Biol Trace Elem Res (2014) 160:56–66 DOI 10.1007/s12011-014-0026-4

Dietary Supplementation of Zinc Nanoparticles and Its Influence on Biology, Physiology and Immune Responses of the Freshwater Prawn, Macrobrachium rosenbergii Thirunavukkarasu Muralisankar & Periyakali Saravana Bhavan & Subramanian Radhakrishnan & Chandirasekar Seenivasan & Narasimman Manickam & Veeran Srinivasan

Received: 6 March 2014 / Accepted: 21 May 2014 / Published online: 31 May 2014 # Springer Science+Business Media New York 2014

Abstract The present study was conducted to assess the influence of dietary zinc nanoparticles (size 50 nm) on the growth, biochemical constituents, enzymatic antioxidant levels and the nonspecific immune response of the freshwater prawn, Macrobrachium rosenbergii post larvae (PL). The concentrations of dietary supplement zinc nanoparticles (ZnNPs) were 0, 10, 20, 40, 60 and 80 mg kg−1 with the basal diet, and the level of Zn in ZnNP-supplemented diets were 0.71, 10.61, 20.73, 40.73, 60.61 and 80.60 mg kg−1, respectively. ZnNP-incorporated diets were fed to M. rosenbergii PL (initial body weight, 0.18±0.02 g) in a triplicate experimental setup for a period of 90 days. ZnNP supplemented feed fed PL up to 60 mg kg−1 showed significantly (P c > d > e > f); some of the mean sharing two superscripts means that it falls into two ranks/columns

renewing aquarium water. The experiment was performed for a period of 90 days. Analysis of Survival, Growth and Nutritional Indices At the end of the feeding trial, the survival rate and food index parameters such as feed intake, length gain, weight gain, specific growth rate, feed conversion ratio and protein efficiency ratio were individually determined by the following equations [14]: Survival rateð%Þ ¼ No:of live prawns=no:of prawns introduced  100 Length gain ðcmÞ ¼ Final length ðcmÞ − initial length ðcmÞ Weight gain ðgÞ ¼ Final weight ðgÞ − initial weight ðgÞ Feed intake g d−1 ¼ Feed eaten ðgÞ = total number of days Specific growth rate ð%Þ ¼log final weight ðgÞ −log initial weight ðgÞ = total number of days  100 Feed conversion ratio ðgÞ ¼ feed intake ðgÞ = weight gain ðgÞ Protein efficiency ratio ðgÞ ¼ weight gain ðgÞ = protein intake ðgÞ

Assay of Digestive Enzymes Activities of digestive enzymes such as protease, amylase and lipase were assayed on initial and final days of feeding trial. The whole flesh except eyestalk, appendages and exoskeleton was homogenized in ice-cold distilled water and centrifuged at 9,300 g under 4 °C for 20 min. The supernatant was used as a crude enzyme source. The activity of protease was estimated by the method of Furne et al. [15]. One unit of enzyme activity represents the

amount of enzyme required to liberate 1 μg of tyrosine per minute under assay conditions. The reaction mixture consisted of 0.25 ml of casein at 1 % (w/v), 0.25 ml of 0.1 M glycine– NaOH buffer (pH 10.0) and 0.1 ml enzyme source. The reaction was incubated for 1 h at 37 °C, then the reaction was stopped by adding 0.6 ml 8 % (w/v) trichloroacetic acid solution and kept for 1 h at 2 °C, then centrifuged at 1,800g for 10 min, and the supernatant absorbance was measured at 280 nm against blank. For the blank preparation, the enzyme source was added at the end of the incubation period, just before adding trichloroacetic acid. Tyrosine solution was used as standard. Amylase activity was assayed by starch hydrolysis method of Bernfeld [16] in which the increase in reducing power of buffered starch solutions was measured. The specific activity of amylase was calculated as milligrams of maltose liberated per gram of protein per hour (mg/g/h). The reaction mixture consisted of 0.125 ml of 2 % (w/v) starch solution, 0.125 ml of 0.1 M citrate–phosphate buffer (pH 7.5) and 0.5 ml enzyme source. The reaction was incubated at 37 °C for 1 h, and the absorbance was measured at 600 nm against a blank. For the blank, the enzyme source was added just after the incubation period. Maltose solution was used as standard. The activity of lipase was assayed by the method of Furne et al. [15]. One unit of lipase activity was defined as the amount of free fatty acid released from triacylglycerol per unit time estimated by the amount of NaOH required to maintain pH constant and represented as mille equivalents of alkali consumed. A solution of 1 % polyvinyl alcohol (PVA) and 5 ml of 0.1 N HCl in 1 l of distilled water was heated to 75– 85 °C, cooled, filtered and adjusted to pH 8.0 with 0.1 N

