Introduction

The main objective for broiler producers is to maximize production with the least cost and best quality. However, due to limited resources, feed prices became more expensive which made it harder for poultry farmers to achieve revenues or even to cover the cost of production, this has directed the farmers to search for several methods to maximize production per rearing cycle. Increasing stocking density is known as one of the practices that farmers adopt to increase their profitability1. Broilers can adjust their physiological, immunity, biochemical, and metabolic responses during stress exposure such as temperature changes out of their thermal neutral zone (TNZ), oxygen shortage, and improper management of feeders and drinkers which are all the main issues with high stocking densities rearing cycles. Stocking density refers to the number of birds or their total live weight in relation to a specific area; increased stocking density will put more stress on birds that can affect the physiological status, productivity, carcass quality, growth performance, immunity, metabolism, and meat quality of broiler chickens2. Also, such a stress factor would initiate an imbalance between oxygen supply and demand of the body3, which will promote the production of free radicals and interrupts the normal body functions4. High stocking density can create oxidative stress due to the imbalance between reactive oxygen species (ROS) or the free radicals and antioxidant defenses. Alanine aminotransferase (ALT) is an enzyme that exists in the liver and is responsible for converting protein into energy for utilization by liver cells. Whenever the liver is damaged, ALT is released into the blood stream along with Aspartate aminotransferase (AST) that aids in the metabolism of amino acids. Like ALT, AST is naturally found in blood at minimal levels. An elevation in AST levels may cause disease, liver and muscle damage5. Corticosterone is a stress hormone indicator that is secreted by the adrenal cortex in response to environmental changes has been increased in HSD6. Myostatin (MSTN) has been known for its negative regulatory role on muscle growth detecting both muscle fiber size and number7. Myogenin (MyoG) is a regulatory factor in muscle. During the elevation of stocking density (MyoG) was negatively affected while MSTN was increased8. On the other hand, Insulin-like growth factor 1 (IGF1) is considered one of several growth factors necessary for normal growth, and skeletal muscle development, the levels of IGF1 was found to be reduced with HSD9. During the last decade using antibiotics was banned10, which made the whole industry pursue other alternatives more trustful and safer to use without affecting human health, and yet can maintain effective production11. Phytogenic feed additives have a reputation for good quality due to different effects on various aspects of poultry production, such additives as the whole part of plants like roots, leaves, herbs, or extractions as essential oils (phytogenic compounds). Essential oils have approved efficiency for immune system and growth performance12. Therefore, it could be hypothesized that phytogenic compounds have a positive role in enhancing growth performance and meat synthesis under stressful situations.

This study was conducted to evaluate the effectiveness of phytogenic compounds when increasing stocking density on carcass traits, growth performance, and evaluate how stocking density can affect the gene expression of muscular growth, biochemical indicators and how phytogenic compounds can have an intervention role in alleviating any negative effect of increasing stocking density.

Materials and methods

Statement of ethics

The experiment was accepted and performed under the guidelines of the Institutional Animal Care and Use Committee of Jordan University of Science and Technology Approval # (16/4/12/508).

The experiment was conducted in accordance with the guidelines set out by Animal Research: Reporting of in Vivo Experiments (ARRIVE).

Location, birds, and experimental design

The experiment was conducted at the Agricultural Research and Training Unit at Jordan University of Science and Technology, Faculty of Agriculture, Department of Animal Production. A total of 784 1-day old mixed-sex broiler chicks Ross 308 were purchased from a commercial hatchery and randomly allocated to 28 floor pens (Each pen was 124 cm in width, 200 cm in length, and 100 cm in height) in open-sided house. The experimental design was conducted using 2 × 2 factorial arrangement to evaluate the stocking density, phytogenic compounds (Digestarom® DC power, Biomin Holding GmbH, Austria, major constituents are oregano oil, caraway oil, carvacrol, methyl salicylate, and menthol.), and their interactions. Chicks were randomly assigned to 4 treatments (Fig. 1) with 7 replicate pens per treatment, as follow:

  1. 1.

    Normal feed (no essential oils [EO] added) and normal stocking as 9 bird/m2, (NSD).

