MATERIALS AND METHODS

Animals, Diets and General Procedures
A total of 180 day-old male Ross-308 broiler chicks were individually weighed, wing banded and distributed as randomly into three treatments with 6 replicate pens and 10 chicks per pen (10 birds m^-2) and fed from 0 to 42 days of age. Floor pen was furnished with fresh wood shavings litter. Three experimental diets based on maize-wheat-soybean were formulated according to NRC (1994). The treatments were: (1) control without any addition (C, ASA0), (2) ascorbic acid (L-ascorbic acid, Rovimix® Stay C®35, Roche, Levent-Istanbul, Turkey) was added 150 mg kg^-1 of diet (ASA150) and (3) 300 mg kg^-1 of diet (ASA300). Fresh feed with ASA was prepared at 7 days intervals. Small amounts of the basal diet were first mixed with the respective amounts of ASA as a small batch and then with a larger amount of the basal diet until the total amount of the respective diets were homogeneously mixed. Chicks were fed a starter diet throughout 0 to 21 days of age and fed a grower diet throughout 22 to 42 days of age. The experimental diets used in this study are given in Table 1. The experiment was performed throughout from June 08 to July 18 2006 at the Ege University, Odemis Vocational School and Poultry Research Unit. The house was artificially heated to provide standard brooding temperatures (28 to 34°C) from hatch to 7 days of age. The lighting schedule was 24L: 0D from 0 to 3 day and 23L: 1D (darkness from 24:00 to 01:00) from 4 to 42 days of age. Feed and water were consumed ad libitum.

Determination of Performance Traits
The individual body weight (BW) of birds was determined at 0, 21 and 42 days of age. Body weight gain (BWG) was calculated from 0 to 21, 22 to 42 and 0 to 42 days. Total feed intake at subgroup base was measured at 21 and 42 day. Mortality was recorded daily and feed intake was adjusted for mortality. Daily feed intake (DFI) and feed conversion ratio (DFI g / BWG g) (FCR) was calculated for 0 to 21, 22 to 42 and 0 to 42 days of age.

Table 1:	The composition of starter diet at 0 to 3 weeks and grower diets at 3 to 6 weeks (g kg-1)
Supplied mg kg-1 of diet: Vitamin A, 15000 I.U; Vitamin D3, 2000 I.U; Vitamin E, 40.0 mg; Vitamin K, 5.0 mg; Vitamin B1 (thiamin), 3.0 mg; Vitamin B2 (riboflavin) 6.0 mg; Vitamin B6, 5.0 mg; Vitamin B12, 0.03 mg; Niacin, 30.0 mg; Biotin, 0.1 mg; Calcium D-pantothenate, 12 mg; Folic acid, 1.0 mg; Choline Chloride, 400 mg; Manganese, 80.0 mg; Iron, 35.0 mg; Zinc, 50.0 mg; Copper, 5.0 mg; Iodine, 2.0 mg; Cobalt, 0.4 mg; Selenium, 0.15 mg. 2Dry matter, crude protein, crude fiber, ether extract and crude ash contents of diets were analyzed, total calcium, available phosphorus, lysine, methionine and metabolizable energy values of diets were calculated

Average ambient temperatures were 28.1±0.47, 33.1±0.27 and 31.7±0.25°C and humidity 50.31±1.03, 38.6±0.81 and 37.03±0.69% for 8 am, 3 pm and 8 pm, between 8 to 42 days, respectively.

Determination of Carcass and Meat Traits
Twelve males were selected according to their BW from each dietary treatment and killed by cervical dislocation at 42 days of age. The carcass, abdominal fat pat (excluding gizzard fat), empty gizzard, liver, heart, kidney weights were recorded individually. Then breast and thigh portions were separated from carcass and weighted. Relative body part weights were obtained by calculation as weight of part *100: total carcass weight. The cold carcass weight was recorded after carcasses were kept at +4°C for 18 h.

Six breast and thigh meat samples each group were collected in plastic trays, weighed and stored in an air tight plastic bag in a freezer (-20°C) until samples were required for analysis. They were homogenized using a blender and analyzed for dry matter, nitrogen, ether extract and crude ash. The dry matter, crude protein, ether extract, crude ash of feeds and meat samples were analyzed according to AOAC (1980). The crude fibre content of feedstuffs was determined by using 12.5% H₂SO₄ and 12.5% NaOH solutions (Naumann and Bassler, 1993). The pH value of the samples was determined with a pH meter (Hanna Instruments-8413) by thrusting the probe into the breast and thigh. The colors of breast and thigh were measured using a Minolta colorimeter (CM508d) to measure CIE Lab values (L* measures relative lightness, a* relative redness and b* relative yellowness).

