LEVERAGE OF SYNTHETIC VITAMINS (C AND E) ON ANTIOXIDANT ACTIVITY IN BROILERS CHALLENGED WITH E. COLI

Rasha F. Muhammad 1 , Aida H. Soliman 1 and Essam S. Soliman 2* . 1. Department of Chemistry, Faculty of Science, Suez Canal University, Ismailia, Egypt 41522. 2. Department of Animal Hygiene, Zoonosis & Animal Behavior, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt 41522. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Vitamin E as a lipid component of biological membranes is known to be a major chain-breaking antioxidant and vitamin C or ascorbic acid is an anti-oxidant which is normally synthesized by the chicken. A comparative study was carried out to demonstrate the influence of synthesized vitamin C and vitamin E on bird performance, biochemical, antioxidant and immunological status. 150 broiler chick was received from day one age; divided into three groups; G1 (supplemented with synthetized vitamin C 3.5 g / 1 L water); G2 (supplemented with synthetized vitamin E 1 mL / 1 L water) and G3 (Control). G1 and G2 were challenged at 2 nd week of age with E. coli O 157 :H 7 1.3 X 10 6 CFU. A total of 180 samples (60 serum, 60 plasma and 60 intestinal swab) were collected during the study period. The results revealed that both synthetized vitamin E and vitamin C were able to enforce a protective effect in the birds last for only 4 week then vanished gradually. Synthetized vitamin E was superior to the vitamin C in enforcing a good protective effect on the birds as vitamin E showed more prominent stimulation of immunoglobulin (IgG; IgM. IgE) and bacterial counts were reduced significantly compared to the influence of vitamin C. The study concluded the necessity of the treatment continuation in order to maintain the enhancing effect on antioxidant enzymatic activity and protective immunological effect.

ISSN: 2320-5407
Int. J. Adv. Res. 5(4), 182-188 183 Vitamin C strongly recommended in poultry industry to alleviate stress. Although the birds were able to synthetize its own requirement of vitamin C, but during stress the requirements may exceed the synthetizing ability of the birds. Vitamin C supplementation in diets have many beneficial influences on blood parameters of broilers raised under heat stress (Ghazi et al., 2015). Vitamin C improved performance traits (body weight, body weight gain, feed conversion ratio, and performance index) and blood metabolites which in turn suppressed the negative effects of heat stress (Rafiee et al., 2016).
The excited environmental stressors as heat stress (Altan et al., 2003), high ammonia concentration, as well as cold stress (Bottje et al., 1998), contributed the shifting of oxidant-antioxidant equilibrium to a pro-oxidative status (Sahin et al., 2002). Depending on this fact; dietary supplementation of broilers with vitamin E, and vitamin C enabled the birds to restore xanthine activity (XO) without affecting NAD (P) H-oxidase (NOX) activity or Nitric oxide (NO) bioavailability. Xanthine activity (XO) may have the ability to produce superoxide and uric acid, and buffered the effects of superoxide in broilers grown under hypoxic condition (Bautista -Ortega et al., 2014).
The present study aims to evaluate the influence of supplementary synthetized vitamins (Vitamin C and E) on antioxidant activity [Superoxide Dismutase (SOD), Glutathione Peroxidase (GSH-Px) and Glutathione Reductase (GSH-Rx)], Blood chemistry and microbial load before and after challenging broilers with E. coli.

Material and Methods:-
Experimental birds and design:-150 broilers (one day old chicks) were purchased and habituated under the same environmental conditions. The birds were divided into three groups (G1, G2, and G3) according to the supplemented growth promoters (vitamins). G1 was supplemented with synthetized vitamin C (3.5 g / 1 L water) and G2 was supplemented with synthetized vitamin E (1 mL / 1 L water), and G3 was kept as control. G1 and G2 were challenged with E. coli O 157 :H 7 strain (ATCC™ 700728). The culture was diluted and added by 1.3 X 10 6 CFU / mL in drinking water. Sampling and sample preparation:-A total of 180 samples (60 serum, 60 plasma, and 60 intestinal swab) were collected in the study period. The samples were collected on a weekly basis starting from 2 nd week of age (zero sample), and three post challenge sample collections (P1, P2, and P3).
Whole blood samples (60 samples) were centrifuged at 4000 rpm / 10 min; aspirate off the plasma and stored at -80 o C. The erythrocytes were washed 3 -4 times with 5 mL saline 0.9% solution with centrifugation after each wash at 4000 rpm / 10 min. Finally the washed erythrocytes were resuscitated in cold distilled water, and the lysate were

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Intestinal swab (60 samples) were prepared by transferring 1 mL aseptically to a test tube containing 9 mL sterile 0.1% buffered peptone water (w / v) to prepare a dilution of 10 -1 , from which a tenfold decimal serial dilution up to 10 -6 were prepared to cover the expected range of contamination which could be easily counted. The dilutions were subjected to bacterial counts including Total Bacterial Count (TBC) and Total Enterobacteriaceae Count (TEC).  (1975,1980). Further identification may be recommended using pure colonies on nutrient slant that were extracted through using five typical colonies were selected and cultured onto Mac-Conkey agar plates, and incubated at 37 o C for 24 hrs, pure colonies on Mac-Conkey agar plates were inoculated onto nutrient slant and incubated at 37 o C for 24 hrs.

