Immune response and antioxidant status of broilers as influenced by oxidized vegetable oil and pomegranate peel

Soybean oil was purchased from the market, was poured into a metal gallon for the oxidation process. In this study, soybean oil was warmed up for 18 hours at 180°C. The oils were also aerated by an air pump during the heating process. After the oxidation time, oxidized soybean oil was stored at −20°C to prevent further changes in oil composition. Previous researches showed that cold storage kept the chemical composition of these products [2]. The chemical characteristics of oxidized and fresh oil were analyzed after the oxidation process of soybean oil. For this purpose, the oil samples were drained into a laboratory bottle, labeled and transferred to the food analysis laboratory. In the laboratory, peroxide values, MDA contents, and anisidine value of oxidized oil and fresh oil were analyzed according to the methods of Harwood and Aparicio [17]. The chemical properties of oils are presented in Table 1.

Pomegranate peels obtained from the pomegranate processing factory were dried, were milled and prepared for the experiment. Before starting the trial, the chemical compositions of peels were measured in the animal food lab of Azad University of Shabestar (Table 2). The gross energy (GE) of pomegranate peel was measured through an adiabatic bomb calorimeter (Parr Instrument Company, Moline, IL, USA) [18]. Methionine, lysine and threonine of pomegranate peels was measured according to the method described by the AOAC [19] and Cooperative [20] using high performance liquid chromatography (HPLC) and the modification of PICO-TAG methods. AOAC methods were used to measure dry matter (DM), CP, ether extract (EE), crude fiber (CF), and ash of pomegranate peels [19]. Besides, the content of total polyphenol, hydrolysable tannins, and condensed tannins of pomegranate peel were estimated utilizing methods reported by Saleh et al. [21]. The pomegranate peels compound are shown in Table 2.

At 54 days of age, 10 roosters from a group of 20 Ross 308 roosters weighing 3,300 ± 50 g were placed in individual cages to determine their metabolizable energy [22]. The roosters were kept in individual metabolism cages at 18°C–24°C. Thirty grams of pomegranate peels were weighed in five replicates and poured into capped plastic dishes. The roosters fasted for 48 h to empty the gastrointestinal tract of food residues. All cages were divided into two parts: in the first part, the peel (30 g) was fed to five roosters using a Sibbald funnel, and the other five roosters fasted to estimate endogenous. Excreta from all cages were collected, weighed, and stored at −18°C for chemical analyses. All excreta were dried in a forced-air draft oven at 70°C for 48 h, and GE and nitrogen were estimated using the AOAC [19] method. The excreta CP, EE, DM, and ash contents were determined according to AOAC [19]. The zero nitrogen retention was corrected using 8.22 kcal/g of retained nitrogen. The pomegranate peel powder apparent metabolizable energy (AME), nitrogen-corrected apparent metabolizable energy (AMEn), true metabolizable energy (TME), and nitrogen-corrected true metabolizable energy (TMEn) were calculated using the following equations, and they are shown in Table 2 [18].

The research protocol utilized in this investigation was approved by the Animal Care and Use Committee of the Islamic Azad University (93/987-2014) [23]. The experiment was designed according to a 3 × 3 × 2 factorial arrangement, with factors i) the pomegranate peel powder (zero, 4 and 8 percent in diets), ii) the oxidized oil (zero, 2 and 4 percent in diets) and iii) the vitamin E (zero and 200 mg/kg). The commercial vitamin E in this experiment (Golbar-Chemi®; www.golbargroup.com, www.Golbar-chemi.com), is a powder vitamin E supplement that contains 5,500 IU vitamin E. Hence, a total of 1,080 day old, male Ross 308 strain broiler chicken, were randomly allocated to 90-floor pens in a completely randomized design with 18 treatments and five replicates with 12 chicks in each replicate. The chicks were fed commercial diets during the ten days of the experiment. At end of 10 d, the chicks were weighed and housed in 1.5 × 1.5 m floor pens each equipped with a pan feeder and manual drinker. Feed and water were available ad libitum. All chicks in the treatment groups were fed a grower (10–24 days) and finisher diet from (25–42 days). All diets were formulated based on nutritional requirements proposed by the NRC [24]. Also, the proximate composition of diets CP, EE and CF were measured according to AOAC [19] methods. The basal diets in this experiment were corn and soybean meal so that different percentages of pomegranate peel powder, oxidized soybean oil, and vitamin E were added to the basal diets. All diets are shown in Tables 3 and 4.