ZnNPs induced growth performance in prawn

NaOH. Virgin olive oil was added to an aliquot of this solution for obtaining 0.1 M substrate concentration. This mixture was emulsified for 5 min. In addition, Mcllvaine buffer was prepared from 0.1 M citric acid and 0.2 M disodium phosphate. The reaction mixture consisted 1 ml of PVA-solutionemulsified substrate, 0.5 ml of Mcllvaine buffer (pH 8.0) and 0.5 ml enzyme source and was incubated at 37 °C for 4 h. To stop the reaction and break the emulsion, 3 ml of 1:1 ethanol-acetone solution was added. To the reaction mixture, phenolphthalein in ethanol 1 % (w/v) was added and titrated against 0.01 N NaOH. For the blanks, the same procedure was followed but boiled enzyme was used. Estimation of Biochemical Constituents and Carcass Mineral Contents Analyses of total nitrogen, crude protein, moisture and ash contents were performed according to standard AOAC procedures [13]. Dry matter was obtained by drying at 105 °C until a constant weight was achieved. Ash content was obtained by burning in a muffle furnace at 600 °C for 12 h. Total nitrogen and crude protein (N*6.25) were analysed after single acid digestion using Kjeldhal apparatus (model: Kelplus DISTYLBS, manufactured by Pelican Equipments Pvt. Ltd. Chennai, India). Concentration of total protein was estimated by the method of Lowry et al. [17], using ethanolic precipitated sample. The blue colour was result of biuret reaction of protein with copper ions in alkali solution and reduction of the phophomolybdicphosphotungstic of Folin reagent by the tyrosine and trphtophan present in the treated protein. Bovine serum albumin (BSA) was used as a standard. This colour intensity was measured at 650 nm against a blank which is devoid of protein sample. Concentration of total amino acid was estimated by the method of Moore and Stein [18]. The total amino acid was extracted with sodium tungstate and H2SO4. When amino acids are heated with ninhydrine, they undergo deamination. The reaction of amino acid-hydratin complex with ninhydrin produced purple colour, which was measured at 540 nm. Leusine was used as standard. Concentration of total carbohydrate was estimated by the method of Roe [19] using TCA extracted sample. Carbohydrates are hydrolysed into simple sugars by diluted HCl in hot acidic medium. Glucose is dehydrated into hydroxyl-methyl furfural. This compound reacts with anthrone and produced a green-coloured product, which was measured at 630 nm. Glucose was used as standard. Total lipid was extracted with chloroform–methanol mixture following the method of Folch et al. [20] and estimated by the method of Barnes and Blackstock [21]. Lipid reacts with vanillin in a medium of H2SO4 and phosphoric acid to form a pink-coloured chromogen, which is proportional to the lipid

