Evaluations of Some Natural Antioxidant Sources in Broiler Diets: 1-Effect on Growth, Physiological and Immunological Performance of Broiler Chicks

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Evaluations of Some Natural Antioxidant Sources in Broiler Diets: 1-Effect on Growth, Physiological and Immunological Performance of Broiler Chicks
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  International Journal of Poultry Science 12 (x): xx-xx, 2013ISSN 1682-8356© Asian Network for Scientific Information, 2013 Corresponding Author:  N.A. Selim, Animal Production Research Institute, ARC, Dokki, Giza, 12618, Egypt 1 Evaluations of Some Natural Antioxidant Sources in Broiler Diets: 1-Effect on Growth, Physiological and Immunological Performance of Broiler Chicks N.A. Selim, S.F. Youssef, A.F. Abdel-Salam and Sh.A. Nada 1   1   2   2  Animal Production Research Institute, ARC, Dokki, Giza, 12618, Egypt 1 Regional Centre for Food and Feed, ARC, Egypt 2 Abstract:  This study was conducted to evaluate the efficiency of aqueous extract of ginger root (GAE),aqueous extract of beetroot (BAE) and tomato puree (TP), as natural antioxidant sources, in broiler dietsduring summer season. Chicks fed on corn-soybean meal basal diets in three phases feeding system.Basal diets were formulated to contain whole strain nutritional requirements including 50 IU of vitamin E/kg.There were two control groups the first fed on basal diet (Con) and supplemented with either 50 IU/kg vitaminE (E-100, to be the second control group), or supplemented with GAE, BAE or TP at levels of 0.5 or 1% from1 to 40 d of age. The total phenols content (in Gallic acid equivalent) of GAE and BAE were 44 and 31 µg/L,respectively and lycopene content in TP was 155 mg/kg. The environmental temperature and humiditysurrounding birds during the experimental period ranged from 35 to 41°C and 30 to 45%. Growthperformance, some plasma constituents and viral and bacterial immunity status at 40 d of age wererecorded. The results indicated that, among examined natural additives, using BAE improved the overall bodyweight gain, while adding GAE to broiler diets decreased feed consumption significantly. However the worstfeed conversion ratio recorded by TP group, that group recorded the best antioxidant status including plasmatotal antioxidant capacity and malondialdehyde. Generally, level 1% of supplementation increased theconsumed feed and depressed the total bacteria count of intestine at 40 d of age. On the other side, titer of immunity against respiratory viral Diseases (ND and IB), hemoglobin, red and white blood cells increasedby adding GAE to diets. Neither antioxidant source, level nor their interactions could change lipids or cholesterol compounds in plasma. The overall results indicated that the best overall broiler performanceduring summer season was obtained by using GAE at level of 1% as natural antioxidant. Key words:  Broiler, antioxidant, ginger, beetroot, tomato, summer season, performance INTRODUCTION During normal metabolism, the antioxidant defensesystem, natural and synthetic antioxidants and theantioxidant enzymes, can mitigate free radicals areproduced. These radicals cause several adverse effectson body if they are present in excessive levels (oxidativestress) under stressful environments such as heatexposure (Sies, 1991). Oxidative stress has beenregarded as one of the major factors negativelyaffecting performance of birds in the Condensedpoultry industry (Lin et al  ., 2006) and as a mainfactor in the pathogenesis of several serious diseases(Kris-Etherton et al  ., 2004). Therefore, supplementationof synthetic antioxidants (e.g., " -tocopheryl acetate or butylated hydroxy toluene) to relieve the oxidative stresshas become a common practice in the poultry industry.Recently Ahn et al  . (2002) showed that, use of plantextracts as natural antioxidants has gained increasinginterest because of the global trend of restriction in useof synthetic substances (Zhang et al  ., 2009). It has beenreported that heat stress increases lipid oxidative stressperoxidation and depresses growth in birds (Sahin andKucuk, 2003). Sahin et al  . (2013) showed