The performance of broilers in terms of body weight gain (BWG), FI, and feed conversion ratio (FCR) at the end of total rearing period (d 11–42) were determined. Production index (PI) for the total rearing period (d 11–42) was calculated [25].

A broiler from each pen was selected at the end of the experiment then five mL of blood samples were collected from the wing vein. All samples were centrifuged at 3,000×g for 15 minutes and serum samples were stored at 20°C for further analysis. The biochemical parameters including glucose, triglyceride (TG), cholesterol, LDL, high density lipoprotein (HDL), urea, uric acid, total protein [26], albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT) [27] and alkaline phosphatase (ALP) were determined by a Technicon RA-1000 auto-analyzer according to the manufacturer’s recommendations (Pars Azmoon, Tehran, Iran). In addition, the serum globulin concentration was calculated by the subtraction between total protein and albumin [23]. Enzyme activities, including serum superoxide dismutase (SOD) [28], glutathione peroxidase (GPx), and total antioxidant capacity (TAC) in serum were analyzed according to the method using the RANSEL and RANSOD kits (Randox Laboratories, Crumlin, Antrim, UK). The serum MDA concentration in the homogenates, was displayed as nmol/g, was determined by the Jo and Ahn [29] method. Furthermore, the catalase (CAT) concentration of blood was measured according to the method of Aebi [30].

On days 42, one broilers from each cage was selected and blood samples were collected from the vein wings. Blood samples were drained into tubes with anticoagulant (EDTA one mg/mL blood). Red blood cell (RBC) and white blood cell (WBC) counts were determined utilizing the method described by Natt and Herrick [31], and pack cell volume (PCV) of blood was measured by hematocrit tubes. Besides, according to the Cyanmethaemoglobin method, as shown by Benjamin [32], hemoglobin [33] was determined [23]. Also, blood smears were made to count lymphocytes and heterophils, which was measured.

Three milliliters of 5% suspension of sheep red blood cells (SRBCs) were injected into two broilers per pen for immunoglobulin G (IgG) and immunoglobulin M (IgM) determination at 35 days, and blood was obtained from the wing vein at the end of the experimental period. All samples were centrifuged, and all sera were properly labeled and stored at −20°C for further studies. To analyze the total anti-SRBC antibodies, all sera were inactivated at 56°C for 30 min [34]. All samples were titrated for whole and mercaptoethanol (ME)-resistant (IgG) anti-SRBC antibody titers. ME-sensitive (IgM) antibody titers were achieved by reducing the level (titer) of the IgG antibodies from the total antibodies, which was defined in terms of Log 2. [2].

At the end of the experiment, one broiler from each pen was slaughtered and the breast muscle was trimmed and stored at −20°C in the freezer. Methods of Ghasemi-Sadabadi et al. [2] were evaluated as the best for measuring the amount of MDA.

Statistical analyses were performed using SAS software. The data were analyzed using a 3×3×2 factorial arrangement design. Significant differences between treatments were separated using the Tukey range test at (p < 0.05). Also, linear and quadratic effects in this experiment were analyzed by using SAS statistical software [35].

The effects of using different level of pomegranate peel powder, oxidized oil and alpha-tocopherol in the diet on performance in broiler chickens are shown in Table 5.

The results showed that feeding 8% pomegranate peel in diets redacted BWG, FI, FCR, and PI compared to other groups (p < 0.05). The results indicated that broilers fed diets with 4% waste oil had significantly lower BWG and FI when compared to broilers fed diets with 2% waste oil (p < 0.05). Whereas, there was no significant difference between the control group and 2% waste oil supplemented treatments. As can be seen in tables, when vitamin E was supplemented into the diets BWG was increased between groups during total rearing period (p < 0.05).

The data demonstrated that when using 8% pomegranate peel in diets, growth performance of broilers. The results were quite similar to the results of [36]. However, no retarded growth was observed when using the lower inclusion level of 4% pomegranate peel. Abbas et al. [10] has reported that 7.5% pomegranate peel inclusion in diets did not affect growth performance of Japanese quail. Growth performance of the birds at high inclusion levels of pomegranate peel may be related to the high amount of tannins and CF of pomegranate peel [21]. It had been showed that polyphenol concentrations such as tannins with the addition of pomegranate peel diminished growth performance in broilers [1].

