MATERIALS AND METHODS

The experimental work of the present study was carried out in the Poultry Production Farm; Center of Agricultural Research and Experiments, Faculty of Agriculture, Mansoura University, Egypt from July to August, 2015.

Birds, management and experimental design: Arbor acres broiler chickens (n = 200), 1 day old with an average body weight of 37 g were divided into 10 treatment groups, each of which included four replicates (cages). A completely randomized block design with a 5×2 factorial arrangement of treatments was used, 5 nutritional densities and two feed forms (mash and pellets). Experimentally diets were formulated to contain nutrient requirements recommend by NRC⁸ (control), two high levels (H1 and H2) and two low levels (L1 and L2) from Metabolizable Energy (ME), crude protein and amino acids (methionine and lysine) that were fed from 1-49 days of age. The estimated means of ME of the experimental diets were 3200, 3345, 3497, 3050 and 2900 kcal kg‾¹ for both the starter and growing periods. Crude protein contents of the experimental starter diets were 23, 24.08, 25.17, 21.93 and 20.85%. In the grower diets, these levels were 19, 19.89, 20.79, 18.12 and 17.22%, respectively. Lysine and methionine levels in the starter experimental diets were 1.1 and 0.52%, 1.15 and 0.55%, 1.19 and 0.59%, 1.06 and 0.49% and 1.03 and 0.46%, respectively. These levels of the two amino acids were 1.0 and 0.57%, 1.04 and 0.59%, 1.07 and 0.61%, 0.96 and 0.54% and 0.90 and 0.50% of grower diets, respectively. All the experimental diets were formulated to have similar calories/CP ratios.

Each replicate group of chickens was reared in a separate compartment in a battery cage measuring 70 cm length, 60 cm width and 40 cm height. Thus, the cage floor area was 0.42 m² (70×60 cm). The number of birds kept in each cage was 5 birds. Feed and water (via nipple drinkers) were provided freely. The composition and calculated analysis of the experimental diets are shown in Table 1.

Performance of broiler chickens: Live Body Weight (LBW), Feed Intake (FI) and Body Weight Gain (BWG) of broiler chickens were measured weekly throughout the experimental period, then Feed Conversion Ratio (FCR) was calculated (Gram feed: Gram gain). Birds of each replication were weighed to the nearest gram in the early morning before receiving any feed or water at the start of study (day-old) ant at weekly intervals thereafter. Mortalities and health status were visually observed and recorded daily throughout the entire experimental period.

Blood sampling and biochemical analysis: Four birds from each treatment were chosen, slaughtered and blood samples were collected in heparinized tubes then centrifuged at 4000 rpm for 15 min and the plasma obtained was stored at -20°C until analysis. Plasma samples were tested colorimetrically using commercial kits according to the procedures outlined by the manufactures. Thyroid hormones [triiodothyronine (T3) and thyroxine (T4)] and insulin like growth factor I in blood plasma were measured by RIA techniques according to the methods of Britton et al.⁹ and Houston and O’Neill¹⁰, respectively. Superoxide dismutase (SOD), catalase were determined by available kit according to Wheeler et al.¹¹ and malondialdehyde (MAD) was determined by available kits according to Uchiyama and Mihara¹². Fatty acids profile was assayed by gas liquid chromatography according to the methods of Radwan¹³.

Statistical analysis: Statistical analysis for the obtained data was performed by two-way analysis of variance using the method of least square analysis of co-variance¹⁴.

Table 1:	Composition and calculated analysis of the experimental diets
#Premix provided the following per kilogram of diet: Vitamin A (retinyl acetate): 2654 μg, Vitamin D3 (cholecalciferol): 125 μg, Vitamin E (dl-a-tocopheryl acetate): 9.9 mg, Vitamin K3 (menadionedimethylpyrimidinol): 1.7 mg, Thiamin mononitrate: 1.6 mg, Cyanocobalamin: 16.7 μg, Riboflavin: 5.3 mg, Niacin (niacinamide): 36 mg, Calcium pantothenate: 13 mg, Folic acid: 0.8 mg, d-biotin: 0.1 mg, Fe (iron sulphate monohydrate): 50 mg, Cu (copper sulphate pentahydrate): 12 mg, I (calcium iodate): 0.9 mg, Zn (zinc oxide): 50 mg, Mn (manganous oxide): 60 mg, Se (sodium selenite): 0.2 mg and Co (cobalt sulphate): 0.2 mg, DSM company

Duncan’s multiple range tests was used to separate significant differences among means¹⁵.