that, althoughexposing to high environmental temperatures can leadto oxidative stress associated with a reduced antioxidantstatus, reflected by increased oxidative damage andlowered plasma concentrations of antioxidants, usingantioxidants in poultry feed including phytochemicalscan alleviate these negative effects of highenvironmental temperatures.Ginger ( Zingiber officinale ) has been widely used as acondiment and as a herbal medicine to treat a widerange of disorders (Ali et al  ., 2008). Ginger roots containa number of biologically active components whichreported to be phenylpropanoid-derived compoundsincluding gingerols and shogaols (Kundu et al  ., 2009).These compounds exert varying biological activities,including antioxidant (Nakatani, 2000; Rababah et al  .,2004), antimicrobial (Sasidharan and Menon, 2010) andvarious pharmacological effects (Ali et al. , 2008; Ling et al  ., 2010). Chrubasik et al  . (2005) presented a review inand suggested that the preparation method of ginger product affected its clinical efficacy. When ginger is driedgingerol undergoes a dehydration reaction formingshogaols, which are about twice as pungent as gingerol(Ling et al  ., 2010).  Int. J. Poult. Sci., 12 (x): xx-xx, 2013 2Ginger has been used in poultry diets in many formsThis study aimed to examine the effect of three naturalsuch as dried ginger powder (Awadein et al  ., 2012;Malekizadeh et al  ., 2012), essential oil (Jang et al  ., 2007)and aqueous extract (Sudarshan et al  ., 2010) andshowed different enhancements affects such as,improving feed conversion ratio by addition of ginger at0.25% to broiler diets (Onu, 2010). Also, Zhang et al  .(2009) reported increased activities of antioxidantenzymes, glutathione peroxidase (GSHPx) andsuperoxide dismutase (SOD) and reduced level of Malondialdehyde (MDA) in broilers fed on dietsupplemented with ginger at the rate of 5 g/kg.Betanines or betalains are natural dye extracted frombeetroot to use in different food industries. Recentstudies have shown that betalains have antioxidant,antimicrobial and antiviral activity (Zhong et al  ., 2005;Pedreno and Escribano, 2001). Although, someresearcher reported that the bioavailability of betalains is at least as high as flavonoids, which arewell-accepted as natural antioxidants (Romero et al  .,2010), using betalains as feed additive has been limitedbecause some factors, such as temperature and pH,influence on the pigment stability (Socaciu, 2008). Sodiet) or one of the natural antioxidant additives (GAE,betalains is exposed to degradation immediatelyafter extraction and the degradation is acceleratedby raising the pH, temperature and water (Sturzoiu et al  ., 2011). Recently, some studies modified theextraction and storage condition of beet root extractto get high concentration and more stable betalains(Socaciu, 2008).Tomato and tomato products are natural sources of antioxidant components (lycopene, ascorbic acid,phenolics and flavonoids (Abushita et al  ., 1997; Agarwaland Rao, 1998), whereas Lycopene is the mostimportant one. Lycopene, a fat soluble carotenoid, is aprecursor of $ -carotene (Sandmann, 1994; Atasoy,2012) and has an effective free radical scavenging Di-calcium phosphate1.841.671.59 activity at least twice that of $ -carotene (Di Mascio et al  .,1989) and a potent antioxidant that has been shown toplay critical role in cancer prevention (Bunghez et al  .,2011) by lowering oxidative damage to DNA incancerous cells (Stacewicz-Sapuntzakis and Bowen,2005). Some workers used isolated lycopene, tomato or tomato products in poultry diets as antioxidant sourceand reported positive effects on growth performance andlipid peroxidation (Sahin et al  ., 2008), while others couldnot detect clear effect of lycopene on immune responseof laying hens (Oslon et al  ., 2008). Another group of investigators found that inclusion of tomato by product(tomato pomace) at level 30% of male chickensimproved seminal quality and decreased lipidperoxidation (Mangiagalli et al  ., 2010; Saemi et al  .,2012). Some reports determined increased availability Sodium0.150.160.17 of lycpene in tomato products which exposed to relativelyhigh temperature during preparation