In this study, the data demonstrated that the supplementation of 4% waste oil in diets significantly reduced BWG and FI. The researchers showed that feeding of 4% waste oil in diets reduced the growth performance of Japanese quail [2]. Previous research has shown that the supplementation of oxidized oil in diets jeopardizes growth performance [5,37,38]. It seems that the negative effect on performance with the inclusion of dietary oxidized oil may be related to higher peroxide value of waste oil [27]. The dietary 200 mg vitamin E improved BWG in the current experiment. Other reports have also shown a beneficial effect of vitamin E supplementation on broiler growth performance.

The effects of using different level of pomegranate peel powder, oxidized oil and alpha-tocopherol in the diet on blood enzymes activity in broiler chickens are shown in Table 6. Also, linear and quadratic effects of pomegranate peel powder, oxidized oil and alpha-tocopherol and blood enzymes activity had shown in Table 7.

Based on the results obtained from this experiment, it was indicated that blood aspartate transaminase (AST), TAC, SOD, GPx, and MDA concentration in broilers were significantly affected by different level of pomegranate peel powder at end of experiment (p < 0.05). Broilers were fed 4% pomegranate peel powder indicated significantly lower AST and MDA compared with 8% pomegranate peel and control group (p < 0.05). The blood TAC, SOD, and GPx significantly increased by using 4% pomegranate peel in diets than other groups (p < 0.05). In addition, regarding the data, the AST, ALT, TAC, SOD, GPx, CAT, and MDA concentration was also affected by dietary oxidized oil (p < 0.05). The inclusion of 4% oxidized oil in diets significantly increased AST, ALT, and MDA in serum (p < 0.05). However, significant reductions were observed in the TAC, SOD, GPx, CAT on 4% oxidized oil treatment than other groups (p < 0.05). Although there was a linear tendency to increased total AST and ALT concentrations, and decreased TAC, SOD, GPx, CAT with oxidized oil supplementation in the diet. Further, the inclusion of 4% oxidized oil in diets quadratically increased MDA concentration in blood. The current study finding indicated that the supplementation of alpha-tocopherol significantly affected blood TAC, SOD, GPx, and MDA concentrations at the end of the experiment (p < 0.05). The addition of 200 mg/kg alpha-tocopherol in diets resulted in higher blood TAC, SOD, and CAT concentration (p < 0.05). Whereas, the concentration of MDA was lower at 200 mg/kg alpha-tocopherol compared to not supplemented group (p < 0.05). Also, the supplementation of the alpha-tocopherol in diets had linearly affected the TAC, CAT, and MDA concentration in broilers (p < 0.05). Overall, the interaction was not observed between pomegranate peel powder, oxidized oil, and alpha-tocopherol in this study.

Serum AST enzyme concentrations were reduced by dietary 4% pomegranate peel powder in this study. A reduction in AST concentration was also reported by Abdel Baset et al. [39] and Abbas et al. [10], where liver enzymes declined in pomegranate peel treatment. Therefore, Abbas et al. [10] reported that the addition of 2.5% pomegranate peel powder to diets reduced liver enzymes in Japanese quails. In contrast, Saleh et al. [21] stated that supplementation with 3 g/kg pomegranate peel powder did not affect glutamic oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) enzymes. A previous study confirmed that phenolic compounds result in a decrease in AST concentrations in birds [2]. Hence, it seems that the antioxidant activity of pomegranate peel powder may protect the liver against oxidative stress, which inhibits liver tissue damage [36].

The results indicated that broilers fed 4% pomegranate peel powder had higher serum TAC, SOD, and GPx concentrations than the other groups. Overall, the body antioxidant defenses such as GPx, SOD, CAT, and other antioxidant products are visible in various tissues and serum at different concentrations and activities [40]. Similarly, Saleh [41] reported a marked improvement in the concentration of SOD in the serum of broilers fed pomegranate peel. In addition, Saleh et al. [21] noted that broilers fed the pomegranate peel diet had higher serum SOD and GPx concentrations than those fed the low-level pomegranate peel powder, although the development rate was not statistically significant. The high concentration of SOD and CAT in the body leads to improved protection of cell membranes against oxidative stress and ROS [40]. Pomegranate peel contains phenolic and antioxidant compounds [10], which may improve the antioxidant status of broilers by increasing antioxidant enzyme activity [2,42]. Verma et al. [43] reported that herbal products include antioxidant compounds that directly inhibit lipid oxidation in tissues. Antioxidant enzymes can neutralize the free radicals of different types of oxygen. Hence, the use of pomegranate by-products in diets reduces protein and DNA damage in the body, which probably prevents the production of oxygen free radicals in broilers [44].