Determination of Bone Traits
All of the chicks left shank length and width were measured with a caliper rule on day 42. After chicks slaughter, twelve left tibia bones each group were removed and cleaned of adheral tissues and then weighed; length and width (at midpoint) were measured with a digital caliper. Bones were frozen at -20°C until analyses. After thawing to room temperature bone breaking strength was determined by Instron Testing Machine and the bones were subjected to test at the midshaft of each bone until they fractured (Norgaard-Nielsen, 1990). The centre of each bone was aligned with the breaking probe (10 mm diameter) which approached at 30 mm min^-1. The supports for each bone were 30 mm apart. The breaking strength was determined from the failure point (peak) of each loading curve. The ash content of tibia bone was determined after heating in a muffle furnace at 550°C for 16 h.

Determination of Blood Components
Twelve blood samples per group were obtained by venepuncture of left wing vein at 42 days of age for blood biochemical analysis. The blood samples were kept on ice and transferred to the laboratory where they were centrifuged at 1500 g for 10 min. and serums were removed and stored at -20°C until analyzed. The serum total protein, albumin, glucose, triglyceride, total cholesterol, high density lipoprotein (HDL), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured using kit from the same manufacturer with an auto analyzer (model BT 3000 plus, Biotechnica, Rome, Italy).

Statistical Analyses
The data were analyzed using the GLM procedure of SPSS (1998). Linear and quadratic polynomial contrasts were used to evaluate treatment effects. Because no biologically significant cubic effects were detected, only the results of linear and quadratic contrasts are reported in tables. The results of statistical analysis were shown as mean values and standard error of the means (SEM) in tables.

RESULTS AND DISCUSSION

Performance Traits
The BW, BWG and FCR of broilers were not statistically influenced by the dietary ASA supplementation at the experimental period (Table 2). However, in the ASA150 group DFI quadratically decreased (p<0.05) and FCR was the best in this group. It is generally assumed that dietary ASA supplementation increased feed intake of birds at the heat stress condition. However in the present study there is no difference in terms of DFI between control and ASA150 or ASA300. On the other hand, DFI in ASA300 group was higher than that of ASA150 group. Dietary ASA300 have more efficient on DFI than ASA150 level. The daily protein consumption of groups were calculated as 10.19, 9.93 and 10.54 g; and 25.93, 24.72 and 26.67 g for control, ASA150 and ASA 300 groups at 0 to 21 and 22 to 42 days of age respectively. Also daily energy consumption of groups were calculated as 0.61, 0.60 and 0.63 MJ kcal/day; and 1.79, 1.70 and 1.84 MJ kcal/day for control, ASA150 and ASA 300 groups at 0 to 21 and 22 to 42 days of age, respectively. Due to lacking marked differentiates in feed intake and feed protein and energy content, consumed daily protein and energy values were similar. It is well known heat stress begins to occur when ambient temperature rises above 27°C in poultry and it causes a decrease in feed intake and BWG (Kutlu, 2001). The present study was carried out under practical condition and daily temperature range of 28.1 to 33.1°C. However, Balnave (2004) reported that birds grown in semitropical and tropical areas have a better acclimate ability than moderate temperature. In these areas, temperatures below 32°C are not considered a seriously stress factor. On the other hand, birds adapt better to a high daily maximum temperature when the night temperature falls to 25°C or lower (Balnave, 2004).

Table 2:	Effects of ascorbic acid levels on body weight, body weight gain, feed intake and feed conversion ratio
ASA0, ASA150, ASA300: ascorbic acid, 0, 150 and 300 mg per kg of diet, *:p<0.05; SEM: standard error of the means (Pooled)

This explains present results, because the experimental area is semitropical and night house temperature around 20 to 25°C. Therefore, chicks consumed enough feed in night and BW is not affected by the diurnal heat stress. Since, it is known that effectiveness of ASA in poultry diets have a rise value in heat stress but not normal condition (Kutlu, 2001). The studies with ASA showed that 0 to 1000 mg kg^-1 doses of ASA supplementation to the diets did not affect body weight, body weight gain and feed conversion ratio in broilers (Pardue et al., 1985; Marron et al., 2001). However, Kutlu and Forbes (1993a, b), Sahin et al. (2003) and Lohakare et al. (2005) reported that dietary ASA (150 to 1000 mg kg^-1) improved performance traits of broilers under heat stress. Contrary to the results above, Njoku (1986) noted that ASA 300 mg kg^-1 reduced the body weights of broilers.

Carcass Traits
Dietary ASA supplementation did not improve carcass and parts, liver, gizzard, heart, kidney and abdominal fat yields (p>0.05) (Table 3). The current results are in agreement with Fletcher and Cason (1991) and Celik and Ozturkcan (2003), who found that ASA supplementation have no effect on carcass and abdominal fat. However, some studies suggested that dietary ASA supplementation increased carcass (Kutlu, 2001; Sahin et al., 2003; Lohakare et al., 2005), breast (Lohakare et al., 2005), liver, heart, spleen and empty gizzard weight (Sahin et al., 2003) and decreased abdominal fat pad (Kutlu, 2001; Sahin et al., 2003).