process of tomatoproducts such as paste and puree than fresh tomato(Anese et al  ., 1999; Agarwal et al  ., 2001; Capanoglu et al  ., 2008).antioxidant sources, ginger root aqueous extract (GAE),beetroot aqueous extract (BAE) and tomato puree (TP),on growth, physiological, microbiological andimmunological performance of broiler chicks duringsummer season. MATERIALS AND METHODS This work has done in Animal Production ResearchInstitute (APRI) and with collaboration with RegionalCenter of Food and Feed (RCFF), ARC, Egypt.Three hundred and twenty 1-d old unsexed Arbor Acresbroiler chicks were individually weighed and divided into8 treatments of 4 replicates each (10 chicks each).Chicks fed on corn-soybean meal basal diets which metthe strain requirements during staring (1-10 d), growing(11-24 d) and finishing (25-40 d) periods (Table 1).There were two control diets the first was controlbasal diet (Con) which contained 50 IU of vit. E/kgof diet and supplemented with either 50 IU/kg vitamin Eas " -tocopherol acetate (E-100, to be the second controlBAE and TP) at levels of 0.5 or 1.0% from 1 to 40 d of age. All birds were kept under similar managementconditions. The environmental temperature and humiditysurrounding birds were recorded daily during theexperimental period (Table 2). Table 1: Composition and calculated analysis of control basal dietStarterGrowerFinisher (1-10 day)(11-24 day)(25-40 day) Composition (per 100 Kg) Yellow corn52.2859.0563.19Soybean meal (44% CP)34.0026.7022.50Corn gluten (60% CP) bean oil3.003.004.00Lime stone1.431.201.10L-Lysine HCl0.320.310.28Dl-Methionine0.260.200.17Sodium chloride0.240.240.24Sodium bicarbonate0.230.230.23Vitamins premix* premix**0.300.300.30Total100.00100.00100.00 Calculated analysis (%) Crude protein23.1721.2519.04Metabolizable energy (Kcal/Kg)310031103207Ether extract5.635.086.88Crude fiber3.803.453.22Calcium 1.040.900.84 Av. Phosphorus0.500.450.43Lysine1.441.241.09Methionine0.680.600.54Methionine+cystine1.060.950.86*Supplied per kg of diet: Vit. A, 11000 IU, Vit. D3, 5000 IU, Vit. E, 50 mg,Vit K3, 3 mg, Vit. B1, 2 mg, Vit. B2 6 mg, B6 3 mg, B12, 14 mcg,Nicotinic acid 60 mg, Folic acid 1.75 mg, Pantothenic acid 13 mg andBiotine 120 mcg**Supplied per kg of diet: Choline 600 mg, Copper 16 mg, Iron 40 mg,Manganese 120 mg, Zinc 100 mg, Iodine 1.25 mg and Selenium 0.3 mg.  Int. J. Poult. Sci., 12 (x): xx-xx, 2013 3 Table 2:Environmental temperatures and relative humidity duringexperimental periodTemperature°CRelative humidity (%)------------------------------------------------------------PeriodMinimumMaximumMinimumMaximumStarting (1-10 day)37392055Growing (11-24 day)36413045Finishing (25-40 day)36373035Overall period (1-40 day)36412055 Antioxidant sources:  Aqueous extract of ginger andbeetroot were prepared in Poultry Nutrition DepartmentLabs before starting the growth trail while TP wasobtained from local commercial company. GAE:  It was prepared according to the method reportedby Kishk and El Sheshetawy (2010) to reach themaximum free radical scavenging activity (94.4%) and0.94 protecting factor, by extracting the dried ginger powder with water (0.72:100, W:V) at 60°C/24.5 min.Total phenols contents of the prepared GAE wasdetermined using Folin-Ciocalteu (FC) assay accordingto the method of Wright et al  . (2000) and Atoui et al  .(2005) and was 44 µg/mL in Gallic acid equivalent. BAE:  It was prepared according the method of Sturzoiu et al  . (2011) by extract the fresh beetroot withwater (1:5 W:V) at acidic media using ascorbic acid(1g/L)+citric acid (2g/L) at 25°C/3 min. Total phenolscontents of the prepared BAE determined by method of Wright et al  . (2000) and Atoui et al  . (2005) and was 31µg/L in Gallic acid equivalent. TP:  The determined value of lycopene content in TP was155 mg/Kg using procedures described by Bunghez et al  . (2011) using UV-Vis technique. Growth traits:  Live body weights and feed consumptionof chicks were recorded at 10, 24 and 40 days of age,then live body weight gains and feed conversion ratioswere calculated. Physiological traitsSlaughtering and Blood sampling:  At the end of theexperimental period (40 d of age), 4 birds/treatment(bird/replicate), around