The phenolic compounds of herbal products directly cleanse free radical species and improve the cell’s other defense systems, which activate antioxidant enzymes such as SOD and CAT in the body and increase antioxidant defense. Generally, it has been suggested that hepatic antioxidant enzymes such as SOD and CAT play an important role in oxidative stress protection, and these enzymes catalytically scavenge ROS in tissue, thereby increasing endogenous health mechanisms [45].

In our investigation, dietary supplementation with 4% pomegranate peel powder resulted in lower MDA concentrations compared with other groups, which is in agreement with earlier studies in birds [21,36]. A previous study demonstrated that serum MDA concentration was reduced in response to antioxidant products in broilers [2]. It has been shown that with enhanced serum SOD and GPx activity, serum MDA concentration was reduced by supplementation of antioxidant compounds into broiler diets [46].

Dietary oxidized oil resulted in higher AST and ALT concentrations in this study, in agreement with Ghasemi-Sadabadi et al. [2], who demonstrated that the addition of 4% high peroxide value soybean oil in broiler diets raised the serum AST and ALT concentrations. Furthermore, dietary intake of 4% oxidized oil has been reported to increase oxidative stress in the body [5]. Oxidative stress can affect liver function because higher oxidative stress causes an imbalance between the defense mechanism of the body and ROS production, which increases the serum AST and ALT concentrations in broilers. Similarly, Hori et al. [47] reported damaged liver tissue in rats exposed to high oxidative stress. They also showed that higher oxidative stress increased the AST and ALT concentrations. Higher AST and ALT concentrations are associated with hepatic or muscular disorders of the liver [48].

The results showed that broilers fed with 4% oxidized oil had lower serum TAC, SOD, and CAT than the other groups. However, the group that received 4% oxidized oil showed increased serum MDA concentration compared to the other groups. The results of various studies have confirmed that oxidized dietary oils reduce the concentrations of serum antioxidants and increase serum MDA concentrations [37,49]. Consistent with the conclusions of previous investigations [7], oxidized oil reduced the activity of blood antioxidant enzymes. Delles et al. [49] observed that the inclusion of highly oxidized oil in broiler diets reduced SOD, CAT, and GPx activity. It seems that the lower antioxidant enzyme activity in this study was due to oxidative stress. In addition, the researchers noted that enzyme activity in the body is associated with stress intensity [50]. Hence, lower antioxidant concentrations in the serum may be directly related to oxidative stress, which is produced by high amounts of oxidized oil [51]. MDA is the second principal lipid oxidation marker that is under the impression of lipid peroxidation and oxidative stress in the body. Similarly, the high serum MDA concentration in broilers fed oxidized oil can be associated with free radicals of lipid peroxidation in birds [7]. Moreover, the researcher demonstrated that the serum MDA concentration was significantly increased by using high peroxide value oil in mice [37].

In the present study, supplementation of 200 mg/kg alpha-tocopherol in diets increased the serum TAC, SOD, and CAT concentrations in broilers. Broilers fed with diets containing alpha-tocopherol were identified by their higher blood antioxidant enzyme concentrations [52]. Alpha-tocopherol is a fat-soluble vitamin, which is commonly appreciated for its ability to prevent free radicals and defend cell membranes from oxidation [53]. Published experimental data show that alpha-tocopherol improves hepatic antioxidant defenses and reduces lipid peroxidation in the body [52]. Lin et al. [40] showed that the use of 160 mg/kg alpha-tocopherol increased the SOD concentration in the brain tissue of birds. In addition, these researchers found higher CAT concentrations in the liver tissue. It has been shown that SOD has an essential function of defending cells against damage from oxidative stress, which converts superoxide to H₂O₂ and oxygen, while H₂O₂ is decreased by CAT and GPx to H₂O and oxygen. Furthermore, GPx is qualitatively necessary for maintaining low cellular H₂O₂ concentrations because this enzyme has a lower Km than the CAT antioxidant [40]. İnal et al. [54] reported a significant increase in the SOD and CAT concentrations by oral alpha-tocopherol therapy (200 mg/day). It was apparent that 200 mg/kg of alpha-tocopherol supplementation reduced the serum MDA concentration in this study. In agreement with this finding, the researchers observed that alpha-tocopherol supplementation decreased the MDA concentrations in birds [36,40]. Tekce et al. [55] demonstrated that the supplementation of phenolic compounds (essential oil) in the water had not affected thiobarbituric acid reactive substance (TBARS) values in the broiler. Alpha-tocopherol is the main antioxidant that protects against peroxidation. This vitamin eliminated hydroxyl, alkoxyl, peroxyl, and superoxide anion radicals and improved membrane health [36]. Sahin et al. [56] indicated that supplementation with alpha-tocopherol in diets reduced the serum and liver MDA concentrations in Japanese quails.

