Submitted22-03-2021|
Accepted26-05-2021|
First Online 21-06-2021|
ABSTRACT
Methods: A total of 240 day-old broiler chicks (Vencob) of mixed sex (avg. BW 47.50±0.26 g) were distributed in a completely randomized design into five treatments each with four replicates of 12 birds (6 of each sex). The dietary treatments involved supplementation of boron at 0 (B-0), 25 (B-25), 50 (B-50), 75 (B-75) and 100 (B-100) mg/kg diet. The birds were offered starter (d 1 to 21) and finisher (d 22 to 42) diet in mash form. At d 42, Whole blood (2 ml) sample was collected for the estimation of total antioxidant status and reduced glutathione by FRAP and DTNB method, respectively. Two birds per replication were selected randomly; sacrificed and right femur bone was collected to measure the bone ash and mineral content.
Result: Boron supplementation enhanced the bone ash, calcium and phosphorus content but decreased the manganese and iron content in bone. Supplementation of Boron significantly enhanced (P<0.05) the total antioxidant capacity but lowered the plasma reduced glutathione level.
INTRODUCTION
MATERIALS AND METHODS
The experiment was conducted at Navsari Agricultural University, Navsari, Gujarat during the month of March and April in the year 2020. A total of 240 one-day old commercial broiler chicks (Vencob) of mixed sex (mean BW 47.50±0.26 g) were used for this experiment. Upon arrival, the chicks were weighed and randomly allotted to floor pens, each representing a replication. Birds were vaccinated against infectious bursal disease virus (GUMBORO I+, Haster Biosciences Limited, Mehsana, India) and Newcastle disease virus (LaSota Strain, Venkateshwara Hatcheries Pvt. Ltd, Pune, India) via drinking water at 10 and 14 d of age, respectively. Each replica was supplied with a floor space of 1.5 m2 (1.5×1.0 m) along with the provision of hanging feeder and waterer. Birds were reared in pens provided with litter material (rice husk and saw dust) to a depth of 5-6 cm. The house was well-ventilated with adjustable windows and every effort was made to reproduce the commercial condition as much as possible. The room temperature was maintained at 33±1°C up to 7 d and gradually decreased to 26±1°C by 21 d. Thereafter, the birds were kept at room temperature up to 6 weeks of age.
Experimental design and diets
The experiment was performed 240 one-day old chicks distributed in completely randomize design with five treatments. Each treatment comprised of four replication with 12 birds (6 males and 6 females) per replicate. The basal diet was a corn-rice-soya based diet formulated to meet or exceed the nutrient requirement of broiler as per the ICAR (2013) recommendations. The birds were offered starter (d 1 to 21) and finisher (d 22 to 42) diet in mash form. The chicks received feed within 12 h of hatching. The ingredient and nutrient composition of basal diet are given in Table 1.
The five dietary treatment were comprised of the basal diet alone (B-0) or with additional boron supplemented at 25 (B-25), 50 (B-50), 75 (B-75) and 100 (B-100) mg/kg. Boric acid (Loba Chemie Pvt. Ltd, Mumbai, India) with 17.48% elemental boron was used as a source of boron. Accordingly, the dietary groups B-0, B-25, B-50, B-75 and B-100 were supplemented with 0, 0.143, 0.286, 0.429 and 0.572 g of boric acid per kg basal diet, respectively. Respective amount of boric acid for each treatment was mixed with the basal diet as a premix prior to feeding the birds.
Sample collection
Whole blood (2 ml) sample was collected in vials with anticoagulant, acid citrate dextrose (300 µl/2 ml blood) and centrifuged at 2000 rpm for 15 min at 4°C with separation of plasma and kept at -40°C and used for the estimation of total antioxidant capacity (TCA), reduced Glutathione and lipid peroxidation. Two birds per replication were selected randomly and sacrificed as per the standard protocol. Thereafter the right femur bone was collected to measure the bone ash and mineral content. The femur bone was excised, all flesh and proximal cartilages were removed. The bone samples were sealed individually in plastic bags and stored at -20°C until the analysis, which was performed within one month after sample collection.