  2. 2.

    Normal feed (no essential oils [EO] added) and high stocking as 14 bird/m2, (HSD).

  3. 3.

    Feed with essential oils (Digestarom DC® 150 g/Ton of feed) and normal stocking as 9 birds/m2, (NSD + EO).

  4. 4.

    Feed with essential oils (Digestarom DC® 150 g/Ton of feed) and high stocking as 14 birds/m2, (HSD + EO).

Figure 1
figure 1

Experimental design.

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Stocking density was calculated based on 20 kg final body weight per m2 for the normal stocking density group and 28 kg final body weight per m2 for the high stocking density group.

Two phases of feeding (starter d 1–14, and grower d 15–35) were fed during the experiment. Birds were provided ad libitum access to feed (Table 1) and water, with 23 h of light and 1 h of darkness per day during the first 3 weeks then natural daylight cycle was used. Broiler feed was formulated to meet the nutritional requirements for broilers suggested by Ross 308 guideline. For each pen there was an automatic bell drinker and one tube feeder. Birds were reared and grown under standard commercial conditions to 35 days of age for all treatments. Temperature was kept at 32 °C on the first day and reduced gradually using cooling fans to reach 21 °C at day 35. The relative humidity inside the house ranged from 45 to 50.

Table 1 Starter and grower diet composition and nutrients analysis.
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Data collection

Growth performance

The period of study was split into two periods; period 1 was 2 weeks in length and period 2 was 3 weeks. At the beginning of each week, a considerable quantity of the diet was weighed and offered for each replicate within each treatment. For each week, the residual feed was weighed and subtracted from the presented amount to find the total feed consumption during the week. All chicks were weighed at the receiving day, as well as for each week of the study. Body weight gain and feed conversion ratio (FCR) were determined each week during the experimental period:

BWG = Final weight—Initial weight/Number of days

FCR = Average feed consumed in gram/Average body weight gain in grams

Carcass traits and meat quality measurements

At 35 days of age, a total of 28 birds (7 birds per treatment) were slaughtered to assess the qualities of the meat. With a knife, both head and shanks were eliminated after the birds were manually exsanguinated by severing both carotid arteries and at least one jugular vein. The bleeding persisted for 120 s. The birds were defeathered after being scalded for 120 s at 60 °C after blood was removed. The viscera were then removed from the carcasses’ cavities after the vent was cut open with a knife and all of the carcasses were eviscerated. The carcasses were cleaned on the inside and outside after the slaughter process was finished. After washing, the carcasses were placed in clean water and cooled for 20 min at 4 °C. Within three hours of the animal being slaughtered, the breast, legs, wings, back, neck, and fat pad were separated and weighed. Cuts were made into the forequarters of carcasses to remove breast cuts. A knife was used to cut the forequarters into wings and breasts. The wings were severed from the forequarter at the proximal ends of the humeral bones, separating them from the carcass at the shoulder and leaving as much meat on the breast as feasible. To remove the meat from the breast, the humeral-scapular joint was severed and pulled downward, separating the breast muscles from the back at the shoulder. The aggregate weight of the parts acquired after cutting determines the percentages of the different parts. Following the recording of the weights of the breast, drumstick, wings, backbone, and neck, each carcass’ whole breast was wrapped in a plastic bag with a label and stored in a freezer at − 32 °C. The frozen breasts were kept in their plastic bags and allowed to thaw overnight at 4 °C in the refrigerator. After the samples were taken out of the bags, they were weighed, and the complete breast samples were manually deboned and chopped up. The pectoralis major muscles on the left and right were removed, and the left muscle was selected for examination of the meat quality. By slicing the humeral-scapular joint and drawing downward to extract the meat from the breast, the muscles were removed. The investigation of meat quality, including cooking loss and Warner–Bratzler Warner–Bratzler shear force values of cooked meat samples, was then limited to the left pectoralis major muscles. The measurements of pH, water holding capacity, and color coordinates were made using the right pectoralis major muscles.

