The effects of vitamin C-polyethylene glycol complex on growth performance and immunity in broiler chickens

This research was conducted to evaluate the efficacy of vitamin C-polyethylene glycol complex (VCPEG) in broiler chickens during heat stress. For a 6-week feeding trial, a total of 330 broiler chickens were allotted to five dietary treatments. The dietary vitamin C (VC) levels (treatments) were, mg/kg: 1. 0 VC (negative control), 2. 200 VC as ascorbate (positive control), 3. 10 VC-PEG, 4. 20 VC-PEG, and 5. 50 VC-PEG. At the end of the experiments nine chickens per treatment were sacrificed for the analyses of VC contents in blood and liver, and meat quality. During the starter period, chickens on negative control group (no VC added) grew slower (P<0.05) than others. During the overall period, chickens fed diets containing 20, 50 mg/kg VC-PEG, and 200 mg/kg ascorbate showed improved weight gain (P<0.05) than those fed 0 or 10 mg/kg VC-PEG. VC concentrations in plasma and liver were higher in chickens fed diets with vitamin C. Bone strength was also higher (linear, P=0.001) as dietary VC level was increased. There was no effect on lymphocyte sub-population. Antibody response to Newcastle disease and infectious bronchitis vaccination was higher in 20 mg/kg VC-PEG fed group as compared with other VC-PEG groups. In conclusion, VC supplementation was beneficial to broilers during heat stress.


INTRODUCTION
Poultry has the ability to synthesize ascorbic acid in their body (McDowell, 2000), and hence there is no recommended requirement established by the NRC (1994). However, environmental and pathological stressors are known to alter ascorbic acid utilization and/or synthesis in the fowl (Pardue and Thaxton, 1986). The chicken is subject to considerable stress conditions, such as rapid growth, exposure to hot or cold temperatures, vaccinations, and diseases in the field condition. High temperature led to reduced growth rate and increased mortality in broilers (Pardue et al., 1985), so it is one of the major concerns in broiler production.
Several researchers observed significant improvement in growth of chickens by the addition of vitamin C (VC) under high temperature (Thaxon and Pardue, 1984;Kassim and Norziha, 1995). It was observed that broilers fed diets containing VC were less stressed by reduced body temperature and respiratory rates (Pardue and Thaxon, 1986;Kassim and Norziha, 1995) and showed increased feed intake (Kutlu and Forbes, 1993;Mckee and Harrison, 1995) than the control birds (no VC).
On the other hand, ascorbic acid is very susceptible to destruction through oxidation (McDowell, 2000). So, coated product is available to improve its stability in the feed. One of the methods to improve the stability is to combine it with other materials like polyethylene glycol. VC-polyethylene glycol complex (VC-PEG) is known to be more stable than any other VC sources during storage at room temperature. This VC-PEG compound was proven to show higher bioavailability and growth promoting effects (LG Chemicals Report, 2000).
Previous substantial reports suggest that under field conditions, treating VC may enhance productivity, immune response, disease resistance, and survivability under stressful conditions (Pardue et al., 1985;Gross, 1988;Zulkifli et al., 1996). The purpose of this experiment was to evaluate the efficacy of the VC in polyethylene glycol complex form on broiler's growth performance and immunity during summer season in Korea.

MATERIAL AND METHODS
The design, birds, sample preparation, chemical and statistical analyses remained the same as explained in detail in vitamin-E polyethylene glycol experiment conducted in parallel with this experiment (Chae et al., 2005). The difference was only in the dietary treatments as the dietary VC levels were, mg/kg: 1. 0 (negative control), 2. 200 ascorbate (positive control), 3. 10 VC-PEG, 4. 20 VC-PEG, and 5. 50 VC-PEG.
The room temperature was also measured after every four hour during the whole experimental period. The vitamin premix used in the control and experimental group in this study contain vitamin E at 10 mg/kg feed. Also after slaughter blood and liver samples were taken to analyse VC contents. Rest experimental procedure remained the same.

