Pomegranate peel as a phytogenic in broiler chickens: Influence upon antioxidant, lipogenesis and hypotensive response

Abstract The aim of this study was to evaluate antioxidant, antihyperlipidemic and hypotensive properties of pomegranate peel (PP) on antioxidant status, fat deposition, lipid peroxidation and pulmonary hypertensive response in broiler chickens. A total of 375 one‐day‐old male broilers (Cobb 500) were randomly assigned to five treatments included dietary PP levels of 0, 2.5, 5.0, 7.5 and 10 g/kg. Supplementation of PP at 7.5 and 10 g/kg resulted in significant upregulation of hepatic catalase (p < 0.004) and superoxide dismutase1 (SOD1; p < 0.05), which reflected in decreased concentration of circulatory malondialdehyde (MDA). Dietary inclusion of PP at 7.5 and 1.0 g/kg significantly decreased serum concentrations of triglycerides (p < 0.004) and cholesterol (p < 0.006) with concomitant decrease in abdominal fat deposition (p < 0.05). The antihyperlipidemic effect of PP was mediated through down‐regulation of peroxisome proliferator activated receptor alpha (PPARα). Hypotensive effect of PP was also observed at 7.5 and 10 g/kg as reduced heart weight and the right‐to‐total ventricular weight ratio (RV/TV) and decreased mortality from pulmonary hypertension. The hypotensive property of PP was associated with increased concentration of serum nitric oxide. In conclusion, this study revealed antioxidative, antihyperlipidemic and hypotensive effects of PP at 7.5 and 10 g/kg in broiler chickens exposed to hypobaric hypoxia. Health‐beneficial effects of PP suggest this product as a promising multi‐functional phytogenic feed additive for broiler chickens.

Broiler chickens accumulate a high degree of polyunsaturated fatty acids (PUFA) in meat lipids and the abdominal cavity (Cui et al., 2012). This is a potential factor that exacerbates oxidative stress because double bonds are the target of ROS (Ahmadipour, Sharifi, et al., 2018;Ahmadipour, Hassanpour, et al., 2018). Oxidative stress has been known to be the principal trigger to the development of pulmonary hypertension syndrome (PHS; also known as ascites) in broiler chickens (Arab et al., 2006;Cawthon et al., 1999;Khajali & Wideman, 2016). High altitude limits oxygen availability and exacerbates oxidative stress by intensifying ROS production and results in faster developing PHS (Balog, 2003). Adverse effect of oxidative stress can be attenuated by inclusion of adequate supply of antioxidants in broiler nutrition. Antioxidant system controls ROS production and maintain the redox (antioxidant/pro-oxidant) balance.
Public choice toward the use of natural products has led to the use of natural feed additives such as natural antioxidants in poultry nutrition. Plant secondary metabolites exert a wide range of health benefits including antioxidant properties. Polyphenols are the biggest phytochemicals with strong antioxidant property (Gessner et al., 2017). Polyphenols also possess several beneficial properties such as antihyperlipidemeic, bactericidal and hypotensive properties (Neyrinck et al., 2013).
Pomegranate peel (PP), obtained from Punicagr anatum, often lefts over from production of the juice. It possesses potential health benefits due to a rich content of antioxidant polyphenols. Pomegranate peel contains the highest contents of polyphenols and flavonoids and the greatest antioxidant capacity compared to the seed and juice (Derakhshan et al., 2018). Sharifian et al. (2019) reported that dietary supplementation with PP extract at 250, 450 and 650 mg/kg linearly reduced the MDA concentration in breast muscle of broiler chickens under heat stress. A review of literature further corroborated that dietary inclusion of PP extract up to 300 mg/kg significantly increased total phenolic contents and antioxidant activity in the breast meat of broiler chickens (Rafiei & Khajali, 2021). An extract of PP has sometimes been added to fruit juices to increase shelf-life of juices (Salgado et al., 2012). Furthermore, PP has been used to improve the shelf life of chicken meat due to antioxidant and antimicrobial effects (Kanatt et al., 2010). In the present study, we used PP as a phytogenic feed additive to investigate its effects on productivity, antioxidant status, hepatic lipogenesis and occurrence of PHS in broiler chickens reared at high land (2100 m).

Birds and experimental
The study was carried out in an area with an altitude of 2100 m above

Treatments
A basal diet was prepared according to the Cobb 500 recommendations for broiler chickens and regarded as the control. The basal diet was then supplemented with 2.5, 5.0, 7.5 and 10 g/kg PP to make treatment groups. In the present study, pomegranate was collected from Ardal district, located in CHB province of Iran. The peels were manually removed, air-dried and finely ground for use in the experimental diets. Chemical composition of PP was assayed and presented in Table 1. Experimental diets were formulated to have similar metabolisable energy and protein contents (Table 2) and offered in mash form.

Measurements
Body weight gain and feed intake were recorded in 1-20, 21-40 and 1-40 days of age periods. Feed conversion ratio (FCR) was also calculated for the same periods. At 40 days, 10 birds were selected from each group for blood collection and processing. The selected birds had body weights within ∼ 5% of the average pen body weight. Blood samples (∼ 3 mL) were collected from brachial vein and centrifuged at 2500 × g for 10 minutes to separate sera. Serum samples were tested for total cholesterol (TC), triglycerides (TG) and malondialdehyde (MDA). TC and TG were measured using commercial laboratory kits following the manufacturer's manuals (Pars Azmoon, Tehran, Iran).
MDA, as biomarker of lipid peroxidation, was assayed according to Nair and Turner (1984). Nitric oxide (NO) was determined by the method described by Behrooj et al. (2012). Birds were then euthanised by CO 2 to determine carcass processing including weights of live body, carcass, heart and abdominal fat. The hearts were dissected to calculate the right ventricular-to-total ventricular weight ratio (RV:TV). Whenever RV:TV exceeded 0.25, it was considered as ascites (Izadinia et al., 2010; Khajali & Khajali, 2014).  . Table 3