RESULTS AND DISCUSSION

Broiler performance: The effects of nutritional density levels and physical form on the weight gain, feed intake and FCR of broiler chickens at 21 and 49 days of age are presented in Table 2. The LBW, BWG, FI and FCR of broilers fed the high nutritional density diets (H1 and H2) were significantly lower compared with the control group but feeding the low nutritional density diets (L1 and L2), produced significantly better means of LBW, BWG, FI and FCR as compared to other treatments. On the other hand, the control group in the starter period was better means of performance production as compared to other groups. This result is in agreement with Roy et al.¹⁶ who observed that LBW, BWG and FCR of broilers fed 19% CP was superior and significantly differed from 18% CP and 20% CP diets. Feed intake of birds during the whole experimental period was significantly reduced with higher level of nutritional density. This result is supported by the findings of Sizemore and Siegel¹⁷ who observed that different ME and CP concentrations, while maintaining a constant ME:CP ratio. An increase in the protein level of diets may increase broiler heat production concomitant with protein digestion, absorption and metabolism. Also, the excretion of surplus AA from the body in the form of uric acid may cause birds to consume more energy and thereby produce more heat. This may exacerbate the growth depression that occurs in hot and humid conditions¹⁸. There were no significant differences between birds fed on mash or pellets for LBW or BWG. On the other hand, FI and FCR for birds fed on mash were significantly lower than those fed pellet diets. This result is in agreement with Chewning et al.¹⁹ who found that birds fed mash diet had lower feed intake and improved FCR. The differences attributable to the particle size in mash feed versus pellet feed may be due in part to the final size of the particles in a pellet, the rate of feed passage because of the feed form and particle size, the distribution of particles within the ground grain and access to litter because the trial was conducted in floor pens. Parsons et al.²⁰ observed that broilers fed diets containing coarse corn had significantly increased FI and decreased feed efficiency compared with birds fed most other mash diets. These results were also supported by findings of Hetland et al.²¹ found that increased FI when feeding diets with high inclusions of whole cereals.

Table 2:	Effect of nutritional density levels and physical form on growth performance of broilers at 21 and 49 days of age
a-cIn each of the main effects, means in the same column with different superscripts differ significantly *p≤0.05, NS: Non significant, FCR: Feed conversion ratio, FI: Feed intake, LBW: Live body weight, BWG: Body weight gain

Actually, recent results showed that pelleting decreases digestibility of starch compared to mash feeding, despite an improvement in feed per gain²². The interaction between nutrient density and physical form in broiler performance was not significant which is in agreement with Brickett et al.¹ who found that there were no significant interactions between nutrient density and feed form in broiler chicks.

Some plasma hormone profile: Thyroxine (T4), triiodothyronine (T3), T4/T3 ratio and insulin like growth factor I (IGF-1) hormones concentrations response to experimental treatments are presented in Table 3. The plasma T4 and T3 levels increased significantly with low levels and high levels of nutritional density compared to the control group and the highest value occurred for L1 treatment group. This finding agrees with results of Irani et al.²³ and Carew et al.²⁴ in which only the level of lysine deficiency was studied. However, in the absence of direct data with different levels of Lys and in view of the well-knowninteraction of Lys with Arg^25,26 other mechanisms of an unknown nature may be involved. Although, based on the T3 data, it is certain that Lys deficiency alters normal thyroid hormone metabolism. Compared with levels of nutritional density and control group, the plasma IGF-I level was significantly higher with low and high nutritional density. However, low nutritional density (L1 and L2) has the highest concentration IGF-1 because low nutritional density was the highest LBW.

Table 3:	Effect of nutritional density levels and physical form on plasma concentrations of T4, T3, T4/T3 ratio and IGF-I hormones (ng mL‾1) in broiler chickens
a-bIn each of the main effects, means in the same column with different superscripts differ significantly *p≤0.05, NS: Non significant