Mineral estimation
The bone samples were ashed in a muffle furnace at 650°C and a mineral extract was prepared from the ash samples for mineral estimation. Mineral content in samples were determined using the Microwave Plasma Atomic Emission Spectrometer (MP-AES) (MP-AES, Agilent, Santa Clara, California, USA) with operating condition (Table 2) suggested by the manufacturer.
Antioxidant status
Total antioxidant activity was measured by ferric reducing antioxidant power (FRAP) assay of Benzie and Strain (1996). Briefly, 100 μl of plasma sample was mixed with 3 ml of working FRAP reagent (Acetate buffer (300 mM pH 3.6), 2, 4, 6-tripyridyl-s- triazine (10 mM in 40 mM HCl) and FeCl3. 6H2O (20 mM) mixed in the ratio of 10:1:1) and absorbance (593 nm) was measured at 0 minute after vortexing. Thereafter, samples were placed at 37°C in water bath and absorption is again measured after 4 minutes. Ascorbic acid standards (100μM-1000μM) were processed in the same way.
FRAP value of sample (μM)
The concentration of reduced glutathione (GSH) in plasma was estimated by 5, 5-dithiobis-(2-nitro- benzoic acid; DTNB) method as per the procedure of Prins and Loos (1969). The lipid peroxides level in the plasma (malonlaldehyde (MDA) was determined by the method of Placer et al., (1966). The concentration of MDA in nmol/ml plasma was calculated using the extinction coefficient of 1.56 × 10-5 L mmol-1 cm-1 (Utley et al., 1967) and expressed in nmol of MDA per ml: LPO (µmol MDA formed/ml) = [(ODT /Î) × (TV / VT) × df x (1/ ml)] x 106; where, ODT: Absorbance of test, Î: Molar extinction coefficient (1.56 × 105)/m/cm, TV: Total volume of reagent with sample taken, VT: Volume of sample taken and df: dilution factor.
Statistical analysis
Data generated in the study were analyzed using the SPSS v. 20.0 (SPSS Inc., Chicago, USA) by one-way ANOVA and comparison of means was tested using Duncan’s multiple range tests (Duncan, 1955). The effects were considered to be significant at P<0.05.
RESULTS AND DISCUSSION
Data pertaining to the antioxidant status of plasma in broilers fed a boron supplemented diet was given in Table 4. Supplementing graded level of boron up to 75 mg/kg diet significantly increased (P<0.01) the TAC (FRAP value) as compared to control. Similar to the present findings, boron supplementation has resulted in significant (P<0.05) increase in the erythrocytic SOD activity and total antioxidant activities (Turkez et al., 2012, href="#turkez_2013"> 2013; Bhasker et al., 2016). Other studies have also indicated the role of dietary boron in ameliorating the toxicity induced by carbon tetrachloride (Ince et al., 2010) and malathion (Coban et al.,2015), reducing the severity of hepatic cell carcinoma in rats by enhancing the SOD activity in liver and improving the antioxidant defense mechanism under oxidative stress condition. Supplementation of boric acid at higher dose (100 mg/kg) is toxic to the cell (Fort et al., 2000) and associated with decreased TAC. In agreement to our assumption, Hu et al., (2014) suggested that 40 mg/L of boron through drinking water increased the antioxidant capacity and that of 80 mg/L decreased the antioxidant capacity of spleens in rat. In the present study, it was found that addition of boron to the diet did not change plasma MDA levels but significantly lowered (P<0.05) plasma reduced glutathione (GSH) levels (Table 4). This shows that the GSH is effective in preventing lipid peroxidation caused by boron supplementation (Sizmaz and Yildiz, 2014). Earlier studies also suggested that boron supplementation enhanced antioxidant defense mechanisms through decreasing lipid peroxidation (Ince et al., 2014).
CONCLUSION
ACKNOWLEDGEMENT
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