through antimicrobial effects on pathogenic microorganisms [
22,
23].
The only edible supplies of anthocyanins are fruits and vegetables [
24,
25]. The anthocyanins levels in fruits are considerably greater than in vegetables [
26]. The highest anthocyanin content was recorded in some berries such as blueberry, huckleberry, chokeberry, and cranberry, while the lowest anthocyanin content was recorded for grapefruit, date, and figs. The richest vegetables in anthocyanins and anthocyanidins are purple potato, purple cabbage, and red cabbage [
27]. However, the total anthocyanin content in fruits and vegetables differs greatly between different genera and cultivars and is greatly influenced by temperature, light, and agronomic factors [
28].
Anthocyanins are currently used as feed constituents due to their potential antioxidant and immunostimulant activities. So far, their impacts on poultry are less known [
29,
30]. Hence, this study investigated the possible effects of anthocyanin-rich roselle extract in broiler chicken diets on the growth, intestinal morphology, blood biochemical parameters, fatty acid composition of breast muscles, antioxidant activity, and immune status.
2. Materials and Methods
2.1. Preparation and Description of the Anthocyanin-Rich Roselle Extract
2.1.1. Anthocyanin-Rich Roselle Extract Preparation
Dehydrated roselle calyces were obtained from the local market, Zagazig City, Sharqia Governorate, Egypt. Dehydrated roselle calyces were ground into a powder with milling; then, the ethanolic extract was prepared according to Zhao et al. [
31]. Then, 10 g of flour was blended with 100 mL acidified ethanol (70%) solution (0.1 M HCl (
v/
v)). These combinations were agitated at 200 rpm for 24 h at room temperature in the dark before being filtered using Whatman No. 42 filter paper (Whatman
® quantitative filter paper, ashless, Grade 42, Merck KGaA, Darmstadt, Germany). Ethanol was separated from the extract using vacuum in a BüCHI B-480 water bath evaporator (Marshall Scientific, Cambridge, MA, USA) at 45°C, then lyophilized in a freeze drier (Thermo-electron Corporation–Heto power dry LL 300 Freeze dryer).
2.1.2. Determination of Bioactive Compounds in Anthocyanin-Rich Roselle Extract
Total anthocyanin concentration was estimated using pH differential protocol [
32] as described in Wu et al. [
33]. The absorbance (
Ab) was calculated by Equation (1).
Total anthocyanin concentration was calculated from the Equation (2), and the results were expressed as mg of cyanidin 3-glucoside/100 g.
where
Ab is absorbance, e is the molar extinction coefficient of cyanidin 3-glucoside (26,900),
L is the cell length (1 cm),
MW is anthocyanins molecular weight (449.2),
D is dilution factor,
V is the final volume (mL), and
G is the dry weight (DW) of roselle flour (mg).
The Folin–Ciocalteu test was used to assess total phenolic content (TPC). The standard curve was made with gallic acid. TPC was measured in mg of gallic acid equivalent (GAE)/100 gm of dry material [
34]. The calibration equation for gallic acid (Equation (3)) was:
where
y and
x are the gallic acid absorbance and concentration in µg/mL, respectively.
The total flavonoids (TFs) were measured according to the procedure outlined before [
35]. The standard curve was created using quercetin, with total flavonoid concentration expressed as quercetin equivalent (QE). Total flavonoids contents were stated as quercetin equivalent (QE), which was calculated based on the calibration curve (Equation (4)).
where
is the absorbance and
is the concentration of quercetin in µg/mL.
2.1.3. Roselle Anthocyanin Estimation by HPLC
The high-performance liquid chromatography (HPLC) analysis was conducted according to Durst and Wrolstad [
36]. Freeze-dried extract in the amount of 10 mg was dissolved with 5 mL of methanol. The Sample was centrifuged at 5000×
g for 5 min, and the supernatant was collected and filtered through a Millipore membrane (0.45 µm). The filtrate was twice diluted with purified distilled water. The analyses were performed on an HPLC (Agilent, Santa Clara, CA, USA) model-LC 1100 series, equipped with a degasser, an autosampler automatic injector, a high-pressure pump, and a UV/Visible detector at multiple wavelengths wave. HPLC experiments were conducted using a reversed-phase C18 column (Prontosil, 250 × 4.0 mm, 5 µm, (Bischoff, Roseville, CA, USA). The mobile phase used was a binary gradient eluent (solvent A, 0.1% trifluoroacetic acid in water; solvent B, 0.1% trifluoroacetic acid in acetonitrile). Acetonitrile used was of HPLC grade (Sigma/Aldrich, Burlington, MA, USA) and was degassed in an ultrasonic bath before use. The water was distilled using a Milli-Q system (Millipore, Sigma/Aldrich, Burlington, MA, USA). The elution program was 5–20% B (0–5 min), 20–35% B (5–10 min), 35–100% B (10–25 min), and 100% B (25–40 min) with a flow rate of 0.8 mL·min
−1. The chromatograms were monitored at 521 nm.
