Synthesis of zinc oxide nano particles:
Synthesis of zinc oxide nanoparticles was carried out using Planetary Ball mill (Retsch – PM 100). To synthesize zinc oxide nano particles different combinations of time duration (7 or 8 or 9 hours), quantity of inorganic zinc oxide (5 or 10 g) and number of balls each having 5 mm diameter (45 or 50 or 55) were used for optimization. The ball mill was operated at 250 RPM speed. Finally, ball milling conditions to synthesize the zinc oxide nanoparticle was optimized with following conditions: Approximately 5 g of food grade zinc oxide was added in the 50 ml capacity zirconium jar with 50 balls each having 5 mm diameter operated for the total running time of 9 hours with 30 minutes interval.
Characterization of zinc oxide nanoparticles
Dynamic light scattering (DLS) and zeta potential analysis were carried out to assess the average size and stability of synthesized nanoparticles (particle size analyzer -Horiba SZ-100). For further confirmation, the particle size and shape were investigated using high resolution transmission electron microscope (Jeol/JEM 2100) operating at 200 kV voltage. The crystalline nature and the average crystallite size of synthesized zinc nanoparticles were determined by X-Ray Diffraction technique (Bruker D8 Advance) with 2θ ranging 0 – 80o (λ=1.5406 A°). The average crystallite size of ZnONPs were calculated using Debye-Scherrer’s equation: D=Kλ/βCosθ, where 0.89 is Scherrer’s constant (K), λ is the wavelength of X-rays, θ is the Bragg diffraction angle, and β is the full width at half-maximum (FWHM) of the diffraction peak. The characteristic functional group of the synthesized zinc oxide nanoparticles was analyzed by Fourier Transform Infra-Red (FTIR) spectroscopy (Thermo Nicolet iS5o) in the region of 4000 cm-1 to 100cm-1 The presence of zinc oxide nanoparticles was confirmed by UV–Vis spectroscopy (Perkin Elmer Lambda 365) in the wavelength region of 200 nm to 1000 nm. The elemental concentration (in percentage) of zinc in synthesized nano particle was estimated using Inductively Coupled Plasma Mass Spectrometry (Thermo Fisher iCAP RQ ICP-MS).
In vitro cytotoxicity Assay:
The cytotoxicity of zinc oxide nanoparticles was assessed by MTT assay using BHK-21, Vero and Primary Chick liver culture cell lines. The cell lines were maintained in 10 % fetal bovine serum and Dulbecco’s modified eagle medium (DMEM) at 37°C with 5 % CO2 incubator. Cells (108 cells/ml) were seeded into 96-well plates and after 24 hours of incubation, the media was discarded. The zinc oxide nanoparticle at different concentrations (6.25, 12.5, 25, 50 and 100 µg /ml) were dispersed in DMEM and added to each well in triplicate and incubated for 72 h. After 72 hours, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide) dye (5 mg / ml) was added to all the wells and incubated at 37 °C for four hours. After the incubation period, the media containing MTT dye was removed from all the wells and about 100 µl of DMSO was added and the absorbance was measured at 570 nm using ELISA reader (Agilent HTX multi-mode reader).
Bird Management and Experimental Design:
A total of 150-day old broiler chicks (Cobb 400) were procured and randomly allocated in a completely randomized design for 5 treatments, each of which had 3 replicates of 10 broiler chicks for 35 days. The dietary treatments consisted of a maize- soyabean meal based diet supplemented with inorganic zinc at 100 % of the requirement as per Bureau of Indian Standards, 2007 was taken as control (T1), basal diet supplemented with organic zinc at 60 mg/kg (T2), basal diet supplemented with nano form of zinc at 60 mg/kg 75 % of the requirement level (T3), basal diet supplemented with nano form of zinc at 40 mg/kg 50% of the requirement level (T4), basal diet supplemented with nano form of zinc at 20mg/kg 25 % of the requirement level (T5). Broiler chicks had ad libitum access to feed and water, and the light program was 23 hours of light per day. The details of experimental design and feed ingredient compositions are furnished in the Table 1 and Table 2, respectively. The experimental rations were formulated as per BIS, 2007 specifications. Based on the nutrient requirement and anticipated growth rate, the experiment was divided into three phases namely, pre-starter phase (0-7 days), starter phase (8-21 days) and finisher phase (22-35 days).
