Does the dietary graded levels of herbal mixture powder impact growth, carcass traits, blood indices and meat quality of the broilers?

Abstract

The present study was conducted to investigate the impacts of dietary supplementation of graded levels of herbal mixture powder (HMP) on growth performance, carcass traits, blood indices and meat quality of broilers. The herbal mixture consisted of (300 g [Capsicum annuum] Hot red pepper + 300 g Thymus vulgaris + 300 g Salvia rosmarinus + 150 g Pimpinella anisum + 150 g Mentha spicata + 300 g Nigella sativa + 300 g garlic [Allium sativum]). A total of 360 chicks of Hubbard broiler one-day-old were randomly distributed into six treatment groups. The groups consisted of the basal diet as a control and a powdered mixture of HMP groups at levels of (2.0, 3.0, 4.0, 5.0 and 6.0 g HMP/kg diet). The chicks fed a basal diet plus 5 g HMP/kg diet had the highest (p < .05) values of live body weight (LBW) and body weight gain (BWG) at five weeks of age. Serum total protein, globulin, liver enzymes and kidney parameter values were reduced in groups treated with different levels of HMP compared with the control. At the same time, values of albumin, A/G ratio, IgM and lysozymes were increased in chicks treated with HMP. The quality of stored meat and antioxidative parameters were improved in groups fed HMP. Thus, it is concluded that HMP could be used as a natural additive to the broiler diet to enhance the growth rate and improve blood parameters and meat quality. Also, the oxidative rancidity was less in the meat of treated chicks during the storage period as compared to control.

Highlights

• Phytogenic additives are used to enhance growth and productive performance.

• Chicks fed 5 g herbal mixture/kg diet had the best growth performance.

• The quality of stored meat and antioxidative parameters were improved in groups fed the herbal mixture.

Keywords:

• Broiler Chicks
• herbal mixture
• performance
• serum metabolites
• meat quality

Introduction

There are a lot of dietary factors that have a major function in animal existence by affecting health, antioxidants and productive performance. Phytogenic additives are used in poultry production as natural feed additives to enhance the growth rate and productive performance. Beneficial impacts of herbs in poultry feeding such as improvement feed intake, appetite, energising secretion of digestive enzyme, stimulation immune response and antioxidant, antimicrobial, antihelminthic, antiviral properties and anti-heat stress modulators (Shewita and Taha 2018; Alagawany et al. 2019, 2020; Gado et al. 2019; Khafaga et al. 2019; Hafez and Attia 2020; Abd El-Hack, Alagawany et al. 2020; Abd El-Hack, Abdelnour et al. 2020; Ashour, Bin-Jumah et al. 2020; Ashour, El-Kholy et al. 2020; Batiha et al. 2020). Several studies reported useful impacts of phytogenic additives on growth performance, nutrient retention, gut health, intestinal microflora, reduced the susceptibility to diseases, enhanced immunity function and improved carcass traits in broiler chickens (Ashour et al. 2014; Abd El-Hack and Alagawany 2015; Alagawany, Farag et al. 2015; Alagawany, Abd El-Hack et al. 2015; Taha et al. 2019; Abo Ghanima et al. 2020; Ashour, Bin-Jumah et al. 2020).

The manufacturing of poultry breeding and enhancement feed efficiency have quickened the use of feed additives in the broiler diet. The feed is a major input for the broiler rearing and contributes about 70–80% of production cost, hence plays a vital role in the broilers economy (Singh et al. 2018). The essential purpose of scientists is to enhance production by the preservation of broiler health. To resistance these problems, herbs and medicinal plant extracts are investigated to be growth promoters in the poultry diet (Alloui et al. 2013). Various researchers tried many medicinal plants; some of them proved the ability of bioactive compounds of plants (phytobiotics) to prevent disease and promote the growth of broilers at the same time. The advantage of phytogenic feed additives over synthetic growth promoters is mainly due to the natural synergistic effect of all agents within the plants. Some traditional medicinal herbs/plants like hot red pepper, Thyme, Al-Ruzmari, anise, mint and Nigella sativa seeds are alone or, in combination, proved for their novel property of phytogenic growth promoter (Attia and Al-Harthi 2015; Attia, Al-Harthi et al. 2017; Attia, Bakhashwain et al. 2017; Attia et al. 2018; Arif et al. 2019; Attia et al. 2019; AL-Sagan et al. 2020). There is no previous reports studied the effect of a herbal mixture containing Capsicum annuum, Thymus vulgaris, Salvia rosmarinus, Pimpinella anisum, Mentha spicata, Nigella sativa and garlic with graded levels on broiler performance. Thus, the present experiment was planned to investigate the effective range of condensations of the current preparation which, consist of mixing natural herbs as phytogenic feed additives of broiler chickens and a natural antioxidant in growing broiler chick diets on the growth, carcass, serum biochemicals and quality of meat in Hubbard chickens.

