Dietary supplementation of green synthesized manganese-oxide nanoparticles and its effect on growth performance, muscle composition and digestive enzyme activities of the giant freshwater prawn Macrobrachium rosenbergii

Accepted Manuscript Title: Dietary supplementation of green synthesized manganese-oxide nanoparticles and its effect on growth performance, muscle composition and digestive enzyme activities of the giant freshwater prawn Macrobrachium rosenbergii Author: Annamalai Asaikkutti Periyakali Saravana Bhavan Karuppaiya Vimala Madhayan Karthik Praseeja Cheruparambath PII: DOI: Reference:

S0946-672X(16)30005-0 http://dx.doi.org/doi:10.1016/j.jtemb.2016.01.005 JTEMB 25740

To appear in: Received date: Revised date: Accepted date:

29-9-2015 10-1-2016 12-1-2016

Please cite this article as: Asaikkutti Annamalai, Bhavan Periyakali Saravana, Vimala Karuppaiya, Karthik Madhayan, Cheruparambath Praseeja.Dietary supplementation of green synthesized manganese-oxide nanoparticles and its effect on growth performance, muscle composition and digestive enzyme activities of the giant freshwater prawn Macrobrachium rosenbergii.Journal of Trace Elements in Medicine and Biology http://dx.doi.org/10.1016/j.jtemb.2016.01.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Dietary supplementation of green synthesized manganese-oxide nanoparticles and its effect on growth performance, muscle composition and digestive enzyme activities of the giant freshwater prawn Macrobrachium rosenbergii

Annamalai Asaikkuttia*, Periyakali Saravana Bhavana, Karuppaiya Vimalab, Madhayan Karthika Praseeja Cheruparambathc

a

Crustacean Biology Laboratory, Department of Zoology, School of Life Sciences,

Bharathiar University, Coimbatore – 641 046, Tamil Nadu, India b

Proteomics and Molecular Cell Physiology, Laboratory, Department of Zoology, School of

Life Sciences, Periyar University, Salem – 636 011, Tamil Nadu, India c

Division of Insect Endocrinology, Department of Zoology, University of Calicut, Kerala-

673 635, India * Corresponding author at: Department of Zoology, Bharathiar University, Coimbatore-641 046, Tamil Nadu, India. E-mail address: [email protected] [email protected]

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Graphical abstract

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Research Highlights  Ananas comosus peel extracts are used to synthesize Mn-oxide NPs. 

 Synthesized Mn-oxide NPs are characterized using HR-SEM, DLS, Zeta potential, XPS and FT-IR analysis.  Mn-oxide NPs improved growth efficiency of Macrobrachium rosenbergii.  Mn-oxide NPs significantly involved in the activity of antioxidant defense enzyme activities of SOD, CAT, GOT and GPT.  It could be used as a potential safe and effective dietary supplementation for the feed fed Prawns.

Abstract The green synthesized Mn3O4 nanoparticles (manganese-oxide nanoparticles) using Ananas comosus (L.) peel extract was characterized by various techniques. HR-SEM photograph showed that manganese-oxide nanoparticles (Mn-oxide NPs) were spherical in shape, with an average size of 40–50 nm. The Zeta potential revealed the surface charge of Mn-oxide NPs to be negative. Further, the Mn-oxide NPs were dietary supplemented for freshwater prawn Macrobrachium rosenbergii. The experimental basal diets were supplemented with Mn-oxide NPs at the rates of 0 (control), 3.0, 6.0, 9.0, 12, 15 and 18 mg/kg dry feed weight. The as-supplemented Mn-oxide NPs were fed in M. rosenbergii for a period of 90 days. The experimental study demonstrated that prawns fed with diet supplemented with 3-18 mg Mn-oxide NPs/kg shows enhanced (P < 0.05) growth performance, including final weight and weight gain (WG). Significant differences (P < 0.05) in feed conversion ratio (FCR) were observed in prawn fed with different diets. Additionally, prawns fed with 3.0–18 mg/kg Mn-oxide NPs supplemented diets achieved significant (P < 0.05) improvement in growth performance, digestive enzyme activities and muscle 3   

biochemical compositions, while, the prawns fed with 16 mg/kg of Mn-oxide NPs showed enhanced performance. Prawns fed on diet supplemented with 16 mg/kg Mn-oxide NPs showed significantly (P < 0.05) higher total protein level. The antioxidants enzymatic activity (SOD and CAT) metabolic enzymes status in muscle and hepatopancreas showed no significant (P > 0.05) alterations in prawns fed with 3.0-18 mg/kg of Mn-oxide NPs supplemented diets. Consequently, the present work proposed that 16 mg/kg of Mn-oxide NPs could be supplemented for flexible enhanced survival, growth and production of M. rosenbergii. Therefore, the data of the present study recommend the addition of 16 mg/kg of Mn-oxide NPs diet to developed prawn growth and antioxidant defence system.