Cooking loss determination

The amount of water lost to shrinkage while cooking was quantified as "cooking loss." In this study, the left pectoralis muscle was deboned, the initial weight of each muscle was noted, and it was then put in a tagged poly-ethylene bag. After that, the bags were cooked for 25 min at 85 °C in a water bath with a thermostat, to reach an interior temperature of at least 80 °C.Following cooking, the bags were opened, and the liquid was drained after being allowed to cool to room temperature. After drying the cooked sample using a paper towel to eliminate any remaining surface moisture, its weight was noted. The weight lost during cooking was divided by the weight of the fresh sample to get cooking loss, which was then reported as a percentage. Because it indicates the muscle tissue’s ability to retain water, cooking loss is a valuable assessment of the quality of meat.

Water holding capacity

The centrifugation method outlined by Warner (2014) was used to measure the amount of expressible juice. Approximately 10 g of muscle samples were weighed and put into polyethylene centrifuge tubes. The specimens underwent a 10-min, 2500×g centrifugation at 4 °C. After carefully pipetting the extracted juice out of each tube, the weight was measured. Expressible juice was calculated as a percentage by dividing its weight by the original weight of the sample.

Tenderness determination

The cooked samples from each left pectoralis major muscle were divided into six cores, each measuring 20 × 13 × 13 mm, after cooking and calculating the cooking loss. The whole depth of each cooked muscle sample was included in the cores, which were cut parallel to a line starting at the humeral insertion and terminating at the location next to the keel. Furthermore, each core was sheared using a Warner–Bratzler meat shear device (G-R Manufacturing Co., 1317 Collins Lane, Manhattan, Kansas, 66,502, USA) equipped with a triangular slot cutting edge, perpendicular to the longitudinal orientation of the muscle fiber. Every sample's peak force (kg) needed to shear was recorded. The average of the maximal force of the six samples from each left pectoralis major muscle sample was the analyzed Warner–Bratzler shear force dependent variable.

pH measurements

Using a solution containing 5 mM iodoacetate reagent and 150 mM KCl, the muscle tissue was homogenized to ascertain the pH value of the right pectoralis major muscle. This procedure is known as the iodoacetate method13. Using a homogenizer (Ultra-Turrax T8, IKA Labortechnik, Janke & Kunkal GmbH & Co., Germany), one to 1.5 g of raw right muscle was added to a plastic test tube together with the iodoacetate reagent and KCl solution. Following the homogenization process, the pH of the mixture was determined using two separate samples and a pH meter (pH spear, waterproof pH/temperature, large screen, tester, double injection, model 35634-40, Eurotech instruments, Malaysia). The electrode was dried with soft tissue paper and rinsed with distilled water before the pH readings were recorded. Since it preserves the pH of the muscle tissue, the iodoacetate method is frequently used to measure the pH of muscle.

Color measurements

A colorimeter (12MM Aperture U 59730-30, Cole-parameter International, Accuracy Microsensors, Inc. Pittsford, New York, USA) was used to measure the raw right pectoralis muscles 24 h after they had thawed. Throughout the study, the colorimeter was calibrated a standard ceramic white colored has been used as reference (CIE L* = 97.91. a* = − 0.68, b* = 2.45). The samples were put on a tray and covered with wax paper to prevent surface drying to prepare them for color measurement. Then, the samples were placed in the refrigerator for roughly two hours to allow for adequate exposure to ambient oxygen before they returned to their natural hue. On the muscular surface next to each sample's skin, three separate random readings were collected in an area devoid of any obvious color abnormalities, such as bruises or burst blood vessels. The color of each sample was then expressed in terms of CIE LAB (Commission Internationale de l’Eclairage) brightness (L*), redness (a*), and yellowness (b*) after the three location measurements were averaged.

Blood sampling

The main approach of blood sampling has been taken randomly from seven birds/treatment at 35 days as age of slaughter. To apply the ELISA technique one blood sample has been drawn from the brachial vein and placed in a heparinized tube. In a frozen refrigerator serum samples have been kept for further examination at − 20 °C.