Materials and methods

Plant materials

Thymus vulgaris (T.V), Pimpinella anisum (P.A), Capsicum annuum (C.A), Mentha spicata (M.S), Salvia rosmarinus (S.R), Allium sativum (A.S), and Nigella sativa (N.S) were bought from the botanicals, spices and herbs market (Zagazig City, Sharqia Governorate, Egypt).

Chemical characterisation

Folin and Ciocalteu’s phenol reagent, (DPPH) 2, 2-Diphenyl-1-picrylhydrazyl, gallic acid, quercetin, methanol, ethanol and aluminium chloride were obtained from Merck (Merck KGaA, Darmstadt, Germany).

Sample preparation

Ten grams of Thymus vulgaris (T.V), Pimpinella anisum (P.A), Capsicum annuum (C.A), Mentha spicata (M.S), Salvia rosmarinus (S.R), Allium sativum (A.S), Nigella sativa (N.S) and its flour were mixed individually with 70% methanol (200 mL), then was stirred for 3 h and then purified through filter paper Whatman No.2. Methanol was removed under vacuum from an extract in a BüCHI- water bath -B-480 evaporator at 45 °C followed by lyophilisation using a freeze-Dryer (Thermo- electron Corporation–Heto power dry LL 300 Freeze dryer). The resulting extract was kept at −20 °C until used for the following analyses (Abd El-Hack et al. 2018; Ashour, Bin-Jumah et al. 2020).

Total phenolic compounds (TPCs) estimation

The TPCs of the methanolic extract (1000 µg/mL) gained from each sample were evaluated by the Folin–Ciocalteu checked as observed by. Gallic acid was a standard phenolic compound at different concentrations (10–1000 µg/mL) for the preparation of the standard curve [y = 0.001x + 0.0563 (R2 = 0.9792)], where y and x are the gallic acid absorbance and condensation in µg/ml, respectively. One millilitre from each sample or standard gallic acid plus 3 ml from diluted Folin–Ciocalteu plus 2 ml from sodium carbonate 7.5% was mixed and kept in the darkness for 0.5 h at 25 °C. Finally, the blend absorbance was recorded at 760 nm by using a spectrophotometer (JENWAY, 6405 UV/Vis, U.K.) (Abdel-Shafi et al. 2019).

Total flavonoids (TFs) estimation

Total TFs of the extract (1000 µg/mL) as recommended by (Ordonez et al. 2006), as explained in (Abdel-Shafi et al. 2019). Quercetin was used as a standard phenolic compound at different concentrations (10-1000 µg/mL) for the preparation of the standard curve [y = 0.0012x + 0.008 (R2 = 0.944)], where y is absorbance and x is concentration of quercetin in µg/mL, respectively. One millilitre from quercetin solution or extract plus One millilitre aliquot of 20 g/L AlCl3 ethanol was mixed. The spectrophotometer has recorded the absorbance of colour at 420 nm.

Antioxidant activity estimation

Antioxidant efficacy of the methanolic extractor of Thymus vulgaris (T.V), Pimpinella anisum (P.A), Capsicum annuum (C.A), Mentha spicata (M.S), Salvia rosmarinus (S.R), Allium sativum (A.S), Nigella sativa (N.S) and its blend flour was determined by their ability to scavenge DPPH-assay (Ramadan et al. 2008; Osman et al. 2014). One millilitre from each sample was blended individually with three millilitres methanolic- DPPH solution and recorded the absorbance at 520 nm (DPPH-assay) after incubation for 30 min using a spectrophotometer. The extract condensation that scavenges 50% of the ABTS and DPPH radicals (SC50) was estimated as described by Abdel-Hamid et al. (2017).