Growth Performances:
Body weights of individual birds were measured on day 1, 7, 14, 21, 28 and 35. The weight gain of individual birds was calculated from weekly body weight data. The feed intake of each replicate was recorded by subtracting the quantity of residual feed (g) from feed offered (g). The FCR was calculated by dividing the respective feed intake of the chicks by weight gain during the respective period.
Carcass characteristics:
At the end of experimental period (35 days of age), six birds (two birds from each replicate) from each treatment group were selected for the carcass characteristic study and were kept off feed for a period of 12 hours prior to slaughter but given ad libitum access to water. The pre slaughter live weights of the birds were recorded. Feathers were then plucked and the birds were eviscerated and weighed to determine the dressing percentage. Further, the pH, Water Holding Capacity and Shear force value of breast muscle meat sample were analyzed.
Serum Biochemistry:
Blood samples were collected from six birds from each group on day 35 and centrifuged at 3000 rpm for 5 minutes to separate serum. The serum was analysed for Glucose, Total protein, albumin, Cholesterol, Alanine transaminase (ALT), Uric acid (UA), calcium and phosphorus using A15 Biosystem random access analyzer.
Concentration of zinc in the serum:
The serum sample was diluted with distilled water (10-fold dilutions) and the concentration of zinc in the serum was analyzed using Atomic Absorptive Spectrophotometer (ZEEnit 700P).
Intestinal Morphometry:
The intestinal morphometry was studied by collecting the intestinal samples from six birds per group from the duodenum, jejunum and ileum, fixed in 10 % of neutral buffered formalin, dehydrated manually, embedded in paraffin wax, cut to 3 µm thick, stained with haematoxylin and eosin. Histological indices measured using an image analyser software (Magvision) included villus length (from the top of villi to the junction of the villus and crypt), crypt depth (defined as the depth of the invagination between adjacent villi) and villus height to crypt depth (V / C) ratio.
Intestinal Tight Junction protein gene expression analyis:
The ileum tissue was collected from different treatment groups and treated with 750 µl of Trizol Reagent (Sigma), mixed thoroughly by vigorous pipetting and incubated at room temperature for 5 minutes. About 200 µl chloroform was added, mixed well, incubated at room temperature for 5 minutes and centrifuged at 12000 rpm for 15 minutes at 4ºC. The upper aqueous layer was collected and equal volume of ice-cold isopropanol was added and incubated at -20 ºC for 20 minutes. Centrifugation was carried out at 12000 rpm for 10 minutes at 4ºC and the supernatant was discarded. The pellet was washed with 1 ml of 70 % of ice-cold ethanol and centrifuged at 10000 rpm for 10 minutes at 4ºC. The supernatant was discarded and pellet was air dried. The pellet was resuspended in 20 µl of NFW, quantified and stored at -80 ºC until further use. About 500 ng of RNA was used for synthesis of cDNA using iScript (TaKaRa) as per the manufacturer’s instructions. The gene expression analysis of intestinal tight junction protein genes (Zona occludens-1 (ZO-1), Mucin-2 & Claudin-3) were carried out by specific primers (Table 3) with the following PCR conditions: an initial denaturation step at 95ºC for 3 min, followed by 40 cycles at 95ºC for 30 sec, the annealing and extension temperature at 60ºC for 30 sec, and a final extension step of 72ºC for 35 sec. The melt curve analysis was carried out to assess the specificity of the amplified products.
Statistical analysis:
All the data of this study was grouped and subjected to statistical analysis by one-way ANOVA using SPSS, version 20.0 statistical package. Pair wise differences in means were computed using Duncan test. The obtained results were expressed as means and standard deviations.