Keywords: Macrobrachium rosenbergii; Mn-oxide nanoparticles; Growth; Antioxidant defence enzymes; Biochemical constituents.

1. Introduction Aquaculture production in the world attained 90.4 million tons in 2012 including 66.6 million tons of food fish (e.g., finfishes, crustaceans, molluscs, and amphibians) for human consumption [1]. The crustacean, giant freshwater prawn, Macrobrachium rosenbergii is a major species by yielding high fecundity, rapid growth, wide range of salinity and possessing unique characters such as temperature tolerance, disease resistance as well as its superior taste and high commercial value etc., [2-4]. The global production of M. rosenbergii has increased from 130,689 tons in 2000 to 203,211 tons in 2011 [5]. There is also representing good nutritional source of proteins, essential amino acids, poly-unsaturated fatty acids and low in fat. Therefore, it can be used as a delicious healthy choice of food for human consumptions [6].

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Manganese (Mn) is an essential micronutrient for growth, reproduction and prevention of skeletal abnormalities in terrestrial animals and fish [7]. The dietary requirement of Mn is species dependent. Different from terrestrial animals, fish can absorb Mn from aquatic environment as well as feed. Dietary supplementation of Mn is often required because Mn absorbed from water is not sufficient. Furthermore, Mn plays significant role in better survival, muscle composition, immune response, antioxidant defense and stress tolerance in some fish and crustaceans [8-13]. In recent years, Manganese oxide have been used as raw materials for fertilizer and as a mineral supplementation in animal feed used in pharmaceutics. Further the preparation of manganese-oxide nano-structures has been the target of scientific interest due to their various intriguing physical and chemical properties. Various synthesis methods have been developed to prepare Mn-oxide nanostructures. However, these common methods involve toxic, corrosive and flammable chemical substances, which are often potentially dangerous to the human health and environment. For this reason, attentions have been recently focused on the development of green chemistry and biological synthetic processes to prepare metal or metal oxide nanostructure by using microorganisms, enzymes, fungi, fruits, plant extracts or even agricultural waste [14-15]. Biological approaches using plants or plant extracts for metal nanoparticle synthesis have been suggested as valuable alternatives to chemical methods. The use of plants for the preparation of nanoparticles could be more advantageous, because it does not require elaborate processes such as intracellular synthesis and multiple purification steps. Several plants and their parts have been successfully used for the extracellular synthesis of metal nanoparticles [16]. Over the past few decades a various precious copy of NPs have been synthesized using green chemistry approach. Previously reported that the preparation of silver nanoplates using the extract of unicellular green alga Chlorella vulgaris [17]. Spherical shape platinum

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NPs were synthesized using extracts of Terminalia chebula [18]. Zirconia nano chains synthesized using Curcuma longatuber extract [19]. In addition, bio-conjugated silver nanoparticles were synthesized using Ocimum sanctum leaves extract [29]. Mn-oxide nanoparticles were synthesized using banana peel extract [21]. According to literature review, few studies have reported the synthesis of silver nanoparticles and gold nanoparticles using Ananas comosus peel extract [15, 22]. Pineapple (Ananas comosus L.), a leading edible member of the botanical family Bromeliaceae, is a perennial herb native to the American tropics [23], which is well known for its freeness from harmful phytochemicals [24]. A. comosus peels, core extracts and crown extract contain sugar especially fructose, sucrose and glucose [25]. A wide range of volatiles (more than 280 compounds) have been identified A. comosus, including esters, terpenes, lactones, aldehydes, ketones, alcohols, hydrocarbons and a group of miscellaneous compounds. The main volatile compounds found in pineapple pulp and cores are esters, followed by terpenes, ketones and aldehydes [26]. Biomolecules with carbonyl, hydroxyl, and amine functional groups have the potential for metal ion reduction and capping the newly formed particles during their growth processes [22, 27]. In view of this knowledge, in the present study, peel extract of A. comosus was used to synthesize Mn3O4 nanoparticles. This abundantly available agricultural waste is composed of polymers such as sugar, pectin, cellulose, polyphenols [15, 25], which can act as both reducing and capping agents in the preparation of Mn-oxide NPs. This novel method has a variety of advantages with low cost, simplicity, potential for large-scale production, green and eco-friendly method without using any toxic chemicals. The green synthesized Mn3O4 nanoparticles were dietary supplemented and its effect were further evaluated on survival, growth performance, feed conversion, activities of digestive enzymes, antioxidant response and biochemical blood parameters in M. rosenbergii.