Experimental design and diets

This investigation was conducted at the Department of Poultry, Faculty of Agriculture, Zagazig University, Egypt. Animal auspices and protection were achieved in conformity to guidelines of the Research Ethics Committee. The institutional committee approved this study. A total number 360 unsexed Hubbard chickens one day old at average 44.89 ± 0.47 g initial body weight were randomly divided into six treatments; 60 chicks in six replicates (6 × 6 × 10). All birds were reared adopting uniform management conditions; chicks were vaccinated against most common viral diseases in Egypt, such as Newcastle disease, Infectious bronchitis, Gambaro and Avian influenza virus according to the recommended veterinarian program. Feed was produced to all experimental groups in mash form. The dietary treatments consisted of the basal diet as a control group and A powdered herbal mixture (HMP) additives groups (2.0, 3.0, 4.0, 5.0 and 6.0 g HMP/kg diet where a herbal mixture consisting of (300 g [Capsicum annuum] Hot red pepper + 300 g Thymus vulgaris + 300 g Salvia rosmarinus + 150 g Pimpinella anisum + 150 g Mentha spicata + 300 g Nigella sativa + 300 g garlic [Allium sativum]) before that, this herbals were mixed and ground together in a powder image. Each group was fed the experimental diet starter (1 day-3 week) and finisher (3–5 weeks, in pellet form) , formulated according to the NRC (1994) recommendation to cover the nutritional requirements of broiler chicks during 1–5 weeks old (Table 1). Offered feed and water were ad-libitum through the period of the experiment, daylight except a hour was preserved during the whole experimental period. Chicks were individually weighted at weekly intervals, then the mean pen body weight cumulatively calculated as experimental unit. Feed consumption was listed weekly until the marketing age on a replicate basis. Consequently, feed conversion (g feed/g gain) and body weight gain were estimated during each experimental period (1–3, 3–5, and 1–5 weeks of age).

Table 1. Composition and chemical analysis of the basal diets as fed.

Items	Basal diets
	Starter (1–3 weeks)	Finisher (3–5 weeks)
Ingredients (g/kg diet)
Yellow corn	571.30	605.30
Soybean meal	316.50	271.50
Gluten meal	65.00	61.00
Di calcium phosphate	17.00	15.00
Limestone	12.40	11.50
Vit-min Premix*	3.00	3.00
NaCl	3.00	3.00
DL Methionine	0.50	0.20
L-Lysine HCl	1.30	1.00
Soybean oil	10.00	28.50
Total	1000	1000
Calculated analysis**:
Dry matter %	91.74	90.43
Crude protein %	23.00	21.00
Metabolizable energy MJ/kg diet	12.35	12.97
Calcium %	1.00	0.90
Phosphorous (Available) %	0.45	0.40
Lysine %	1.20	1.05
Methionine + Cysteine %	0.83	0.74
Crude fibre %	3.56	3.31

*Growth vitamin and Mineral premix Each 2.5 kg consists of: Vit A 1,20,00,000 IU; Vit D3, 20,00,000 IU; Vit. E. 10 g; Vit k3 2 g; Vit B1, 1000 mg; Vit B2, 49 g; Vit B6, 105 g; Vit B12, 10 mg; Pantothenic acid, 10 g; Niacin, 20 g, Folic acid, 1000 mg; Biotin, 50 g; Choline Chloride, 500 mg, Fe, 30 g; Mn, 40 g; Cu, 3 g; Co, 200 mg; Si, 100 mg and Zn, 45 g.

**Calculated according to NRC (1994).

Table 2. Total phenolic (TP; mg GAE g⁻¹ extract) and total flavonoid (TF; mg QE g⁻¹ extract) contents and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) activity (SC₅₀; µg mL⁻¹) of the methanolic extract acquired from Thymus vulgaris (T.V), Pimpinella anisum (P.A), Capsicum annuum (C.A), Mentha spicata (M.S), Salvia rosmarinus (S.R), Allium sativum (A.S), Nigella sativa (N.S) and its blend.

Samples	TP (mg GAE g^−1 extract)	TF (mg QE g^−1 extract)	DPPH activity (SC50; µg mL^−1)
T.V.	9.7	2.4	150
P.A.	12.15	4.9	100
C.A.	4.9	0.9	270
M.S	10.23	3.9	118
S.R.	5.2	1.8	230
A.S.	6.6	2.4	200
N.S.	4.5	1.3	300
Blend	25	16	13

Carcass characteristics

At the end of the experiment, six birds were randomly selected from each group (3 males and 3 females); they fasted overnight, weighted and then slaughtered. After that, birds were scalded and de-feathered. The heart, gizzard, liver, thigh, and breast were weighted and expressed as g/kg of slaughter weight (SW). The characteristics studied of the carcass were a carcass, giblets (liver, heart and gizzard) and dressing (dressed percentage = carcass weight inclusion giblets weight/a live weight × 100).