Tissue sampling

On day 35, samples weighing one hundred milligrams were taken from the thigh, and pectoral tissues. There were 2 tissue samples from 7 birds in each of the 4 treatment groups, for a total of 56 samples assayed. Semi-quantitative real-time polymerase chain reaction (RT-PCR) analyses were performed on the mRNA expression levels of muscle marker genes MyoG and muscle growth factors (IGF-1, MSTN) in the pectoral and thigh muscles using a real-time thermal cycler (CFX96 Touch IM, USA, California) and BIO-RAD RT-PCR.

Primers design

Sequences of broiler chicken for IGF-I, MyoG and MSTN, were obtained from the National Library of Medicine Prime-Blast are shown in (Table 2). By using the prementioned sequences, primers were established for the desired genes RT-PCR analysis, as all primers has been constructed using primer-blast, as the following Table shows:

Table 2 Primers used for gene expression analysis using quantitative real-time PCR.
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RNA isolation and semi-quantitative RT-PCR analysis

After homogenizing muscle tissue samples, supernatants were removed, and total RNA was extracted using the TRIzol/chloroform/isopropanol technique. Next, diethylpyrocarbonate (DEPCH-treated RNase-free water; Ambion, Austin, TX) was used to dissolve the RNA pellet. After removing the DNA using a DNase I kit from Ambion in Austin, Texas, the RNA samples were examined for purity and concentration using the absorbency at 260 and 280 nm. In a reaction mixture, 2 µg of RNA were reverse transcribed into cDNA using the iScript DNA Synthesis Kit (BIORAD, California, USA). Then, the cDNA was used for semi-quantitative RI-PCR (CFX96 Touch TM Real time PCR, BIO-RAD IQ5, California, USA). Using the Advanced IM SYBR Green Supermix kit (BIO-RAD, California, USA), the semi-quantitative RI-PCR was carried out. In summary, 10 μL of the master mix, 2 μL forward primer (10 pmol), 2 μL reverse primer (10 pmol), 2 μL sample DNA, and 4 μL of nuclease-free water were used to prepare the 20 L reaction mix. The cycling parameters included 2 min at 50 °C, a total of 15 min at 95 °C, forty cycles at 95 °C for 10 s, 30 s at 55 °C, 10 s at 72 °C, and 20 s at 95 °C for melting. Using CFX management TM software V3.1 (BioRad, Hercules, CA, USA), duplicates from each cDNA were examined, emission of fluorescence was determined, and relative quantification was computed automatically.

Biochemical and hormonal analysis

Specifically, a microplate reader (AccuReader, M965, Nangang, Taiwan) was used to measure corticosterone, mTOR, IGF-1, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) using commercial assay kits from (SUNLONG Biotech Co., LTD, Hangzhou, China).

Statistical analysis

Data collected were analyzed using the PROC GLM procedure of SAS (SAS Inst., Inc., Cary, NC). For growth performance and blood indicators models included stocking density, essential oils, and their interaction as the fixed effect. For relative expression of mRNA levels of IGF-I, MSTN, and MyoG model included the stocking density-essential oils interaction as the main effect. The level of significance (probability of a wrong rejection) was set at alpha 0.05, and differences between treatment means were assessed in pairwise comparisons with a Tukey–Kramer adjustment applied to P values to correct for multiple testing.

To evaluate the traits of the carcass and meat quality, the models included stocking density, essential oils, and their interaction as a fixed effect and carcass weight as a linear covariate.

Results

Growth performance

Results are presented as main effects and the interactions between essential oils (with and without) and stocking density (low stock density and high stocking density).

Feed intake

Results are presented in Table 3. Stocking density has shown a significant effect on feed intake from 1–14, and 1–35 days of age. High stoking density groups consumed more feed (P < 0.05) during the first 2 weeks, while the normal stocking density groups showed higher (P < 0.05) cumulative (1–35 d) feed consumption. On the other hand, the addition of essential oils did not affect (P > 0.05) birds’ feed intake. There were significant differences (P < 0.05) in cumulative feed intake over the 1–7, 1–14, and 1–35 days as shown. At 1–7 days of age, the lowest cumulative feed intake was in HSD + EO where HSD had the highest (P < 0.05) intake, at 1–14 days NSD + EO had the lowest cumulative feed intake followed by NSD, while both HSD + EO and HSD were similar. At 1–35 days, HSD and HSD + EO had the lowest cumulative feed intake, followed by both the normal stocking density groups.