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2. Materials and Methods 2.1.Preparation of Ananas comosus peel extract The waste Ananas comosus (L.) peel is collected from local market and washed with sterile distilled water thoroughly. After the cleaned peel are dried at room temperature in a sterile room for 10 days, the 20 g air dried peel are cut into small pieces and mixed with 500 mL double distilled water stirred with magnet for 4-5 hours at room temperature. The A. comosus (L.) Peel Extract (AcPE) was filtered using Whatman filter paper. Then it was stored at room temperature and resultant extract is stored at -4°C for further experiment. 2.2. Synthesis of Mn3O4 nanoparticles A. comosus peel extract (5 mL) was added drop wise to 50 mL of 0.02 M KMnO4 (Sigma Aldrich) aqueous solution under magnetic stirring at room temperature. After continuous stirring for 5 h, the dark brown product was collected, washed by ethanol several times and then dried in air at 60°C overnight. 2.3. Characterization of Mn-oxide nanoparticles The morphology was examined by scanning electron microscopy (SEM) (Hitachi 7000H, Tokyo, Japan). The nanoparticles solutions were sonicated for 1 min to produce better particle dispersion and to prevent nanoparticles agglomeration on the copper grid. After this, drop of the nanoparticles solution was spread onto a carbon-coated copper grid, which was then dried at room temperature. The sample was then examined and photographed under the microscope. The particle size distribution and zeta potential were measured by photon correlation spectroscopy (PCS) with a zeta potential analyzer add-on unit (Nicomp Zetasizer 380ZLS, Urbana, IL, USA). Samples were diluted to the desired concentration and two 5 min cycles were run for each sample with water prior to the measurements. Intensityweighted size distributions are reported. For zeta potential measurements, three 30s cycles were run for each sample.

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2.4. Feed formulation The branded feed basal ingredients (BI), such as fish meal (25%), soybean meal (20%), groundnut oilcake (20%) and wheat bran (10%) were taken in powder forms and thoroughly mixed. Sunflower oil (2%) was used as lipid source. The Manganese oxide (Mn3O4) nanoparticles were individually incorporated with BI in seven different concentrations each at 0, 3.0, 6.0, 9.0, 12, 15 and 18 mg/kg by replacing the right quantity of BI. Tapioca flour (15%) and egg albumin (7%) were used as binding agents. The dough was steam cooked and cooled at room temperature. Vitamin B-complex forte with vitamin C (1%, BECOSULES® CAPSULES, Pfizer Ltd, Navi Mumbai, India) was also mixed. Sterilized water was adequately added for maintaining the mix in moist and paste form. This mix was pelletized in a manual pelletizer (Kolkata, India) fixed with 3 mm diameter mesh. The pellets were dried in a thermostatic oven (M/s Modern Industrial, Mumbai, India) at 40ºC until they reached constant weight and stored in airtight jars at room temperature. The basal diet formulated contained more or less 40% protein, 32% carbohydrate and 7% lipid. The content of these biochemical constituents was determined by methods of total protein [28], carbohydrate [29] and lipid [30]. 2.5. Experimental animals The giant freshwater prawn M. rosenbergii (De Man, 1879) PL (PL-5) (Syn: Palaemon rosenbergii) were procured from Aqua Hatchery, Koovathur (Latitude 12.44° N; longitude 80.10° E), Kanchipuram District, Tamil Nadu, India. They were safely transported to the laboratory in plastic bags half filled with hatchery water and well-oxygenated. The prawns were acclimatized in crustacean biology laboratory (Bharathiar University, Coimbatore) condition with ground water in cement tanks (1000 L, 6×3×3 feet) for 3 weeks. The ground water satisfied the required physico-chemical parameters (temperature 28 °C; pH, 7.11 ± 0.20; total dissolved solids, 0.95 ± 0.02 g L−1; dissolved oxygen, 7.24 ± 0.311 mg L−1;