the average body weight wereslaughtered and three blood samples were collectedfrom each bird in heparinized tubes (2 complete bloodsample and 1 plasma sample/bird). One completeblood sample/slaughtered bird was used to determineblood hematological parameters, while the secondsample sent directly to the regional veterinary lab toexamine the immunological status. The heparinizedplasma samples kept at -20°C until the time of chemicalanalyses. Plasma chemical constituents:  One plasmasample/bird was used to determine Total Lipids (TL)and Total Cholesterol (TC), Low Density Lipoprotein(LDL), High Density Lipoprotein (HDL), Triglycerides(TG), Total antioxidant capacity (TAOC) andMalnodialdehyde (MDA) by colorimetric methods usinganalytical kits produced by Biodiagnostic Company. Immunological response:  At 40 d of age one wholeblood sample/bird (4 samples/treatment) were used toexamined the immune response to Newcastle DiseaseVirus (NDV) and Infectious Bronchitis Virus (IBV) bymeasuring titer against these viruses using preventingfrom hemangontinasion method and using commercialElisa Kits, respectively. These examinations werecarried out in Reference Laboratory for Veterinary QualityControl on Poultry Production, Egypt. Microbiological traits:  At the End of the experimentsmall intestine of slaughtered birds (4 birds/treatment)were collected and bacteriologically examined for detection of pathogenic bacteria, i.e., Escherichia Coli,Campylobacter and Salmonella  according toCollins et al  . (1998), Oosterom et al  . (1983) and Ellis et al  . (1976), respectively. Also count of total bacteria and Staphylococcus aureus  in small intestine were carriedout according to Gouda (2002). Statistical analyses:  Data of GAE, BAE and TPtreatments were analyzed by using two way analysis of variance to detect the effect of antioxidant source andlevel. Data of all experimental treatments, includingCon and E-100, were analyzed by using one wayanalysis of variance to detect the differences betweenthem. Variables showed significant differences at F-test(p = 0.05) were compared to each other’s usingDuncan's Multiple Range Test (Duncan, 1955). Thestatistical procedures were computed using SAS (1997). RESULTS AND DISCUSSION Growth performance:  The recorded environmentaltemperatures and relative humidity % during starting,growing and finishing periods, Table 2 showed thatthere were permanent exposing to high temperatureduring the overall experimental period and thiscontinuous stress had effect on the chicks’ performanceand their antioxidant system. Results of broiler growthperformance in Table 3 and 4 showed that there wereclear reduction in final body weight and feedconsumption during the experimental period, comparedto the ideal growth performance of Arbor Acres broiler chicks reported in strain catalog. This result iscommonly due to exposure chicks to high environmentaltemperature, while using of natural antioxidant sourceseither at 0.5 or 1% enhance growth performancesignificantly. Among examined sources of antioxidant,chicks fed diets supplemented with BAE recordedthe highest values of final live body weight (1731 g),  Int. J. Poult. Sci., 12 (x): xx-xx, 2013 4 Table 3:Effect of ginger aqueous extract (GAE), beetroot aqueous extract (BAE) and tomato puree (TP) on body weight (BW) and body weight gain(BWG) of broilersBWBWBWBWGSBWGGBWGFTBWG14 d (g)24 d (g)40 d (g)1-14 d (g)15-24 d (g)25-40 d (g)1-40 d (g) Main factorsAntioxidant source GAE17076416771295949131636 bbbabb BAE18378717311416049441689 aaaaa TP17376116561325888951615 bbbbb Supplemental level 0.5%17876816741365909061632   1.0%17377317021326009291661   P-value  Antioxidant 0.00370.19980.00480.00710.52070.04800.0054Level0.12920.71070.14450.12700.39100.15870.1450Interactions0.00800.00290.01380.00710.00750.22510.0135Mean of P-SE±1.676.539.891.675.78.329.88 Treatments Con16669015351245248451493 cddcccd E-10017074216601285729181618 bccbcbcbabbc GAE 0.516673016271255648961585 ccdccbbcc GAE 1.017579717281346229311687 bcabababcaabab BAE 0.518780317221456129191681 aaabaaabab BAE 1.017977017401375919701698 ababcaababaa TP 0.518177116741405909031633 ababcabcaabbcabc TP 1.016555216381245868871597 