Table 3 Means for feed intake (g) of broilers as affected by stocking density and essential oils (EOs).
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Adjusted feed conversion ratio (AdjFCR)

Adjusted feed conversion ratio is shown in (Table 4). Normal stoking density groups have shown lower (P < 0.05) AdjFCR during the starter phase compared to their high stocking density counterparts. The addition of essential oils has improved AdjFCR from 1–7 and 1–14 days of age, where supplemented essential oils groups have shown lower (P < 0.05) AdjFCR. For the 1–7, 1–14, and 1–35 days of age there were significant differences (P < 0.05) as the lowest AdjFCR were observed at HSD + EO group (1.32) compared to the highest HSD (1.53) for 1–7 days. For the 1–14 days of age, NSD + EO had the lowest AdjFCR (1.15), while NSD and HSD + EO were similar and lower than HSD. For 1–35 AdjFCR, the lowest value was for NSD + EO (1.52), while NSD and HSD + EO were similar, and HSD had the highest AdjFCR.

Table 4 Means for Adjusted feed conversion ratio of broilers as affected by stocking density and essential oils (EOs).
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Body weight and cumulative body weight gain

Body Weight and Cumulative Body Weight Gain (cBWG) are presented in (Table 5). Cumulative BWG and the final BW were lower (P < 0.05) for the high stocking density groups. The effect of essential oils was significant during the starter phase, EO supplemented groups gained more (P < 0.05) weight compared to non-supplemented groups. Final body weight was higher for the NSD and NSD + EO groups (P < 0.05) compared to HSD and HSD + EO groups. For the first 7 days of age the highest cBWG was for HSD + EO, followed by both NSD and NSD + EO, then HSD as the lowest. For 1–14 days of age there was no significant difference on cBWG among all groups. The cBWG at 1–35 days of age was higher (P < 0.05) for NSD + EO and NSD compared to HSD + EO, and HSD groups.

Table 5 Means for body weight, and cumulative body weight gain (cBWG)g of broilers as affected by stocking density and essential oils (EOs).
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Carcass and meat quality traits

Carcass traits

Carcass traits are presented in (Table 6). Stoking density showed a significant effect on Leg, wings, and fatpad percentages. The Leg % was higher (P < 0.05) for the normal stocking density groups, while wings and fatpad percentages were higher (P < 0.05) for the high stocking density groups. No effect of the essential oils’ addition was detected for carcass traits. The Legs % were the highest for NSD group followed by NSD + EO and HSD + EO, and HSD respectively. Lastly, there was significant difference (P < 0.05) on fatPad % as HSD + EO had the highest percentage while NSD, HSD, and NSD + EO were similar.

Table 6 Means for carcass traits of broilers as affected by stocking density and essential oils (EOs).
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Meat quality traits

Results of meat quality are presented in Tables 7 and 8. Stocking density showed no effect on all meat quality traits. The essential oils receiving groups showed lower (P < 0.05) cooking loss compared to non-supplemented groups. Looking at the interaction effect, there were no significant differences in Breast Fillet %, pH, l, a, b, Hue, and Chroma. However, there was a significant difference in CL % as both HSD + EO and NSD + EO had the lowest percentage. On the other hand, there was significant difference in WHC % as HSD + EO, NSD had the highest percentage, followed by NSD + EO, and HSD. The SF there was the highest (P < 0.05) for HSD group where all groups were the same.

Table 7 Means for meat quality traits of broilers as affected by stocking density and essential oils (EOs).
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Table 8 Means for color coordinates of broilers as affected by stocking density and essential oils (EOs).
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Blood biochemical indicators

The blood biochemical indicators results are shown in Table 9. Stocking density has significantly affected Serum IGF-1. The normal stocking groups had higher (P < 0.05) serum IGF-1 compared to high stocking density groups. The essential oil supplemented groups had higher levels of serum mTOR and AST (P < 0.05). There was a significant difference (P < 0.05) on serum IGF-1 between HSD + EO and HSD. Also, levels of mTOR and AST were higher in the HSD + EO and NSD + EO groups compared to HSD, and NSD. Whereas there were no significant differences in ALT and CORT between all groups.