Serum biochemical analysis

At the end of the experiment, six birds per group were chosen randomly slaughtered; blood samples were collected into clean tubes without coagulate. Serum samples were separated from blood samples (Sitohy et al. 2013). By using commercial diagnostic kits provided by Bio Diagnostic Co. (Giza, Egypt), determined urea, creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein and albumin. Globulin was calculated from the following equation (Abdel-Hamid et al. 2020): $$\mathrm{Globulin}=\text{Total protein}-\mathrm{Albumin}$$

IgM estimated as described by (Amer et al. 2020). Lysozyme is estimated according to (Ellis 1990).

Meat quality samples

The TBA test

Lipid oxidation is estimated, according to Fernandez-Lopez et al. (2005), with a little modulation. Meat samples 10 g was dissolved with 100 mL distilled water each group for 2 min. The sample pH regulates to 1.5 by supplementing a few drops of 4 N HCl and then transported to a distillation tube. 50 mL of the distilled mixture was gathered. 5 mL of 0.02 M 2- thiobarbituric acid in 90% acetic acid and added (TBA reagent) to 5 mL of the distillate in a flask then well mixed. To develop the chromogen, the flask was covered and put for 30 min in a boiling water bath. After that, samples were refrigerated to the temperature room. At 538 nm, absorbance was estimated, prepared the blank against 5 mL TBA-reagent and 5 mL distilled water, using a Spectrophotometer. Numbers of TBA were calculated as malondialdehyde/kg sample according to the following equation: $$\text{TBA}\mathrm{ }\text{number}\mathrm{ }\left(\text{kg}\right)\mathrm{ }=\mathrm{ }\text{Absorbance}\mathrm{ }\text{at}\mathrm{ }538\text{nm}\mathrm{ }\times \mathrm{ }7.8$$

Measured pH values of beef burger and minced meat samples Fernandez-Lopez et al. (2005) Measurement colour of poultry meat was performed to Rao et al. (2011).

Statistical analysis

By using SPSS (2008) of GLM procedures, statically analysed the present data according to a completely randomised design with the following model: $${\mathrm{Y}}_{\text{ij}}=\mathrm{ }\mu \mathrm{ }+{\text{ T}}_{\mathrm{i}}+{\text{ e}}_{\text{ij}}$$ where Y_ij observed value; µ, overall mean; T_i, treatment effect (C: control; MIX1, MIX2, MIX3, MIX4 and MIX5: basal diet + 2, 3, 4, 5 and 6 g herbal mixture/kg diet, respectively); e_ij, random error. The test of student Newman keuls was determination the differences between means. Significant is depending on p < .05.

Results

Chemical characterisation

Total phenolic (TP; mg GAE g⁻¹ extract) and total flavonoid (TF; mg QE g⁻¹ extract) contents and DPPH activity (SC₅₀; µg mL⁻¹) of the methanolic extract acquired from Thymus vulgaris (T.V), Pimpinella anisum (P.A), Capsicum annuum (C.A), Mentha spicata (M.S), Salvia rosmarinus (S.R), Allium sativum (A.S), Nigella sativa (N.S) and its blend are presented in Table 2. The total phenol amount was 9.7, 12.15, 4.9, 10.23, 5.2, 6.6, 4.5 and 25 mg GAE g⁻¹ extract for TV, PA, CA, MS, SR, AS, NS and its blend, respectively. While the total flavonoid amount was 2.4, 4.9, 0.9, 3.9, 1.8, 2.4, 1.3 and 16 mg QE g⁻¹ extract for TV, PA, CA, MS, SR, AS, NS and its blend, respectively. The activity of antioxidant is expressed as SC₅₀ (µg mL⁻¹) of the extract. Low SC₅₀ values indicate high antioxidant activity (Osman et al. 2019). In DPPH-radical scavenging activity assay, the antioxidant activity (SC₅₀) was 150, 100, 270, 118, 230, 200, 300 and 13 µg mL⁻¹ for TV, PA, CA, MS, SR, AS, NS and its blend, respectively. Antioxidant ability of different foods inclusive coffee, is backed to polyphenol aggregation (Acidri et al. 2020).

Growth performance

The growth performance, as impacted by herbals mixture powder (HMP), is clarified in Table 3. LBW and BWG of broilers fed a diet enriched with HMP was influenced (p < .05) during the studied period. Whereas chicks received 5.0 and 6.0 g HMP/kg had the highest BW and BWG values throughout 3 and 5 weeks of age for body weight or (0–3, 3–5 and 1–5 weeks of age) for BWG compared to control and another trail groups. Data on feed intake in Table 3 cleared insignificant impacts of added HMP on broiler FI among most treatment periods except the first term (1–3 wks of old) FI (p < 0.01) changed without a constant trend. Chicks fed 5.0 g/kg diet of HMP recorded the highest FI (61.27 g/day) while the group fed 2.0 g/kg HMP had the lowest one (55.67 g/day). Impacts of a diet enriched with HMP on FCR of chicks were not significant in the current study throughout variation intervals. The best FCR recorded in chicks fed HMP diet at level 5.0 g/kg diet, while the worst FCR was recorded in group fed HMP at level 3.0 g/kg diet during the total term (0–5 wk of old).

Table 3. Growth performance parameters of broiler chicks as affected by graded levels of dietary herbal mixture supplementation.

Items	LBW (g)			BWG (g)			DFC (g/day)			FCR (g feed/ g gain)
	0 wk	3 wk	5 wk	0–3wk	3–5wk	0–5wk	0–3Wk	3–5wk	0–5wk	0–3 wk	3–5 wk	0–5wk
C	45.33	948.67^b	1938.46^a	43.02^b	70.70^a	56.86^a	58.99^ab	148.43	103.71	1.37	2.10	1.82
MIX1	46.15	927.00^bc	1893.07^a	41.95^bc	69.01^b	55.47^a	55.67^c	143.03	99.36	1.33	2.08	1.79
MIX2	42.20	873.50^c	1742.39^b	39.59^c	62.06^ab	50.83^b	56.87^bc	142.46	99.67	1.44	2.31	1.96
MIX3	44.00	944.25^b	1882.02^a	42.87^b	66.99^ab	54.93^ab	58.24^bc	141.17	99.71	1.36	2.10	1.81
MIX4	46.40	1014.0^a	2011.75^a	46.08^a	71.27^a	58.67^a	61.27^a	151.44	106.35	1.33	2.13	1.81
MIX5	45.30	954.17^b	1963.57^a	43.28^b	72.10^a	57.69^a	57.78^bc	152.07	104.92	1.34	2.11	1.82
SEM	0.47	11.84	25.29	0.56	1.20	0.79	0.51	1.69	0.98	0.02	0.03	0.02
P value	0.550	0.005	0.014	0.007	0.030	0.027	0.007	0.236	0.115	0.247	0.253	0.07

C: control; MIX1, MIX2, MIX3, MIX4 and MIX5: basal diet + 2, 3, 4, 5 and 6 g herbal mixture/kg diet, respectively.

LBW: live body weight; BWG: body weight gain; DFC: daily feed consumption; FCR: feed conversion ratio.

SEM: standard error mean.

Different letters within one column are significantly different (p < 0.05).

Carcass characteristics

Carcass characteristics as affected by the addition of dietary HMP are offered in Table 4. Carcass, dressing, thigh, breast, and liver were not differed (p > .05) in trail diet groups. The highest (p < .05) relative weight of gizzard and heart is occurred in chicks fed 2.0 g/kg HMP and in the control group, respectively. Otherwise, the lowest (p < .05) values of the aforementioned were offered in groups fed 4.0 and 2.0 g/kg diet HMP, respectively.

Table 4. Carcass characteristics of broiler chicks as affected by graded levels of dietary herbal mixture supplementation.

Items	Carcass traits (relative to pre-slaughter weight %)
	Carcass	Dressing	Breast	Thigh	Liver	Heart	Gizzard
C	72.32	76.28	44.43	27.80	2.24	0.50^a	1.21^ab
MIX1	72.71	76.72	44.36	28.29	2.05	0.38^b	1.58^a
MIX2	73.39	77.36	46.45	26.20	2.35	0.48^a	1.15^b
MIX3	73.40	77.19	46.22	27.07	2.35	0.48^a	0.95^b
MIX4	72.87	76.77	43.95	28.70	2.19	0.40^b	1.31^ab
MIX5	73.18	76.99	45.37	27.92	2.22	0.45^ab	1.14^b
SEM	0.17	0.16	0.35	0.29	0.07	0.01	0.06
P value	0.441	0.526	0.160	0.132	0.839	0.013	0.045

C: control; MIX1, MIX2, MIX3, MIX4 and MIX5: basal diet + 2, 3, 4, 5 and 6 g herbal mixture/kg diet, respectively.

SEM: standard error mean.

Different letters within one column are significantly different (P < 0.05).

Blood parameters

The effects of dietary plus herbal mixture powder on serum metabolites of broiler chicks are stated in Table 5. Concentrations of serum ALT, AST, urea, creatinine, total protein and globulin reduced (p < .05) in all chick groups trail with HMP whereas, the highest concentrations of ALT, AST, urea, creatinine, total protein and globulin (10.81 IU/L, 123.67 IU/L, 44.89 mg/dL, 6.62 g/dL and 2.90 g/dL respectively) presents chicks fed the basal diet. Data for creatinine show that there was no significant effect (p < .05) of the treatments (MIX2 and MIX4) compared to control. Albumin concentration and A/G ratio values enhanced (p < .05) in the serum of trail broilers by HMP. In the present work, IgM and lysozyme enhanced (p < .05) with increasing levels of treatments.

Table 5. Biochemical parameters of broiler chicks supplemented with different concentrations of herbal mixture.

Biochemical parameters	C	MIX1	MIX2	MIX3	MIX4	MIX5
ALT (IU/L)	10.81^a	10.58^b	10.28^bc	10.13 ^cd	9.98 ^cd	9.80^d
AST (IU/L)	123.67^a	121.50^b	119.50^c	118.50^c	116.50^d	115.50^d
Urea (mg/dL)	44.89^a	35.50^d	39.50^b	36.75^c	35.s15^d	33.50^e
Creatinine (mg/dL)	0.54^a	0.43^c	0.56^a	0.48^b	0.53^a	0.42^c
Total protein (g/dL)	6.62^a	6.45^b	6.38^bc	6.40^bc	6.31 ^cd	6.25^d
Albumin (g/dL)	3.23^bc	3.16^c	3.33^a	3.28^ab	3.33^a	3.35^a
Globulin (g/dL)	3.38^a	3.29^a	3.06^bc	3.12^b	2.98 ^cd	2.90^d
A/G ratio	0.96^d	0.96^d	1.09^bc	1.05^c	1.12^ab	1.16^a
IgM (µg/mL)	217.67^f	221.00^e	226.50^d	230.50^c	244.00^b	250.50^a
Lysozymes (µg/mL)	11.45^e	12.80d	13.50^d	15.50^c	18.50^b	24.00^a
SEM	0.03	0.06	0.25	0.24	0.02	0.52
P value	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001

C: control; MIX1, MIX2, MIX3, MIX4 and MIX5: basal diet + 2, 3, 4, 5 and 6 g herbal mixture/kg diet, respectively.

ALT: alanine aminotransferase; AST: aspartate aminotransferase; A/G: albumin/ globulin ratio ; IgM: Imunonoglubulin M.

Different letters within one row are significantly different (p < .05).

Meat quality samples

pH values

pH values of chicken meat through cold storage (4 ± 1 °C for 14 days) were given in Table 6. All samples showed increases in pH values during cold storage. This increase in pH values throughout meat storage was lead to the growth micro-organisms on the meat surface, stimulating the decomposition of different meat elements such as amino acids and fat (Zhao et al. 2019).

Table 6. Effect of herpes mixture on PH of broilers samples during cold storage (±4 °C).

Items	pH
	0 days	7 days	15 days
C	5.83^a	5.99^a	6.24^a
MIX1	5.75^b	5.90^c	6.12^c
MIX2	5.83^a	5.98^a	6.16^b
MIX3	5.76^b	5.93^b	6.15^b
MIX4	5.62^c	5.86^d	6.09^d
MIX5	5.62^c	5.82^e	6.03^e
SEM	0.02	0.01	0.02
P value	<0.000	<0.000	<0.000

C: control; MIX1, MIX2, MIX3, MIX4 and MIX5: basal diet + 2, 3, 4, 5 and 6 g herbal mixture/kg diet, respectively.

SEM: standard error mean.

Different letters within one column are significantly different (p < .05).

Thiobarbituric acid reactive substances (TBARS)

The effect of herpes mixture powder addition to broiler diet on the oxidative constancy of stored meat chicken in the refrigerator clarified in Table 7. Throughout the storage period, TBARS was increased in all studied samples. However, rancidity oxidative was lowered in storage meat chicken of broiler fed diet containing HMP compared to samples of the control group.

Table 7. Effect of herpes mixture on TBA (mg MD/Kg) of broilers samples during cold storage (±4 °C).

Items	TBA
	0 days	7 days	15 days
C	0.34^a	0.49^a	0.85^a
MIX1	0.24^c	0.41^c	0.70^b
MIX2	0.26^bc	0.37^d	0.52^d
MIX3	0.33^a	0.43^b	0.69^c
MIX4	0.16^d	0.28^f	0.41^e
MIX5	0.27^b	0.34^e	0.51^d
SEM	0.01	0.02	0.04
P value	<0.000	<0.000	<0.000

C: control; MIX1, MIX2, MIX3, MIX4 and MIX5: basal diet + 2, 3, 4, 5 and 6 g herbal mixture/kg diet, respectively.

SEM: standard error mean.

Different letters within one column are significantly different (P < 0.05).

Meat colour evaluation

The changes in meat colour values of chickens treated with different levels of HMP throughout storage cold were illustrated in Table 8. The L* and a* grade values lowered in all samples slowly through cold storage. In control, values of L* and a* grads of samples were lower than treatment groups.

Table 8. Effect of herpes mixture on meat colour of broiler samples during cold storage (±4 °C).

Items	Meat colour
	0 days			7 days			15 days
	L*	a*	b*	L*	a*	b*	L*	a*	b*
C	53.35^a	5.27^e	13.56^a	52.52^a	5.05^d	13.31^c	52.46^b	6.70^d	12.55^c
MIX1	53.72^a	8.74^d	12.57^b	51.52^b	6.57^b	12.34^e	49.86^c	8.13^a	14.15^a
MIX2	41.79^e	8.43^a	13.91	47.69^e	6.57^b	13.88	55.29^a	7.38^c	11.80^e
MIX3	51.56^b	6.41^c	12.68^b	50.64^c	5.34^c	12.28^e	48.32^d	4.61^f	13.17^b
MIX4	48.41^c	7.14^e	12.63^b	48.09^d	5.44^c	13.65^b	45.70^f	5.93^e	10.37^f
MIX5	47.19^d	8.11^b	13.62^a	46.82^f	7.55^a	13.13^d	47.40^e	7.51^b	12.40^d
SEM	1.00	0.32	0.39	0.52	0.21	0.15	0.78	0.28	0.28
P value	<0.000	<0.000	<0.000	<0.000	<0.000	<0.000	<0.000	<0.000	<0.000

C: control; MIX1, MIX2, MIX3, MIX4 and MIX5: basal diet + 2, 3, 4, 5 and 6 g herbal mixture/kg diet, respectively.

SEM: standard error mean; L: lightness; a: redness; b: yellowness.

Different letters within one column are significantly different (p < .05).

Discussion

Chemical characterisation

The total phenol amount was 9.7, 12.15, 4.9, 10.23, 5.2, 6.6, 4.5, and 25 mg GAE g⁻¹ extract for TV, PA, CA, MS, SR, AS, NS and its blend, respectively. While, the total flavonoid amount was 2.4, 4.9, 0.9, 3.9, 1.8, 2.4, 1.3 and 16 mg QE g⁻¹ extract for TV, PA, CA, MS, SR, AS, NS and its blend, respectively. Low values of SC₅₀ mean antioxidant activity higher (Osman et al. 2019). In DPPH-radical scavenging activity assay, antioxidant activity (SC₅₀) was 150, 100, 270, 118, 230, 200, 300 and 13 µg mL⁻¹ for TV, PA, CA, MS, SR, AS, NS and its blend, respectively. The antioxidant ability of different foods inclusive coffee is backed to polyphenol aggregation (Acidri et al. 2020).

Growth performance

This noticeable improvement in LBW at the whole experimental period (1–5 wks of age) might be due to improved digestion and absorption of diet nutrients by some components of the phytogenic additives (Tables 2 and 3). However, that may be contributed to enhancing the utilisation of feed consequence, enhancing the growth rate. Prohibition of used antibiotics in poultry production due to a widely used of herbs and plant medicines as feed additives to improve growth condition, its induced saliva secretion and improve digestion processes (Suganya et al. 2016). Phytogenic additives may also reduce the environmental problems dangerous that are produced by using antibiotics as feed additives such as bacterial resistance (Perić et al. 2009). Fotina et al. (2013) reported that to preserve broiler growth rate, quality of meat and immune responses, its necessary added optimal antioxidant level to diets.

On the other hand, Marzoni et al. (2014) cleared that the growth rate did not impact adversely by adding a mix of natural antioxidants in the broiler diet. Results also showed no statistical differences in FI due to the dietary supplementation of HMP except the term of age 1-3 wk. In agreement, Cho et al. (2014) claimed that feed intake and FCR of broiler chickens did not affect by the addition of phytogenic additives to broiler diet compared to the control. In contrast, Perić et al. (2009) and Scheuermann et al. (2009) stated that additives of phytogenic could improve the consuming and transformation of nutrients, but the action mechanism is not yet clear. The positive effect of these additives leads to BWG improvement, as presented in Table 3. However, this improvement may be due to the synergetic effect of chemical ingredients present in HMP (Table 2). Kohlert et al. (2000) reported that phytogenic additives theoretically enhance digestion and absorption by hyperactivation of intestinal villi and fastly body metabolised. It stimulates modification in membrane permeability and penetration characteristics, such as proteins synthesis linked with the function of cytoskeletal, consequence enhance absorption in small intestine surface (Khajuria et al. 2002).