ccbccabbcc Mean of P-SE±1.415.858.761.415.197.358.76P-value0.00010.00010.00010.00010.00010.00320.0001Table 4:Effect of ginger aqueous extract (GAE), beetroot aqueous extract (BAE) and tomato puree (TP) on feed intake and feed conversion ratio of broilersFeed Intake (g) Feed Conversion Ratio-----------------------------------------------------------------------------------------------------------------------------------------------------------StarterGrowerFinisherOverallStarterGrowerFinisherOverall Main factorsAntioxidant source GAE1671081139625811.291.751.551.59 bbbccbb BAE1971025147827001.391.741.601.61 aabaaaabb TP1731038145926711.311.791.651.66 baabbaa Supplemental level0.5%1841020140526091.341.761.571.61 abbba 1.0%1741034148426921.321.761.631.63 baaab P-value  Antioxidant 0.00010.01610.00010.00010.00010.46970.03680.0064Level0.02970.00940.00010.00010.00010.87910.07930.1573Interactions0.00680.00010.00010.17240.00010.55950.02640.0237Mean of P-SE±2.373.095.846.690.0030.0170.0150.0098 Treatments Con1801011132025101.451.991.621.68 bcbddaaabab E-1001601041134525451.251.871.491.59 daddgabbc GAE 0.5162984138525311.301.771.571.61 dccdebcbc GAE 1.01711053140626311.281.731.541.57 cdabccfbcbc BAE 0.52031037143226721.401.721.591.60 aabbbcbc BAE 1.01911013152327231.391.771.611.63 abbaacbcbbc TP 0.51861039139826231.331.791.571.62 bcaccdbcbc TP 1.01611036152227191.301.791.731.71 daaaebcba Mean of P-SE±2.002.835.656.630.0030.0170.0140.008P-value0.00010.00010.00010.00010.00010.00320.00600.0003 body weight gain (1689 g) and feed consumptionsources at levels of 1.0 % to broiler diets increased feed(2700 g) at the end of experimental period (40 daysconsumption during the experimental period significantlyof age) compared to GAE and TP groups, calculated(p = 0.0001). Results of one way analysis of variancefeed conversion ratio of GAE group was (1.59) asbetween all experimental treatments showed that, livesignificant (p = 0.0064) as that calculated for BAE groupbody weight, body weight gain and feed conversion ratio(1.61) because GAE group consumed the lowestof broiler chicks were improved significantly by addingquantity of feed (2581 g) during the experimental period.any source of antioxidant except those of chicks fed dietOn the other hand the calculated feed conversion ratio ofsupplemented with 1% TP. Using GAE at level 1.0% inTP group was the worst value (1.66). Adding antioxidantbroiler diets resulted in the best feed conversion ratio  Int. J. Poult. Sci., 12 (x): xx-xx, 2013 5(1.57), without significant different with the calculatedsignificant reduction of Hb, RBCs, WBCs and L values,value of Vit.E-100 group which was 1.59 during theoverall experimental period.The aforementioned researches and reviews proved theadverse effects of high environmental temperature onnutrients digestibility, growth, health, welfare and survivaland product quality of poultry (Mashaly et al  ., 2004; Khan et al  ., 2012a,b; Sahin et al.,  2013) and the ability of some antioxidant sources to reduce these problems(Sahin et al  ., 2008, 2011). The reported improvement of growth performance had observed previously by usingginger or tomato powder as a source of antioxidant. Onu(2010) found that inclusion of ginger in broiler diets atlevel 0.25% increased the final body weight andimproved feed conversion ratio, also Tekeli et al  . (2011)reported the same trend of growth when using ginger at120, 240 or 360 ppm in broiler diets. In other researches, no significant improvements were detectedin body weight, feed intake or feed conversion ratio byusing ginger in broiler diets within range between 0.5and 4.5% (Ademola et al  ., 2009; Zhang et al  ., 2009;Kehinde et al  ., 2011). Also, Sahin et al  . (2008) reportedincreased growth performance of quail exposed to highambient temperature when fed on diets supplementedwith graded levels of tomato powder (0,0, 2.5 and 5%)and the improvement was correlated with thesupplementing level. The increasing in consumed feedof natural antioxidant supplemented groups in this studywas explained previously by improvement of palatabilityand quick digestive effect of the natural products byincrease secretion and activity of