Statistical analysis
The ANOVA procedure of SAS (2007) software was adopted to analyse the data in a completely randomised design. Treatment means were separated by the Duncan's multiple range test. Linear, quadratic and cubic effects of dietary PP inclusion levels were studied using orthogonal polynomial contrasts to compare the control with PP groups.  Table 5 indicates serum variables of broilers received different levels of PP in the feed. Regression analysis indicated a significant linear response for serum variables when PP included in broiler diets. Broilers received PP at 7.5 and 10 g/kg had significantly (p < 0.05) lower concentrations of MDA, TC and TG than the control. However, inclusion of PP at 7.5 g/kg significantly increased circulatory level of NO compared to the control. They also deposited less fat in the abdomen compared to their counterparts in the control group. Orthogonal contrast between the control vs. PP groups was significant for serum variables.

RESULTS
The effects of PP supplementation on carcass characteristics and ascites mortality of broiler chickens are shown in PPARα, peroxisome proliferator activated receptor alpha; CAT, catalase; SOD1, superoxide dismutase 1. 1 Orthogonal contrast.
The expression of PPARα, SOD1 and catalase genes in the liver of broilers was affected by dietary PP (Table 7). PPARα, catalase and SOD1 genes were linearly overexpressed in broilers fed PP at 7.5 g/kg relative to the control.

DISCUSSION
Reduced feed intake of birds when PP included at 7.5 and 10 g/kg could be expected, and it is associated with high crude fibre content of PP. Improved FCR in birds fed with 7.5 g/kg PP could be explained by reduced feed intake and compromised weight gain. This finding is in line with those of Sarica and Urkmez (2016)  α. PPAR-α is a transcription factor and it is a major regulator of lipid metabolism in the liver (Ahmadipour, Sharifi, et al., 2018;Fruchart, 2009). PPAR-α activation has shown to decrease triglycerides (TG) by increasing fatty acid β-oxidation and hepatic lipoprotein lipase expression (Fruchart, 2009). Aviram and Fuhrman (2001) also reported that pomegranate extract attenuated low-density lipoprotein oxidation. In birds, the liver is the main site of lipogenesis (Steven, 1996). Esmaillzadeh et al. (2006) reported that pomegranate extract decreased total cholesterol and LDL-cholesterol by modulating the activity of HMG-CoA reductase. In agreement with our results, Xu et al. (2009) demonstrated over-expression of PPARα by feeding pomegranate extract to rats.
Birds received PP at 7.5 and 10 g/kg had significantly lower heart weight and RV:TV. The RV:TV ratio is an index of right ventricular hypertrophy and reflects pulmonary hypertension (Ahmadipour, Hassanpour, et al., 2018). These results clearly indicate hypotensive effect of PP in broiler chickens, which could be associated with bioactive components of PP. Punicalagin, a bioactive component of PP, has shown to modulate vascular damage resulting from oxidative stress in hypoxic pulmonary hypertension (Shao et al., 2016). Furthermore, Pomegranate has been reported to induce endothelial nitric oxide synthase gene regulation in human coronary endothelial cells (Stowe, 2011). In the present study, serum NO concentration was significantly increased when PP included at 7.5 and 10 g/kg. This observation suggests the hypotensive effect of PP mediated through changes in circulatory level of NO. Khajali and Wideman (2010) have reviewed the role of NO in prevention of pulmonary hypertension in broiler chickens.
Consequently, mortality from pulmonary hypertension has been significantly decreased in the corresponding treatment groups compared to the control (6.6 vs. 14.6%).
Inclusion of PP at 7.5 and 10 g/kg was associated with reduced level of MDA. MDA is an index of lipid peroxidation and oxidative damage caused by ROS. ROS are highly influential factor in the pathogenesis of pulmonary hypertension (Zuo et al., 2014). Although ROS formation naturally occurs through various metabolic processes in cells, excessive amount of ROS is detrimental to the cell and subsequently the entire organism. This study revealed that PP is a strong free radical scavenger because of its antioxidant constituents such as polyphenols and anthocyanins. It has been shown that total phenolics content of PP extract was nearly 10-fold as high as that of pomegranate pulp extract (Li et al., 2006). This finding clearly indicates that PP contains more antioxidants than does flesh tissue of pomegranate. Antioxidant properties of PP to prevent lipid peroxidation (as reflected in reduced MDA level) may be associated with over-expression of antioxidant genes including Catalase and SOD1. PP at 7.5 g/kg significantly resulted in upregulation of catalase and SOD1. In line with this finding, Hasan et al. (2020) reported that Supplementing pomegranate extract to rabbit diet at 100, 150 and 200 mg/kg significantly increased the activity of SOD and GSH-Px compared to the control group.

CONCLUSION
Supplementation of PP at 7.5 g/kg could result in reduced hepatic lipogenesis through upregulation of PPRAα. Dietary inclusion of PP at 7.5 g/kg prevents lipid peroxidation and pulmonary hypertension in broiler diets. PP seems to be a promising natural antioxidant to be included in broiler diets.

ACKNOWLEDGEMENT
This research was funded by Shahrekord University, Shahrekord, Iran.

AVAILABILITY OF DATA AND MATERIALS
Date are ready upon request.

CONFLICTS OF INTEREST
The authors declare that there are no conflicts of interest.

ETHICS STATEMENT
The Ethical Committee of Shahrekord University Research Council approved all procedures used in the study in accordance with the standard of 1964 Declaration of Helsinki.