Table 9 Means for blood biochemical indicators of broilers as affected by stocking density and essential oils (EOs).
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Gene expression

The stocking density-essential oil interaction effect on gene expression is presented in Fig. 2. the expression of MSTN was upregulated in the HSD group and was significantly higher than NSD, NSD + EO, and HSD + EO groups. On the other hand, IGF-1 and MYO-G were both downregulated in the HSD group and their expression was lower (P < 0.05) than NSD, NSD + EO, and HSD + EO groups.

Figure 2
figure 2

Relative fold change of broilers as affected by stocking density and essential oils. Treatment: NSD = stocking density 9 birds/m2, HSD = 14 birds/m2, NSD + EO = stocking density 9 birds/m2 + essential oils 150 g/Ton, HSD + EO = stocking density 14 birds/m2 + essential oils 150 g/Ton. a,b,cMean values with different superscripts differ significantly (P < .05).

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Discussion

The main physical effect of stocking density is less space available for feed, respiration, and water, but what is hidden behind this effect is how the internal body responds to such competitive surroundings, which will result in variant cascades of several parameters. Most broiler businesses aim to develop unique solutions away from antibiotics as growth promoters, the use of essential oils for optimal broiler performance has been widespread. Essential oils were found to have a beneficial effect on growth and stress alleviation14. In the current study, high stocking density birds supplemented with essential oils showed lower feed intake, and FCR, and greater body weight gain than high stocking density without essential oils during the first 7 days of the rearing period. For both days 1–14 and 1–35 of the rearing period, the FCR in the HSD + EO treatment group was lower than that in the relevant group, which did not receive EO; moreover, the FCR in the HSD + EO group was similar to that in the NSD group. These findings agree with those of15 who also reported that supplementing phytogenic extracts improved the FCR in the high stocking density group. These findings are more related to the effect of plant passed products, in which it can enhance digestibility and allow the best utilization of nutrients by boosting the production of digestive secretions, stimulating the blood circulation, and decreasing the abundance of pathogenic bacteria16. In contrast17, reported no significant differences in growth performance in broilers supplemented with EO. These differences from our findings might be due to differences in the breeds of the broilers raised, as they have used Cobb 500, as well as differences in the composition of the essential oils used.

In the present study, the main differences in carcass traits were found in legs %, and fat pad between HSD + EO and HSD whereas there were no significant differences on carcass weight, dressing percentage, or any other carcass traits. In contrast18, reported a significant difference in carcass weight, as for legs %, they stated that legs % was affected when stocking density was increased compared to normal stocking density. in the current findings HSD + significantly affected leg percentage compared to HSD. Conversely, supplementing essential oils did not affect the fat pad percentage19,20.

In the current findings, both groups that received EO had lower cooking loss and greater water holding capacity than did those that received NSD or HSD, which supports the positive effect of EO in enhancing cooking loss and water holding capacity even when stocking density is increased4. Contrary to the current results, supplementing EO did not alter the water loss rate even at increased stocking densities21, which might be due to the difference in stocking densities used in the experiment, as they used 10 and 20 birds/m2 compared to the stocking density used in the present study. Furthermore22, reported that the use of certain EOs lowered cooking loss and enhanced water holding capacity when added to a broiler diet. However23, reported no difference in water holding capacity when using EO, which might be related to the composition of the EO used in their trial. Increasing stocking density did not affect Warner–Bratzler shear force and cooking loss, cooking loss and water holding capacity2,24. In the present study, the addition of EO and change in stocking density did not affect the color parameters; these results indicate that the use of stocking density did not alter blood circulation, which could affect the color coordinates of the meat. Additionally, there was no significant difference in the effect of adding EO on color coordinates (l, a, and b) when the same EO was used19.