59

content of the sample and measured at 540 nm. Olive oil was used as standard. The carcass mineral contents including trace elements were analysed using the atomic absorption spectrophotometer (AAS) (PerkinElmer, model 2380) in air acetylene flame by adopting triple acid digestion method [13]. Total and Differential Haemocyte Counts At the end of the experiment, 100 μl of haemolymph was withdrawn from the ventral sinus in the first abdominal segment using a 26-gauge hypodermic needle on a 1-ml syringe. Each syringe was pre-filled with 200 μl of anticoagulant (10 mM Tris-HCl, 250 mM sucrose, 100 mM sodium citrate, pH 7·6). More anticoagulants were added to make the volume up to 1 ml in the anticoagulated haemolymph. Further, a volume of 200 μl anticoagulated haemolymph was fixed with an equal volume of formalin (10 %) for 30 min. The fixed haemolymph was used to THC and DHC determinations. For THC, 100 μl of fixed haemolymph was diluted at 1:2 ratio (v/v) with ice-cold phosphate-buffered saline (PBS, 20 mM, pH 7·2). The diluted haemolymph was stained with 20 μl of Rose Bengal strain (1.2 % Rose Bengal in 50 % ethanol) and incubated at room temperature for 20 min. THC was determined by haemocytometer (Neubauer improved, Germany) under the light microscope at RP 1000× (Labomed, CXR2).  Counted cells  depth of chamber  dilution factor THC 106 cells ml−1 ¼ Number of 1−mm square

For DHC, fixed haemolymph was stained with Rose Bengal solution (10 %) for 10 min and smeared on a slide. The numbers of differential haemocytes, such as hyalinocytes, semigranulocytes and granulocytes, were characterised according to Tsing et al. [22], and 350–400 cells from each smear were counted under a Trinocular Inverted Microscope (model number INVERSO 3000) RP 1000×. Activities of Enzymatic Antioxidants and Lipid Peroxidation The muscles and hepatopancreas of test prawns were individually homogenised (10 %w/v) in ice-cold 50-mM Tris buffer (pH 7.4), centrifuged at 9,300g for 20 min at 4 °C, and the supernatant was used to assay the enzyme activities. Soluble protein concentration was determined by the method of Lowry et al. [17] using BSA as the standard. Superoxide dismutase (SOD) activity was measured using pyrogallol (10 mM) autoxidation in Tris buffer (50 mM, pH 7.0) [23]. The assay mixture contained 1.2 ml of sodium pyrophosphate buffer, 0.1 ml of phenazine methosulphate (PMS), 0.3 ml of nitro blue tetrazolium (NBT), 0.2 ml of the enzyme prepared and water accounting to a total volume of 2.8 ml. The reaction was initiated by the addition of 0.2 ml

60

NADH. The mixture was incubated at 30 °C for 90 s and arrested by the addition of 1.0 ml glacial acetic acid. The reaction mixture was then shaken with 4.0 ml n-butanol, allowed to stand for 10 min and centrifuged. The intensity of the chromogen in the butanol layer was measured at 560 nm using spectrophotometer. The specific activity of the enzyme was expressed in U/mg protein. Catalase (CAT) activity was measured using H2O2 as the substrate in phosphate buffer [24]. The reaction was initiated by the addition of 1.0 ml of phosphate buffer (0.01 M, pH 7.1), 0.5 ml of H2O2 (0.2 M) and 0.4 ml of distilled water successively to 0.5 ml of tissue homogenate. After 60 s, the reaction was stopped by the addition of 2.0 ml of dichromate-acetic acid reagent. Further, the tubes were kept in a boiling water bath for 10 min and cooled at room temperature. The absorbance of the chromophore was read at 620 nm. A system devoid of enzyme served as the control. The activity of catalase was expressed as micromoles of hydrogen peroxide consumed per minute per milligram of protein. Lipid peroxidation (LPO) in the tissue homogenates was measured by estimating the formation of thiobarbituric-acidreactive substances (TBARS) [25]. The tubes containing 1 ml of tissue homogenate (10 %w/v in 50 mM phosphate buffer, pH 7.4) was subsequently mixed with 1 ml of Tris buffer (0.02 M, pH 7.5), 1 ml of 10 % tricholoro acetic acid and 1.5 ml of thiobarbituric acid (1.5 %). The reaction mixture was boiled for 15 min in the boiling water bath and cooled at room temperature. The content was centrifuged at 100g for 20 min and the supernatant was collected. The absorbance of the supernatant was measured at 535 nm against the reagent blank. TBARS was expressed as nanomoles of malondialdehyde (MDA) per milligram of protein. Activities of Metabolic Enzymes The metabolic enzymes such as glutamic oxaloacetate transaminase (GOT) and glutamic pyruvate transaminase (GPT) were analysed according to the method of Reitman and Frankel [26] using a media source kit (Medsource Ozone Biomedicals Pvt. Ltd. Haryana, India). A total of 100 mg of muscle and hepatopancreas tissues was homogenized in 0.25 M sucrose and centrifuged at 3,300g for 20 min in a high-speed cooling centrifuge at 4 °C. The supernatant was used as the enzyme source. GOT Analysis The substrate solution, L-aspartic acid (500 μl, pH 7.4) was added with 100 μl sample and incubated at 37 °C for 1 h. Then, 500 μl of 2,4-dinitrophenyl hydrazine was added and allowed to stand for 20 min at room temperature, then 3 ml of freshly prepared 4 N sodium hydroxide solution was added to the above solution. The colour development was read at