The chemical composition and pH of meat samples are shown in Table 3. Dry matter, crude protein and crude ash content of the thigh and meat chemical composition were not statistically affected by the dietary ASA supplementation (p>0.05). However, dietary ASA supplementation linearly decreased (p<0.05) crude fat content of thigh meat. Low fat content meat is preferred due to costumers demand. These results in agreement with Kutlu (2001), who found that dietary ASA supplementation decreased fat content in broilers. The lightness (L*), redness (a*) and yellowness (b*) values of breast were not affected by the dietary ASA supplementation (p>0.05, Table 3). However, L* values linearly increased (p<0.05) and b* value quadratically changed but a* values did not statistically affected by the treatments. While Lohakare et al. (2004) noted that dietary ASA did not affect colour of broiler meat, but their one study, Lohakare et al. (2005) found ASA positively affected meat color and caused an increase in L* and a* values but low b* values in broilers. In this study, pH values of breast and thigh meat did not influenced by the dietary ASA levels. Young et al. (2003) reported that providing ASA before slaughtering did not affect meat pH and colour of broilers.

Table 3:	Effects of ascorbic acid levels on slaughter and carcass weight (g) and yield (%) and carcass part weights (g) and relative weights1 (%) of parts of male broilers at 6 weeks of age
ASA0, ASA150, ASA300: ascorbic acid, 0, 150 and 300 mg per kg of diet, P: probability, *:p<0.05; **:p<0.01; SEM: standard error of the means (Pooled).; L*: lightness, a*: redness, b*: yellowness

Table 4:	Effects of ascorbic acid levels on shank and tibia measurements
ASA0, ASA150, ASA300: ascorbic acid, 0, 150 and 300 mg kg-1 of diet, SEM: Standard error of the means (Pooled)

Bone Traits
Shank length and width and tibia weight, length, breaking strength and ash were not affected by the dietary ASA supplementation (Table 4, p>0.05). Similarly, Roberson and Edwards (1994), Franchini et al. (1993) and McCormack et al. (2001) reported that dietary ASA supplementation did not affect bone properties in broilers. In contrast, Lohakare et al. (2005) showed that dietary ASA addition increased tibia breaking strength and ash. It is known that ASA plays a crucial role in the hydroxylation of proline residues necessary for the synthesis of procollagen, (Leeson and Summers, 2001) and also it is required for bone development as a cofactor for the bioconversion of vitamin D₃ to its active form of 1,25 (OH) D₃ (McDowell, 2000). However, Orban et al. (1993) reported that effect of ASA in bone metabolism is limited to hydroxylation of proline for formation of collagen and bone matrix during the growth phase of bone. Moreover, Thornton (1968) reported that ASA seemed to enhance the reduction in ossification of the epiphysical plate, mineralization of new bone and the reduction in the degrees of calcification of both types.

Table 5:	Effects of ascorbic acid levels on blood serum constituents
*: p<0.05, SEM: Standard error of the means (Pooled)

So, this might partially explain why bone traits were not appreciably affected by dietary ASA supplementation in the present study.

Blood Parameters
Dietary ASA supplementation did not affect serum total protein, albumin, glucose, triglyceride and total cholesterol levels of broilers (p>0.05, Table 5). However, dietary ASA supplementation caused a linear increase in serum HDL concentration (p<0.05). Similar results were obtained by Clegg et al. (1976) and McKee et al. (1997), who reported that dietary ASA supplementation did not affect blood cholesterol and plasma triglyceride (McKee et al., 1997) in broilers and albumin, creatine and glucose (Seyrek et al., 2004) in quails. On the contrary these findings the reduction of blood cholesterol (Kutlu and Forbes, 1993a; Sahin et al., 2003), triglyceride (Clegg et al., 1976) and glucose (Kutlu and Forbes, 1993a; Sahin et al., 2003) concentrations by feeding ASA have been demonstrated in broilers and quails (Gursu et al., 2004; Sahin et al., 2002). Also, Kutlu and Forbes (1993a) and Sahin et al. (2003) reported that dietary ASA supplementation increased serum protein in broilers. In the current study, dietary ASA supplementation caused a quadratic change in the AST and a linear and quadratic decrease in the serum ALT levels (p<0.05). The ALT catalyzes the transfer of an amino group from alanine to α-ketoglutarate, the products of this reversible transamination reaction being pyruvate and glutamate. It is used as a liver function test and elevated levels monitored liver malfunction (Murray et al., 1990). On the contrary to those results, Sahin et al. (2002) and Gursu et al. (2004) reported that dietary ASA did not affect serum ALT and AST levels in quails.

In conclusion, this experiment results suggested that additional ASA in broiler diets had some beneficial effect on some of examined parameters such as crude fat of meat, colour of thigh in broiler chicks, but most of parameters did not influenced by the dietary ASA supplementation. However, there were a number of fundamental differences among reports, including the basal diets, genetic stocks used and environmental conditions of the experiments. Further experiments should need to be conducted to determine whether the effect of ASA at different condition in broilers.

ACKNOWLEDGMENT

The study was supported by the Ege University, Scientific Research Projects Directorate, Izmir, Turkey.