RESULTS
Growth performance of broiler chickens as affected by dietary VC levels is presented in Table 2. During the starter period, chickens in negative control group (no VC added) grew slower (P<0.05) than others. Feed intake was also reduced (P<0.05) in chickens fed the negative control diet compared to others except 10 mg/kg VC-PEG fed group. Resultantly, there were linear effects on weight gain (P=0.03). Dietary VC levels did not affect weight gain and feed intake during finisher period. During the overall period, chickens fed diets containing 20, 50 mg/kg VC-PEG and 200 mg/kg VC showed improved weight gain (P<0.05) than those fed 0 mg/kg VC. No differences were found in feed intake and F/G among dietary treatments.
In the nutrient digestibility, energy digestibility was not different among treatments both in starter and finisher diets (Table 3). However, the digestibilities of protein and ether extract were higher (P<0.05) in 50 mg/kg VC-PEG and 200 mg/ kg VC groups than others during starter phase. Protein digestibility in the finisher diet was lower (P<0.05) in 0 mg/kg VC group than others, while no difference was found in fat digestibility among treatments.
Bone strength was higher linearly (P=0.0001) as dietary VC level was increased (Table 4). Ash contents in 20 and 50 mg/kg VC-PEG fed groups were higher (P<0.05) than 0 mg/kg VC group. Calcium and phosphorus contents in tibia from chickens fed diets containing VC were higher than control, though it was not statistically significant.
As shown in Table 5, there were no differences in dressing percentage, breast meat and abdominal fat percent among treatments. In chicken meat colours, 'a' value (redness) was significantly (P=0.03) increased, and 'b' value (brownness) was linearly (P=0.001) decreased as dietary VC level was increased in the diet.
VC concentrations in plasma and liver were higher in chickens fed diets added the vitamin, showing linear effects (P=0.02 for plasma and P=0.05 for liver) ( Table 6).  The percentage of CD4, CD8, TCR1, TCR2, and B cells were not affected significantly by VC treatment (Table 7). However, the percentages of CD4 lymphocytes tended to increase with increasing levels of dietary VC supplementation. Apparently, dietary VC supplementation had no effect upon the immune cell populations in chickens.  (Table 8) when compared with control. ELISA titer to ND of chicken in 20 mg/kg VC-PEG group was significantly (P<0.05) higher than other groups at 14 th day.