Anthocyanin’s identification and peak assignments are based on their retention durations, UV–VIS spectra comparisons, and published data. The cyanidin 3-O-galactoside was used to measure anthocyanin levels.
2.2. Birds, Experimental Design, and Diets
This research was conducted in a poultry research unit in the faculty of veterinary medicine, Zagazig University, Egypt, to assess the effect of dietary supplementation of different levels of anthocyanin-rich roselle extract (Hibiscus sabdariffa L.) (ARRE) on growth performance, intestinal histomorphology, immune status, antioxidant activity, the fatty acid profile of breast muscles, and blood biochemical parameters of broiler chickens. All experiment procedures were approved by the Institutional Animal Care and Use Committee (ZU-IACUC) of Zagazig University, Egypt (Approval No. ZU-IACUC/2/F/17/2022).
In total, 250 1-day-old chicks (Ross 308 broiler) were obtained from a commercial chick producer. Before starting the experiment, birds were submitted to a 3-day adaptation period to reach an average body weight of 87.85 gm ± 0.32
. Then they were randomly allotted to five experimental groups with five replicates for each (10 chicks/replicate). Birds were fed on basal diets supplemented with five levels of ARRE: 0, 50, 100, 200, and 400 mg Kg
−1 for 35 days. The proximate chemical composition of the basal diet is shown in (
Table 1). The managerial conditions and the experimental diets were conducted following Ross 308 broiler nutrition specifications AVIAGEN [
37].
2.3. Growth Performance
The birds were individually weighed on the fourth day of age to obtain the average initial body weight; then, the body weight was recorded at 10, 23, and 35 days to calculate the average body weight of the birds in each group.
The body weight gain (BWG) was calculated by Equation (5).
where
is the final body weight at the intended period, and
is the initial body weight in the same period.
Feed intake (FI) of each replicate was recorded as the difference between the weight of the feed offered and residues left and then divided by the number of birds in each replicate to find out the average feed intake per bird.
The feed conversion ratio (FCR) was calculated by Equation (6).
The relative growth rate (RGR) was calculated using Equation (7) described by [
38].
where
= the initial live weight (g),
= the live weight at the end of the considered period (g).
Protein efficiency ratio (PER) was determined by Equation (8) according to [
39].
2.4. Carcass Traits
At the end of the experiment, ten birds from each treatment were chosen for carcass traits evaluation, according to Amer et al. [
2].
2.5. Determination of the Chemical and Fatty Acid Composition of the Breast Muscle
At the end of the experiment, breast muscle samples (5 samples/group) were taken. Oils from the breast muscle were extracted using a solvent mixture of chloroform/methanol (2:1,
v/
v) [
40]. Fatty acids in the extracted oil and the chemical composition of the breast muscle (dry matter, fat, crude protein, ash content %) were determined according to AOAC [
41].
2.6. Sample Collection and Laboratory Analyses
At the end of the feeding period, blood samples (two aliquots) were randomly collected after slaughter (two birds/replicate, ten birds/group). The chicks were euthanized using cervical dislocation, according to the American Veterinary Medical Association guidelines [
42]. The first aliquot of blood was placed in tubes containing dipotassium salt of Ethylene diamine tetra acetic acid (EDTA )as an anticoagulant for hematological analysis by Hemascreen 18 Automatic Cell Counter (Hospitex Diagnostics, Sesto Fiorentino, Italy) according to Harrison et al. [
43]. The differential leukocytes count was estimated as Schalm et al. [
44] described. The second aliquot of blood was collected without anticoagulant, left to clot at room temperature, centrifuged for 15 min at 3500 rpm for serum separation, and stored at −20 °C in deep freezing until biochemical analysis. Samples from different parts of the small intestine were taken for histomorphology examination. Spleen samples were taken for immunohistochemistry.
2.6.1. Blood Biochemical Indices
Chicken ELISA kits (My Biosource Co. San Diego, CA, USA) of CAT. NO. MBS269454, MBS265796, MBS025331, and MBS266317 were used for Triiodothyronine (T3) and Thyroxine (T4), leptin, and growth hormones determination, respectively, following the instructions of the enclosed pamphlets of each kit.
The serum levels of glucose, creatinine, and uric acid were measured by an automatic biochemical analyzer (Robotnik Prietest ECO Ambernath (W), Thane, India) [
45,
46,
47]. The method of Reitman and Frankel [
48] was used to estimate serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT).