gastrointestinalenzymes (Zhang et al  ., 2009; Zhao et al  ., 2011). Physiological performance:  The presented results of Physiological Performance in Table 5 showed thatneither antioxidant source nor supplemented levelshad an effect on plasma lipid profile of broiler chicks at40 days of age. The determined values of plasma TLof chicks in Con and E-100 groups were lower thanthose of other groups. Results of plasma antioxidantstatus clearly showed that both supplementalantioxidant source and level had significant effect onplasma TAOC, while supplemental levels did notchange plasma MDA determined values. The bestresults obtained by using TP as natural antioxidantsource and this improvement increased by increasingthe level of supplementation from 0.5 to 1%. Amongexperimental treatments, Con group showed the worstantioxidant status, while E-100 group was as significantas TP groups. Adding GAE to broiler diets increased Hb,RBCs, WBCs and Lymphocytes (L) and decreasedHeterophil (H) of chicks significantly than determinedvalues for BAE and TP groups. Using antioxidants atlevel of 1.0% decreased H and H/L determined valuesand increased L values significantly. Among allexperimental treatments, birds of Con group showedwhile significant increase of H and H/L determinedvalues were obtained compared to some other treatments.Some researchers could not detect significant change inblood lipid profile (Nasiroleshami and Torki, 2010) or blood hematological parameters (Ademola et al  ., 2009;Kehinde et al  ., 2011) by using ginger in broiler diets. Onthe other hand, some workers recorded reduction of blood TC, LDL or HDL when used ginger extract at levels0.4 and 0.6% to drinking water for broilers (Saeid et al  .,2010), when used ginger in broiler diets at levels of 0.1and 0.2% (Mohamed et al  ., 2012) or when used ginger in layer diets at levels 1 and 3% (Malekizadeh et al  .,2012). The increase of RBCs in birds fed on BAE dietswas in agreement of those obtained in a study ondistribution of betalin pigment in red blood cells and theresistance of the cells to ex vivo induced oxidativehemolysis in humans. Results of micromolar basissupported the concept that betalin may offer antioxidativeprotection to the cells (Tesoriere et al  ., 2005). Ademola et al  . (2009) reported increase of serum WBCs of broiler fed on diet contains ginger at 1, 1.5 and 2%. Khan et al  .(2012) showed in his review that heat stress reducenumber of WBCs and the H/L ratio has been used as areliable indicator of stress in birds (Mashaly et al  ., 2004),also McFarlane and Curtis (1989) supported the viewthat the H/L increased under heat stress in broiler chicks. So the obtained significant increase in WBCsand reduction of H and H/L values in experimentalgroups compared with Con group indicating that thesetreatments are effective ways to reduce the harmful effectof heat stress on chick’s immunity and GAE is the bestsource of antioxidant when used at 1% of broiler diet.The reported enhancement of the antioxidant status andreducing plasma MDA level by using antioxidantsconfirmed the previous results of using antioxidantphytochemicals as ginger (Zhang et al  ., 2009; Akbarian et al  ., 2011; Saied et al  ., 2011) and tomato products(Sahin et al  ., 2008, 2011), which showed decreasedconcentrations of serum and liver MDA and increasedthe activities of antioxidant enzymes (GSHPx and SOD).Sadeghi et al  . (2012) reported that inclusion of ginger (0.75%) in broiler diets resulted in improvement of TAOCand decreased plasma MDA of challenged andunchallenged broiler chicks with Salmonella. The effectof diet supplemented with red beet leafs on antioxidantstatus of plasma and tissue was investigated in mice byLee et al  . (2009). Their results showed that levels of antioxidants (glutathione and $ -carotene) and theactivities of antioxidant enzyme (glutathione peroxidase)in plasma were considerably increased, suggesting thatantioxidant defenses were improved by red beet leafsdiet and increased resistance of lymphocyte DNA tooxidative damage.However most of previous researches could not detectthe smode of action of antioxidant phytochemicals
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