DISCUSSION
There was a trend to increase weight gain when VC was added in the broiler diets in present study. The results were not in agreement with the results of Sifri et al. (1977) who did not demonstrate improved growth of chickens with the addition of VC in broiler's diet, but Thaxon and Pardue (1984) obtained positive responses especially immediately after heat stress, and Kassim and Norziha (1995) also reported that ascorbic acid supplementation in broilers improved weight gain and feed efficiency in the natural hot humid tropical climate (20-35 o C).
As previously stated, VC is synthesized by poultry. Therefore, its requirement is not established by NRC (1994). However, biosynthesis of VC was limited in very young birds (Horning and Frigg, 1979), and several stressors were known to alter the biosynthesis and/or utilization of ascorbic acid (Pardue and Thaxton, 1986). In terms of environmental stress, high temperature is a major concern in broiler production in summer. In our study, improvement in weight gain was clearer during the starter period than the finisher period. This result supported the reports of Horning and Frigg (1979) that young chickens have lower synthetic ability of ascorbic acid. Also, we conducted this experiment in hot season in Korea.
Under high environmental temperature, bird's heat-associated mortality is increased, and the effect of VC is also increased. In a field trial (Pardue et al., 1985), the mortality was markedly reduced when 4-week old broiler chickens were exposed to 38 o C fed with VC. Reduced mortality was also found in our study when VC was added in the diet (Table 2). It had been suggested that ascorbic acid might reduce glucocorticoid levels (Mckee and Harrison, 1995), and suppress heat-induced corticosteroids (Edens and Siegel, 1975) during heat stress.
In addition, improved weight gain of chickens fed diets added VC might partially be attributed to feed intake and nutrient digestibility in this study. Feed intake was linearly (P=0.01) increased as dietary VC levels were increased. Feed intake is related to body temperature in chickens. This is consistent with the results of some researchers, who reported increased feed intake in chickens fed ascorbic acid under heat stress (Kutlu and Forbes, 1993;Kassim and Norziha, 1995;Mckee and Harrison, 1995).
The digestibilities of protein and fat were higher (P<0.05) in VC added groups (50 mg/kg VC-PEG and 200 mg/kg VC) than others during starter phase in our study. The crude protein and fat digestibility was also higher in 10 and 20 mg/kg VC supplemented diets, when compared to non-supplemented group in our earlier studies conducted during finisher phase in broilers (Lohakare et al., 2004) There is, however, no evidence why nutrient digestibility is improved when VC is added in the diet. Sahin and Kucuk (2001) also reported greater (P<0.02) digestibility of DM, OM, CP and EE when fed higher dietary vitamin C at the level of 200 mg/kg diet to Japanese quails reared under chronic heat stress (34 o C). Sahin and Sahin (2002) found increased N retention and decreased N excretion of laying hens reared at a low ambient temperature (7 o C) when fed VC at a level of 250mg/ kg diet when compared with non-supplemented diet. Scanty reports are available with respect to effect of VC on nutrient digestibility.
VC is necessary for bone development and eggshell quality, as a cofactor for the bioconversion of vitamin D 3 to its active form of 1,25(OH) 2 D 3 (McDowell, 2000). In the present study, bone strength was higher (P=0.0001) as dietary VC level was increased. Calcium and phosphorus contents in tibia from chickens fed diets containing VC were higher than control, though it was not statistically significant. Sergeev et al. (1990) reported first time that ascorbic acid plays a critical role in vitamin D metabolism and binding in guinea pigs. The effects occur both at a level of vitamin D hormone formation in kidneys and it's binding in target tissue. VC is a cofactor, for several key enzymes, involved in procollagen synthesis (Tillman, 1993). More specifically, VC aids the enzymes required to convert peptide bound proline and lysine into hydroxyproline and hydroxylysine, respectively. These conversions are necessary for controlled formation of collagen. The collagen fibril network, which is required for proper bone formation has to be mineralized with hydroxyapatite. This mineralization process is under the direct influence of the active metabolites of vitamin D 3 . Metabolism of calcium has also been reported to be influenced by dietary ascorbic acid (Thornton, 1970;Dorr and Balloun, 1976;Orban et al., 1993) and the binding capacity of calcium binding proteins was significantly improved with VC supplementation. The increased collagen synthesis and increased mineralization must have increased the bone breaking strength in VC supplemented diet at 200 mg/kg level in our study. This was also confirmed from the crude ash content estimated which was higher. Since the intake and digestibility of the nutrients was higher in VC supplemented groups the bioavailability and its content increases in the bone and it was also reflected in the plasma and liver content of VC (Table 6).
There were no differences in dressing percentage, breast meat and abdominal fat percentages among treatments. In chicken meat colours, however, 'a' value (redness) was significantly (P=0.03) increased, and 'b' value (yellowness) was linearly (P=0.001) decreased as dietary VC level was increased in the diet. This means that the colour stability was improved by the addition of VC as an antioxidant agent. It took 6 h to prepare and measure the colour after slaughtering the birds, suggesting lipid oxidation was somewhat progressed. Lipid oxidation affects the formation of oxidized pigment such as metmyoglobin. This meaned that the colour stability was improved in present study by the addition of VC as an antioxidant agent and the higher values in non-supplemented group is suggestive of lipid peroxidation has somewhat progressed. The muscle oxymyoglobin oxidized in the presence of oxidizing lipids and the undesirable brown discolouration results, which was prevented by supplemental VC levels.
VC concentrations in plasma and liver were linearly increased as dietary VC levels were increased (P=0.02 for plasma and P=0.05 for liver; Table 6). Plasma ascorbic acid level in the fowl is easily influenced by exogenously administered ascorbic acid (Pardue and Thaxton, 1986). The data of VC content in plasma revealed significantly (P<0.05) higher levels in VC supplemented diets as compared with non-supplemented one. In our study, as the level of supplementation increased, the levels in plasma and liver also increased linearly. A significant positive trend was noticed with the levels supplied and their levels in plasma and liver. Because ascorbic acid is actively transported into tissues and its utilization increases during period of stress like heat, the birds synthetic capacity may become inefficient reducing plasma ascorbic acid concentrations which was found in non-supplemented groups. The efficacy of supplementing birds with VC under stressful conditions therefore depends on its ability to elevate plasma ascorbic acid concentrations, thereby preventing tissue depletion. Ascorbic acid either decreases heat load by lowering heat production or increase heat loss by influencing avenues of thermal exchange between the body and the environment as suggested by Chang et al. (1993).
The CD4, CD8, TCR-I, TCR-II and B cells were not affected by VC supplementation. The B cells population was lowest as compared to other lymphocyte cells in chickens. Apparently, dietary VC supplementation had no effect on lymphocyte populations when compared to non-supplemented one. The possible effect of less impact of supplementation in our study may be the age as we conducted study at 4 weeks of age, since Erf et al. (1998) found less apparent effects of vitamin E supplementation on T cell differentiation than at 7 weeks old chickens. Scanty reports are available with respect to effect of VC on lymphocyte subpopulation. Further studies needs to be done to see the effect of VC supplementation on lymphocyte subpopulations.
The IB and ND vaccination was done at 18 days of age and the titers were measured after 14 and 28 days post vaccination in 6 chickens per group. The ELISA titers to IB were higher significantly (P=0.04) at 14 th day of measurement in 200 mg/kg VC fed group and the lowest in non-supplemented group (Table 8), but it was not significantly higher in 200 mg/kg VC fed chickens at 28 th day. The ND titers were significantly influenced at 14 th day of measurement by VC supplementation at 20 mg/kg VC-PEG level. The results are in agreement with that of Tuekam et al. (1994) who found increased antibody titers to IB vaccine in chickens fed 500 mg/kg VC. Similar response of increased antibody response of chickens to sheep red blood cells and infectious bursal disease at 1000 ppm supplementation was also noted by Pardue et al. (1985) and Amakye-Anim et al. (2000). Dieter and Breitenbach (1971) postulated the possibility of increasing antibody to vaccination in VC supplemented chickens compared to non-supplemented one, may be due to speeding up of differentiation of lymphoid organs by increasing the activity of hexose monophosphate pathway, thus increasing circulating antibody.
Overall it could be concluded that VC supplementation at higher levels (200 mg/kg) is beneficial for broilers during heat stress to improve the performance and immunity. Also VC supplemented in polyethylene glycol complex form at 50 mg/kg was proven beneficial to improve the performance at par with 200 mg/kg VC levels.

CONCLUSIONS
The present study was conducted to evaluate the effect of VC under heat stress, when given in either polyethylene glycol complex form and as ascorbate at 200 mg/kg, on the performance, meat quality and immunity of broilers. The PEG form at 50 mg/kg performed at par with that of 200 mg/kg VC fed group. The study revealed that supplementation of VC is beneficial to broilers during heat stress and it also maintained the meat quality and immunity of broilers.