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BOD, 10.60 ± 1.15 mg L−1; COD, 65.0 ± 2.00 mg L−1; ammonia, 0.018 ±0.003 mg L−1; zinc level in the water flowing into the rearing system was 27.00 ± 2.41 μg L−1). During acclimatization period they were fed with boiled egg albumin, Artemia nauplii (Artemia salina) and control feed prepared with basal ingredients alternatively three times (at 6 pm, 6 am and 12 pm respectively) per day and 80% of aquarium water was renewed daily at 6 am. 2.6. Feeding experiment In the study, seven groups of M. rosenbergii PL (1.42 ± 0.35 cm length; 0.18 ± 0.02 g weight) were assigned for this experiment in triplicate for 90 days. One group served as control and was fed with‘0’concentration of Mn oxide NPs supplemented diet. The remaining six groups were fed with 3.0, 6.0, 9.0, 12, 15 and 18 mg/kg supplemented diets respectively. Each group consisted of 40 PL in an aquarium maintained with 40 L of ground water. The water medium was changed every 24 h by siphoning method with minimum disturbance to the prawns and aerated adequately. The experimental prawns were fed with these feeds at 10% of body weight twice per day. During the feeding trial, the unconsumed feed, feces and molts were removed on a daily basis while renewing the aquarium water. 2.7. Analysis of survival, growth and food indices At the end of the feeding trial, the survival rate, growth (length gain and weight gain) and other food parameters such as feed intake, specific growth rate, feed conversion ratio and protein efficiency ratio were individually calculated by the following equations [31]. Survival (%) Length gain (cm)







100



Final length

Weight gain (WG)

Initial length











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100



Specific growth rate (SGR) Food conversion ratio (FCR)











100



Protein efficiency ratio (g) Feed intake g day-1













2.8. Assay of digestive enzymes Activities of digestive enzymes (protease, amylase and lipase) were assayed on the initial and final days of the feeding experiment. The whole digestive tract and hepatopancreas were homogenized in ice cold double distilled water and centrifuged at 9300 g under 4°C for 20 min. The supernatant was used as a crude enzyme source. Total protease activity was determined by the casein-hydrolysis method of Furne [32], where 1 unit of enzyme activity represents the amount of enzyme required to liberate 1μg of tyrosine per minute under assay conditions. Amylase activity was determined by the starch-hydrolysis method of Bernfeld [33]. The specific activity of amylase was calculated as milligrams of maltose liberated per gram of protein per hour. Lipase activity was analyzed by the method of Furne [32]. One unit of lipase activity was defined as the amount of free fatty acid released from triacylglycerol per unit of time estimated by the amount of NaOH required to maintain pH constant and represented as mille equivalents of alkali consumed. 2.9. Estimation of muscle biochemical composition and carcass mineral contents Analysis of total nitrogen, crude protein, moisture and ash contents was performed according to the standard procedures of AOAC [34]. 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 analyzed after single acid digestion using a Kjeldhal apparatus (model: Kelplus DISTYL-BS manufactured by Pelican Equipments Pvt. Ltd. Chennai, India). Concentrations of muscle total protein [28], total amino acids (Moore and Stein, 1948) and total carbohydrate [29] were analyzed following standard methods. The total lipid was extracted by the method of Folch [29] and estimated by the method of Barnes [35]. 10   

The carcass mineral contents such as Cu, Zn, Fe, Ca, Mg, Na and K were analyzed using the Atomic Absorption Spectrophotometer (AAS) (Perk in -Elmer; Model 2380) in air acetylene flame by adopting the triple acid digestion method [34]. To achieve this, sacrificed prawns were digested in 9:3:1 ratio of HNO3, H2SO4 and HClO4 using a hot plate at 400°C for 2 h. The digested samples were allowed to cool to room temperature and diluted with double distilled water. 2.10. Activities of enzymatic antioxidants Muscles and hepatopancreas of test prawns were individually homogenized (10% w/v) in ice-cold 50 mM Tris buffer (pH 7.4) centrifuged at 9300 g 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 AOAC [30]. Superoxide dismutase (SOD) activity was measured using pyrogallol (10 mM) autoxidation in Tris buffer (50 mM, pH 7.0) (36) and 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 [37] and the activity of catalase was expressed as μmoles of hydrogen peroxide consumed/min/mg protein. 2.11. Activities of Metabolic Enzymes The metabolic enzymes such as glutamic oxaloacetate transaminase (GOT) and glutamic pyruvate transaminase (GPT) were analyzed according to the method of Reitman and frank [38], using a med source kit (Medsource Ozone Biomedicals Pvt. Ltd. Haryana , India). 100 mg of muscle and hepatopancreas tissues were homogenized in 0. 25 M sucrose and centrifuged at 3300 g for 20 min in a high speed cooling centrifuge at 4°C. The supernatant was used as the enzyme source. For GOT analysis, the substrate solution, L – