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.


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. 2019Gado 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;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.

Total phenolic compounds (TPCs) estimation
The TPCs of the methanolic extract (1000 mg/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 mg/mL) for the preparation of the standard curve [y ¼ 0.001x þ 0.0563 (R2 ¼ 0.9792)], where y and x are the gallic acid absorbance and condensation in mg/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 mg/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 mg/mL) for the preparation of the standard curve [y ¼ 0.0012x þ 0.008 (R2 ¼ 0.944)], where y is absorbance and x is concentration of quercetin in mg/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.

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 Â 6 Â 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 þ 300 g Thymus vulgaris þ 300 g Salvia rosmarinus þ 150 g Pimpinella anisum þ 150 g Mentha spicata þ 300 g Nigella sativa þ 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).

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 ¼ carcass weight inclusion giblets weight/a live weight Â 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): Globulin ¼ Total protein À 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: TBA number kg ð Þ ¼ Absorbance at 538nm Â 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: where Y ij observed value; m, overall mean; T i , treatment effect (C: control; MIX1, MIX2, MIX3, MIX4 and MIX5: basal diet þ 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.

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 Table 2. Total phenolic (TP; mg GAE g À1 extract) and total flavonoid (TF; mg QE g À1 extract) contents and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) activity (SC 50 ; mg mL À1 ) 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. (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).

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.

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.

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).

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.

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 Ã and a Ã grade values lowered in all samples slowly through cold storage. In control, values of L Ã and a Ã grads of samples were lower than treatment groups.
In DPPH-radical scavenging activity assay, antioxidant activity (SC 50 ) was 150,100,270,118,230,200,300 and 13 mg mL À1 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 (Peri c 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, Peri c 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.

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 andJia et al. 2012). Also, the expression of antioxidant activity as SC 50 (mg mL À1 ) of the extract. Low SC 50 values lead to high antioxidant activity ). The blend material contains 13 DPPH activity (SC 50 ; mg mL À1 ) 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 Ã 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 Ã value. The formation of MetMb and high lipid oxidation are the major elements responsible for the differences in b Ã 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.
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