The effects of using different level of pomegranate peel powder, oxidized oil and alpha- tocopherol in the diet on blood biochemical parameters in broiler chickens are shown in Tables 8 and 9. Also, linear and quadratic effects of pomegranate peel powder, oxidized oil and alpha-tocopherol and blood biochemical parameters shown in Table 10.

The serum glucose, total protein, globulin and LDL concentration were significantly affected by feeding pomegranate peel powder in diets (p < 0.05), although there was a linear tendency to decrease these blood parameters. Further, the results indicated that the use of 8% pomegranate peel in diets had significantly lower serum glucose, total protein, and globulin concentration compared to other groups in this study (p < 0.05). Also, the addition of 4% pomegranate peel powder in diet reduced LDL concentration than control group. Results showed that serum glucose, triglyceride, cholesterol and LDL concentration had significantly affected by the utilization of oxidized oil in diets (p < 0.05). The serum glucose concentration tended to decrease linearly with increasing oxidized oil level in the diet, and also significantly lower serum glucose concentration had shown at 4% oxidized oil than the control group. The serum triglyceride and cholesterol concentration increased linearly at 4% oxidized oil treatment. Further, the LDL concentration showed quadric differences among oxidized oil treatment. Results mentioned that the addition of 4% of oxidized oil in diets significantly had higher blood lipids than other groups (p < 0.05). Both cholesterol and LDL concentration results were significantly affected by dietary alpha-tocopherol, hence the supplementation of 200 mg/kg alpha-tocopherol in the diet significantly decreased serum cholesterol and LDL concentration in broilers (p < 0.05). Besides, the supplementation of the alpha-tocopherol in diets had linearly affected the serum LDL concentration in broilers (p < 0.05). According to data, an interaction was not found between dietary pomegranate peel powder, oxidized oil, and alpha-tocopherol for the blood biochemical parameters. But, serum cholesterol concentration showed interaction affect between dietary pomegranate peel powder, oxidized oil, and alpha-tocopherol.

In the present study, serum glucose, total protein, and globulin concentrations decreased in response to the high amount of pomegranate peel powder, which is in agreement with the reports of previous studies These experimental results agree with those of Abass et al. [10], who reported a decline in the serum glucose concentration associated with 7.5% pomegranate peel powder in Japanese quails. Furthermore, Huang et al. [57] reported that supplementation with pomegranate peel extract decreased the blood glucose concentration in rats. A reduction in the blood protein was also found by Abdel Baset et al. [39], where the serum total protein concentration in the broilers decreased with 4 g of pomegranate peel powder. In contrast with our findings, Abass et al. [10] reported an improvement in the serum total protein concentration when using pomegranate peel powder in quail diets. Furthermore, it has been shown that pomegranate peel contains a high amount of CF and tannins, which may reduce gastrointestinal absorption and consequently diminish food intake [58]. It seems that low gastrointestinal absorption reduced blood metabolites, such as glucose and blood protein, in this experiment. According to previous research, the physiological and biochemical systems are changed by tannins [59]. Therefore, these compounds obtained from pomegranate peel have a negative effect on nitrogen balance, intestinal absorption of sugars and amino acids, and protein catabolism [60]. It has also been previously stated that tannins cause major changes in protein bioavailability in the body, which may decrease blood protein concentrations.