Carcass characteristics

Positive impacts were observed on gizzard (p > .01) and heart (p <.05) yields, while carcass, dressing, breast and thighs yields were adversely influenced (p > .05 and .01) by dietary HMP supplementation. In a partial agreement with our findings, Marzoni et al. (2014) cleared that broiler carcass yields did not impact by adding a natural antioxidant mixer to chick's diet. In disagreement with our results, Cabuk et al. (2006) concluded that a herbs mixture were not had significant impacts on body weight or internal organs of the broilers at the marketing age.

Blood parameters

Blood biochemical estimations are usually expressed to bird health conditions. These estimations are a good index of nutritional, physiological and pathological conditions of the bird and have the possibility of starting used to illustrate the influence of feed ingredients and additives dietary provided. This present finding is positively confirmed by Swain and Johri (2000) and Biswas et al. (2010). Our results are agreeing with Suganya et al. (2016), who showed that the herbs, flavonoids, carotenoids and vitamin C were useful for the immune system. Also, Lavinia et al. (2009) revealed that dietary additives of essential oils of medicinal plants enhance duodenum mucosa and immune response due to improving animal performance.

Meat quality samples

pH values

The glycolysis rate after slaughter causes meets acidity that responsible for the primary variety of meat quality. The pH values increasing in storage meat responsible for the growth micro-organisms on storage meat surface, stimulating fat and amino acid decomposition, Shengming Zhao et al. (2019). The lower pH in chicken meat treatment with herpes mix may be responsible for inhibition of the integration of growth microorganism deterioration.

Thiobarbituric acid reactive substances (TBARS)

Food oxidative rancidity is estimated by using the methods of thiobarbituric acid reactive substances. The changes of treated samples or control were not significant at zero time, which indicates that oxidative deterioration of broilers lipid occurred during storage time (Table 7). During the storage period, TBARS values tended to significantly (p ≤ .05) increases, and all samples recorded values lower than the critical limit (0.9 mg malonaldehyde/kg sample), as reported by Greene and Cumuze (1982). The inhibitory effect of herpes mix of lipid oxidation may be linked by the contents of biochemical constituents and phenolic components that mostly participate in the activity of antioxidant (Zhang et al. 2010 and Jia et al. 2012). Also, the expression of antioxidant activity as SC₅₀ (µg mL⁻¹) of the extract. Low SC₅₀ values lead to high antioxidant activity (Osman et al. 2019). The blend material contains 13 DPPH activity (SC₅₀; µg mL⁻¹) as reported in Table 2 that means that it has a strong antioxidant activity.

Meat colour evaluation

Cold chicken marketing during refrigerated storage period depends on chicken surface colour, which indicates to its freshness and commercial acceptability. Reducing water retention leads to less surface light reflecting which, responsible for L* value decreasing (Hughes et al. 2014). Metmyoglobin (MetMb) cumulating on the storage meat surface participate in the discolouration of storage meat (Bekhit et al. 2007), which clears the differences in a* value. The formation of MetMb and high lipid oxidation are the major elements responsible for the differences in b* value (Xiong, 2000).

Conclusions

It is concluded that HMP could be used as a natural additive to the broiler diet to enhance the growth rate and improve blood parameters and meat quality. Chicks fed 5 g herbal mixture/kg diet had the best growth performance. Also, the oxidative rancidity was less in the meat of treated chicks during the storage period as compared to control samples.

Ethical approval

All procedures of the current study were followed according to the Directive 2010/63/EU of the European Parliament and the Council of September 22, 2010, on the safety of animals used for scientific purposes. The experimental protocol regarding the care and handling of animals had been approved by the Ethics Committee of Department of Poultry, Faculty of Agriculture, Zagazig University, Egypt.

Disclosure statement

The authors declare that there is no conflict of interest associated with the paper. The authors alone are responsible for the content and writing of this article.

Correction Statement

This article has been republished with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

The authors thank the Researchers Supporting Project for their funding this work number (RSP-2020/232) at King Saud University, Riyadh, Saudi Arabia.