An increase in AST levels in the blood can indicate liver damage and lesions in bile ducts, among other conditions. However, the synthesis of AST from other tissues, such as muscles, should be considered25. AST is considered to be a transaminase enzyme that catalyzes the reversible transfer of an α-amino group from aspartate to α-ketoglutarate to create oxaloacetate26. The formation of oxaloacetate can be used in gluconeogenesis via phosphoenolpyruvate carboxykinase (PEPCK), which is catalyzed in the liver or returned to the mitochondria as malate27. Malate has a key role in increasing serum glucose levels28 which can decrease feed intake by increasing the amount of available glucose for the body and hence improve the FCR. In the current findings, AST was significantly greater in both groups that received essential oils, which suggested that such an increase could be related to increased glucose availability, which aided in improving the FCR, especially in the HSD + EO group. Also29, reported that high stocking density increases AST in broilers, which contradicts our recent findings, as increasing stocking density did not significantly affect AST. This finding is more related to the stocking density used in our trial, which was 14 birds/m2, than to the density at which birds were subjected to 22 birds/m2, which indicates that 14 birds/m2 cannot negatively affect the AST metabolic pathway. Additionally, this increase in AST can be related to how it increased whether metabolic supportive additives, such as essential oils, enhanced the body’s response to such positive impacts, which might be due to the mitochondrial use of oxaloacetic acid as malate when these essential oils were used or negatively related to extensive stocking, as accumulation in the liver due to less oxygen, water and feed would be available, creating oxidative stress and more muscular damage. There were no significant differences in the corticosterone or ALT concentration between the treatment groups. However15, reported a significant difference in high stocking density, which can be related to the higher stocking density used The present findings revealed that the serum and mRNA expression levels of IGF-1 were the same in the HSD + EO, HSD-positive, and NSD groups compared to the HSD group, which had the lowest serum and mRNA expression levels of IGF-1. These findings confirm the benefit of adding essential oils in escaping the adverse effects of stocking density on growth even at increased stocking density and enhancing the genetic expression of IGF-1. Concurrently30, reported that supplementing EO increased the serum IGF-1 concentration, which can enhance protein synthesis. Similarly15, reported that adding curcumin increased the mRNA expression of IGF-1 even when birds were subjected to a high stocking density, which agrees with the current work showing that EO has a positive impact on growth-promoting effects on skeletal muscular growth, as EO increased the percentage of legs in the HSD + EO group compared to its relevant form. One of the key signaling pathways that can affect muscle synthesis is the mTOR pathway, which interferes with muscle development in combination with IGF-131, as mTOR is a serine/threonine kinase that induces different intercellular and environmental changes, such as changes in energy status and nutrient availability, and organizes several cellular processes, such as cell growth, survival, and differentiation32. According to the current findings, the serum levels of mTOR and IGF-1 were significantly greater in both groups that received EO, which confirmed that EO enhances the signaling pathway involved in protein synthesis. In the present study, the mRNA expression of MSTN was the highest in the HSD group, while no differences in MSTN expression were detected among the other treatment groups, which agrees with the findings of15, who also reported that supplementation with curcumin reduced MSTN gene expression in different groups of HSDs. Concurrently, MyoG expression was the lowest in the HSD group, while the other groups did not differ. These findings support the role of EO in improving MyoG mRNA expression by lowering MSTN levels at high stocking densities, as both are antagonistic to each other33. In addition8 used sodium butyrate in the HSD group, and their findings showed that the supplement lowered MSTN and increased MyoG.

Conclusion

In summary, the resulted investigations showed that, EO did improve some of growth parameters in terms of feed conversion ratio and legs percentage in terms of carcass trait even when stocking density is increased and alleviate negative impact of stocking density on both prementioned parameter, as for meat quality trait WHC, CL, and SF were both enhanced by adding EO with HSD. In addition, blood biochemical indicators such IGF-1 and mTOR were significantly differed when stocking is increased by adding EO synchronically with skeletal muscles of legs percentage enhancement. Also, AST were highest in groups had EO which can declare the method of alleviation of such parameter can be related of mechanism by which is raised, while no difference on CORT for oxidative stress’s sake with HSD of 14 birds/m2, whereas adding EO to such stocking did downregulate gene expression of MSTN, and up regulate IGF-1, and MyoG. More interaction between the intensity of stocking and EO must be investigated to understand how broiler body responds by evaluating the prementioned parameters.