By examining the results, it can be concluded that the replacement of 4% pomegranate peel powder in the grower and finisher diets reduced the serum LDL concentration of broilers at 42 days of age. These outcomes are in agreement with earlier investigations reporting that the addition of pomegranate peel to diets decreased blood lipid concentrations in broilers [10,61]. Similarly, serum lipids decreased in broilers fed with a diet containing 7.5% of pomegranate peel powder Abbas et al. [10]. It is generally accepted that the phenolic compounds of pomegranate peel, such as punicalagin, punicalin, gallic acid, and elegiac acid, may affect serum lipid concentrations and other blood metabolites [62]. Furthermore, pomegranate peel contains powerful antioxidants that play an important role in the oxidation of lipids in the body [10]. Phenolic compounds protect serum lipids against oxidation [63]. In addition, pomegranate peel may accelerate cholesterol metabolism by modifying cholesterol transport via HDL; therefore, it reduces serum cholesterol and LDL concentrations in the blood [33]. Similarly, the researcher suggested that pomegranate peels had hypocholesterolemic and hypolipidemic impacts. They also state that pomegranate peel hindered lipid digestion, absorption, and metabolism [61]. It is similar to a high-fat diet with gallic acid supplementation, as phenolic compounds reduced triglyceride concentrations [64]. The low serum lipid concentrations in this study may be associated with active phenolic compounds in pomegranate peel powder, which have an inhibitory effect on pancreatic lipase activity inhibits fat absorption from the intestinal tract, and reduces fat aggregation in lipoid tissues [8,65]. Bayraktar et al. [66] indicated that the essential fatty acid mixture application was found to have no effect on the serum levels of very low-density lipoprotein (VLDL), glucose, total bilirubin, and TG in broilers.

In the current study, the serum glucose concentration decreased with the addition of 4% oxidized oil to the diet, which is in agreement with the reports of earlier studies performed on birds. Ghasemi-Sadabadi et al. [2] and Tavárez et al. [38] reported that the feeding of high peroxide value oil decreased serum glucose concentration in birds. Besides, Ghasemi-Sadabadi et al. [2] reported that the addition of 4% oxidized oil with 45.18, 101.99, and 146.03 meq/kg peroxide values in the diets reduced the serum glucose concentration than fresh oil. It seems that the low serum glucose concentration observed in this study may be associated with oxidative stress. Thus, the use of oxidized oil raises ROS counts, which react with proteins, lipids, and fat-soluble vitamins that reduce the nutrient value of food [38]. In addition, researchers have shown that the use of oxidized oil in diets has deleterious effects on body metabolism in birds [2]. In contrast, Açıkgöz et al. [27] observed no differences in blood glucose concentrations after the treatments when broilers were fed oxidized oil during the experiment. These differences in the reports of the studies may be attributed to the various concentrations of oxidized oil in diets and different peroxide values of oil [2].

The results demonstrated that by utilizing 4% oxidized oil in diets, the serum triglyceride, cholesterol, and LDL concentrations of broilers increased at the end of the experimental period. These conclusions are consistent with other experimental results, in which oxidized oil supplementation increased serum triglyceride, cholesterol, and LDL concentrations [67]. These results match those of Khan-Merchant et al. [67], who confirmed that oxidized oil enhanced serum cholesterol, triglyceride, LDL concentrations, and the rate of atherosclerosis. Kishawy et al. [8] reported that the inclusion of oxidized oil in broiler diets increased cholesterol, triglyceride, and LDL concentrations. In general, the increase in serum lipid concentration in broilers fed with oxidized oil was expected because oxidized oil increased oxidative stress in birds, which is directly related to the higher serum lipid concentrations [67]. It seems that the higher plasma triglyceride concentration is a consequence of the lower availability of polyunsaturated fatty acids (PUFAs). Furthermore, the increase in serum cholesterol concentration can reduce the liver uptake of cholesterol [27].

The results showed that supplementation with 200 mg/kg alpha-tocopherol in the diet decreased the serum cholesterol and LDL concentrations in broilers at 42 days of age. These findings are in agreement with those of previous studies reporting that alpha-tocopherol supplementation reduced cholesterol and LDL concentrations in broilers [68]. Similarly, Sahin et al. [56] discovered that alpha-tocopherol supplementation reduced blood cholesterol concentrations in Japanese quails under heat stress. Arslan et al. [68] concluded that raising the concentrations of alpha-tocopherol decreased the serum cholesterol concentration, but this reduction was not statistically significant. In addition, Franchini et al. [69] stated that the inclusion of 325 ppm alpha-tocopherol decreased serum cholesterol and triglyceride concentrations in broilers. Previous studies have demonstrated that the cholesterol concentration was low in turkeys by using alpha-tocopherol supplementation [69]. The addition of alpha-tocopherol to diets prevented atherosclerosis in poultry. However, they reported that the serum cholesterol concentration was not affected by alpha-tocopherol [70].

The effects of using different level of pomegranate peel powder, waste vegetable oil and alpha-tocopherol in the diet on blood hematology in broiler chickens are shown in Table 11. Also, linear and quadratic effects of pomegranate peel powder, waste vegetable oil and alpha-tocopherol and blood hematology shown in Table 12.