These result agreement with McGuinness and Cogburn²⁷ who found that chicken relative growth rate and age-related changes in plasma concentrations of insulin-like growth factor 1 (IGF-I) and thyroxin are highly correlated. The peaks of these hormones in broilers have been reported to occur between 3 and 6 weeks of age. Rosebrough et al.²⁸ determined that plasma IGF-I concentrations matched both growth and relative breast muscle size, which suggests that this hormone regulate lean tissue development. Rosebrough et al.²⁹ found that dietary fat levels had little influence on plasma IGF-I, T3 and T4. Plasma IGF-I and T4 were greater and T3 less in birds fed the higher level of CP. Substitution of CP but no fat, for carbohydrate increased plasma IGF-I and T4. In contrast, this same substitution regimen (CP for carbohydrate) decreased plasma GH. The substitution of CP for carbohydrate resulting in a CP content of 285 g and 1,140 kcal kg‾¹ gave the only consistent decrease in plasma T3. No significant effect of physical form on plasma concentrations of T4, T3 and IGF-I hormones.

Antioxidant status: The effect of nutritional density and physical form on plasma SOD, catalase and MDH concentration are presented in Table 4. There was no significant effect of nutritional density on MDH. However, nutritional density significantly increase SOD and Catalase with low levels and high levels of nutritional density compared to the control group. These results are in agreement with other studies^25,23 which administrated that excess different dietary lysine the effect of arginine requirement of broiler chickens. The arginine is a nutritionally substantial amino acid and plays multiple physiologic functions in animals. One of these functions is increasing antioxidant ability. No significant effect of physical form on plasma SOD, catalase and MDA.

Fatty acids profile: The effect of nutritional density and feed form on blood plasma fatty acids of broiler chicks is illustrated in Table 5. It is clear from the results that the total saturated fatty acids of chicks that fed mash diets was lower for the control, H1 and H2 treatment groups but it was greatly increased in L1 and L2 fed chicks. On the other hand, the total Saturated fatty acids content in blood plasma of chicks that fed pelleted diets was lower in the control, H1, H2 and L2 treatments, whereas L1 group recorded the greatest value. Concerning total unsaturated fatty acids chicks that fed mash diets had higher levels of plasma total unsaturated fatty acid, especially in H1 and H2 than those fed L1 and L2 diets. Similarly, total unsaturated fatty acids was also higher for birds fed pellet diets with higher ME (H1 and H2) levels and surprising L2-fed chicks, however, the lower total unsaturated fatty acids level was recorded for birds fed L1 pellet diet.

Table 4:	Effect of nutritional density levels and physical form on plasma concentrations of SOD, catalase and MDA in broiler chickens
a-bIn each of the main effects, means in the same column with different superscripts differ significantly *p≤0.05, NS: Non significant

Results showed also that the percentages of total unsaturat to saturated fatty acids were higher for the control, H1, H2 and L2 diets but lower for both L1 and L2 diets. This was also observed for n-6 to n-3 ratio which was low for the two L1 diets, H2 pellet diet and then L2 pellet diet. It appease from the present results that the fatty acids profile of blood plasma in broiler chicks showed inconsistent trend especially when both total unsaturated to total saturated and omega-6 to omega-3 ratios are considered. This difference is a reflection of the higher levels of both dietary levels of crude protein, added vegetable oils, lysine and methionine levels in the diets. It may be that the differences in fatty acid profile are a function of dietary contents of fats, oils and (or) their sources. In this concern, use of plant sources of linolenic and α-linolenic acids is of great importance as a source of n-3 fatty acids. They are primarily found in highly abundant level in canola, safflower, corn and sunflower oils³⁰. It is claimed that, fatty acid profile difference of blood in the present study is due mainly to the great variation in both ME and CP level in the experimental diets. Although, the C:P ratio was nearly similar for all diets, the percentage of oils content in each diet was greatly different.

Table 5:	Fatty acids profile in blood plasma of broiler chicks

Since, the impact of ME, protein and fat (%) on growth performance and body composition of broiler chickens has been widely investigated. In general, diets with high ME levels but (normal protein levels) promote fat deposition without negative effect on body weight gain³¹. Moreover, widening of the ME to protein ratio due to a reduction in protein level results is not only a higher fat deposition but also reduced growth rate. This is in close agreement with the findings by Donaldson³² and Alvarenga et al.³³.

CONCLUSION

The findings of the present study suggest that feeding low level of nutrient density have about 90% of recommended of ME, CP, lysine and methionine a positive effect on productive performance and antioxidative status of broiler chickens.

ACKNOWLEDGMENT

Great thanks to Prof. Dr. I.E. EL-Wardany and Department of Poultry Production, Faculty of Agriculture, Mansoura University, Mansoura, Egypt for their help in this study.