Muralisankar et al.

505 nm using spectrophotometer within 15 min. Sodium pyruvate (160 U/l) was used as a calibrator. The activity of GOT was expressed as units per litre (U/l). GPT Analysis Buffered L-alanine, 2-oxoglutarate substrate (500 μl; pH 7.4) were added with 100-μl sample and incubated at 37 °C for 20 min. With this, 500 μl of 2,4-dinitrophenyl hydrazine was added and allowed to stand at room temperature for 30 min, then 3 ml of freshly prepared 4 N sodium hydroxide solution was added. The colour development was read at 505 nm using spectrophotometer within 15 min. Sodium pyruvate (170 U/l) was used as a calibrator. The activity of GPT was expressed as units per litre (U/l). Statistical Analysis The data were analysed by one-way analysis of variance (ANOVA) using SPSS (16.0), followed by Duncan’s multiple range test to compare the differences among treatments where significant differences (P f); some of the mean sharing two superscripts means that it falls into two ranks/columns. SR, survival rate; LG, length gain; WG, weight gain; SGR, specific growth rate; FCR, feed conversion ratio; PER, protein efficiency ratio

60 mg ZnNPs kg−1 incorporated diet fed PL. Whereas, the PL fed with 80 mg ZnNPs kg−1 incorporated diet acts as negatively influenced on activities of these enzymes. The activity of protease was found to be significantly higher when compared with control, but significantly lower when compared with 60 mg ZnNPs kg−1 incorporated diet fed PL. The activity of amylase was found to be lower in 80 mg ZnNPs kg−1 incorporated diet fed PL when compared with control, but this was not statistically significant, whereas the decrease was statistically significant when compared with 60 mg ZnNPs kg−1 incorporated diet fed PL. In the case of lipase activity, insignificant difference was seen between control and 80 mg ZnNPs kg−1 incorporated diet fed PL, whereas the activity of lipase in 80 mg ZnNPs kg−1 incorporated diet fed PL showed a significant decrease when compared with 60 mg ZnNPs kg−1 (Table 2).

amino acids, total carbohydrate, total lipid and ash were significantly increased (P e); some of the mean sharing two superscripts means that it falls into two ranks/columns THC total haemocyte count, DHC differential haemocyte count

level of LPO, showed significant elevations (P

Dietary supplementation of zinc nanoparticles and its influence on biology, physiology and immune responses of the freshwater prawn, Macrobrachium rosenbergii.

The present study was conducted to assess the influence of dietary zinc nanoparticles (size 50 nm) on the growth, biochemical constituents, enzymatic ...
249KB Sizes 0 Downloads 3 Views