2.6.2. Serum Lipid Profile and Proteinogram
Colorimetric diagnostic kits of spectrum-bioscience (Egyptian Company for Biotechnology, Cairo, Egypt) were used for measuring the serum total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C), following the methods of Allain et al. [
49], McGowan et al. [
50], and Vassault et al. [
51], respectively. The low-density lipoprotein cholesterol (LDL-C) level was calculated following the Iranian formula LDL-C = TC/1.19 + TG/1.9 − HDL/1.1 − 38. The very low-density lipoprotein cholesterol (VLDL-C) was measured using the turbidimetric method described by Griffin and Whitehead [
52].
The serum level of total protein was determined according to Grant [
53]. The serum albumin level was evaluated according to Doumas et al. [
54]. The serum globulin level was calculated mathematically by subtracting albumin values from total proteins [
55].
2.6.3. Antioxidant Activity
The serum total antioxidant capacity (TAC) was estimated as mentioned by Rice-Evans and Miller [
56], catalase (CAT) was calculated according to Aebi [
57], superoxide dismutase (SOD) activity was evaluated according to Nishikimi et al. [
58], and the Malondialdehyde (MDA) level was determined according to Mcdonald and Hultin [
59].
2.6.4. Immune Indices
The serum level of interleukin 10 (IL10) was determined using chicken ELISA kits of MyBioSource Co. of CAT.NO. MBS701683. Meanwhile, the serum complement 3 level was determined using a sandwich enzyme-linked immunosorbent assay (ELISA) kit (Life Span Biosciences, Inc., Seattle, WA, USA) of CAT. NO. LS-F9287). The serum lysozyme activity was determined according to Lie et al. [
60].
2.7. Histological Examination of the Small Intestine
Two-centimeter samples (3 samples/group) were taken from each part of the small intestine (the duodenum, jejunum, and ileum) and preserved in 10% neutral buffered formaldehyde (NBF) for 72 h, then processed for dehydration and clearing, and embedded in wax. Histological study was performed on 5 µm thick transverse sections (cut by a microtome), fixed on slides, and stained with hematoxylin and eosin [
61]. The villous height (VH) was measured from the tip (with a lamina propria) of the villus to the base (villus-crypt junction), and the crypt depth (CD) was calculated from the villus-crypt junction to the distal limit of the crypt.
2.8. Immunohistochemical Procedures
At the end of the experiment, spleen samples (3 samples/group) were collected for examination of immunoexpression of immunoglobulin G (IgG) according to Saber et al. [
62]. Briefly, samples were directly trimmed and immersed in neutral buffer formalin. Fixation of samples was conducted for four days. Routine histological techniques were performed on all the samples, including the previous steps used in histological sections such as dehydration, clearance, embedding, and cutting by microtome. Tissue ribbon was mounted on positively charged slides to avoid separation during the autoclaving step. Then slides were rehydrated through immersion in xylene, alcohols, and water. The antigen retrieval step aimed to remove methylene bridges on the protein caused by formalin. Therefore, it is too essential to unmask the antigen epitopes to allow the antibodies to bind. This step was carried out by immersion of the samples in a solution of 0.05 M citrate buffer, pH 6.8. Inhibition of the endogenous cellular enzymes to avoid nonspecific binding of horseradish peroxidase (HRP) or alkaline phosphatase (AP). Thus, samples were put in 0.3% H
2O
2 and protein block with sera of the animal spp. of the secondary antibody at room temperature for 30 min. After that, slides were incubated with a goat anti-Chicken IgG (Cat. No. NBP1-72720, Novus Biologicals, Briarwood Avenue, USA). The slides were rinsed with PBS three times for 10 of each. Slides were visualized with a DAB kit (3,3′-Diaminobenzidine) and eventually stained with Mayer’s hematoxylin as a counterstain. The staining intensity was assessed by positive areas per area using ImageJ ecosystem (IJ 1.46r, 2012, National institutes of health NIH, WA, USA), and data were expressed as the percent of positive area. Labeling indices were performed by counting positive cells in 1000 cells.
2.9. Statistical Analysis
Data were analyzed with a one-way analysis of variance (ANOVA) using the GLM procedure in SPSS (SPSS Inc., Chicago, Illinois, USA) after Shapiro–Wilk test was used to verify the normality and Levene’s test was used to verify homogeneity of variance components between experimental treatments. Tukey’s test was used to compare the differences between the means at 5% probability. Variation in the data was expressed as pooled SEM, and the significance level was set at p < 0.05. The broken-line regression with Tukey’s test considered information on BWG, FCR, growth hormone, and thyroxin hormone for determining the optimum supplementation level of ARRE.