Results:-
Proteinogram as shown in Table 1, revealed that Total Protein and Globulins had a non-significant decrease (P<0.05) in G1, G2 compared to control; while Albumin showed a significant increase (P≤0.05) in G2 and a highly significant increase (P<0.01) in G1 compared to control. The liver and kidney profile in Table 2; revealed a nonsignificant decrease (P<0.05) in ALT in G1 and a non-significant increase (P<0.05) in G2 compared to control, while AST showed a significant increase (P≤0.05) in G1 and a significant decrease (P≤0.05) in G2 compared to control. On the other hand Urea showed a non-significant decrease (P<0.05) in G2 and a non-significant increase (P<0.05) in G1 compared to control, while Creatinine showed a non-significant increase (P<0.05) in all groups to control. Table 3; revealed a non-significant increase (P<0.05) in Triglycerides in G2 and a non-significant decrease (P<0.05) in G1, while Cholesterol showed a non-significant increase (P<0.05) in G1, G2 compared to control. Glucose showed a non-significant decrease (P<0.05) in G1 and a non-significant increase (P<0.05) in G2. Table 4; revealed that Glutathione Peroxidase had a non-significant increase (P<0.05) in G1 and a non-significant decrease (P<0.05) in G2; Glutathione Reductase showed a non-significant increase (P<0.05) in G1, G2 compared with control, while Superoxide Dismutase showed a significant decrease (P≤0.05) at G1 and a significant increase (P≤0.05) in G2 compared to control. Immunoglobulin IgG showed a significant increase (P≤0.05) in G1 compared with control in Table (5) and a highly significant increase (P<0.01) in G2, IgM showed a highly significant decrease (P<0.01) in all groups compared to control and IgE showed a non-significant decrease (P<0.05) in G1 and a non-significant increase (P<0.05) in G2 compared to control. In Table 5; TBC showed a highly significant increase (P<0.01) in G1 and a non-significant decrease (P<0.05) in G2 compared to control; while TEC showed a non-significant increase (P < 0.05) in all groups compared to control.

Antioxidant in
Vitamin C correlations revealed in Table 6 185 Vitamin E revealed in Table 7; TBC showed a     .637 a G1: birds supplemented with synthetized vitamin C (3.5 g / L). G2: birds supplemented with synthetized vitamin E (1 ml / L). G3: control group Means carrying different superscripts in the same column are significantly different at (P≤0.05) or highly significantly different at (P<0.01). Means carrying the same superscripts in the same column are non-significantly different at (P<0.05).   .237 a G1: birds supplemented with synthetized vitamin C (3.5 g / L). G2: birds supplemented with synthetized vitamin E (1 ml / L). G3: control group Means carrying different superscripts in the same column are significantly different at (P ≤ 0.05) or highly significantly different at (P < 0.01). Means carrying the same superscripts in the same column are non-significantly different at (P < 0.05).

Discussion:-
Vitamin C can be synthetized easily as the enzyme systems responsible for its production were formed early enough during the development of life process on earth, that is why the most prominent source of vitamin C on earth are of plant and animals origin. The quantity and titre of L-ascorbic acid in the human body clears its daily requirement and vitality as nutrients for healthy maintenance; Ascorbic acid (vitamin C) is primarily synthesized in broiler through the kidneys. This synthesis become insufficient during the presence of some stressors as heat stress causing suppressed immunity, lowered feed intake and consequently the performance traits, to some extend lowered body temperature, lowered carcass weight and increase mortality rates ; Hu, et al., (2015). The present study showed the improvement caused by the enhancing effects of vitamin C on broilers performance traits (body weight gain, feed conversion ratio and performance index), but this enhancement was retreated as the broiler advances in age due to the failure of immune barrier imposed by vitamin C.
Broilers are usually adapted the surrounding conditions, but any changes might contributed a great drop in their performance and meat quality; that is why broilers are highly sensitive to the change in the surrounding 187 microclimatic conditions (Nolan et al., 1999). A special reference was given to the elevations in ambient temperature; that contributed depressed feed intake, lower weight gain and increased mortality rates; (Ayo et al., 1996). Based on these facts the dietary supplementation of broilers with vitamin C increased adrenal cortex secretion and can alleviate the heat stress influences.
Vitamin E is a fat soluble vitamin of plant origin that can act as a lipid-soluble antioxidant. It acts in synergism with glutathione peroxidase, catalase, and superoxide dismutase to get rid of the metabolites which were formed during free radical chain reaction; Herrera and Barbas, (2001). Thus, vitamin E was able to prevent the increase accumulation of free radicals and avoid the damage of protein and DNA as well as the fatty acids (poly unsaturated) present on cell membrane; Mézes et al., (1997). Vitamin E has proven the ability to induce immunoregulatory effect, which could be carried out through the modification of cyclooxygenase and lipoxygenase pathways causing exert of a control on the synthesis of prostaglandins and leukotrienes (Leshchinsky and Klasing, 2001).
The present article revealed the superiority of vitamin E compared to vitamin C in the immune stimulating effects and antibacterial action. Rice and Kennedy, (1988) suggested that the advantage of higher levels of dietary vitamin E will be witnessed mainly when the group is exposed to some kind of stressor. The absence of differences in the growth performance among the 5 dietary treatments in this study indicates that all the birds might have been exposed to stress below the threshold level to get a beneficiary effect from higher levels of dietary vitamin E on growth performance and yield.

Conclusion:-
Synthetized vitamin E and vitamin C were able to enforce a protective effect in the birds which last for only 4 week then vanished gradually. Synthetized Vitamin E was superior to vitamin C in enforcing a good protective effect on the birds. A final recommendation is advised to maintain the supplementations on a higher levels and longer period to maintain its influences on the birds.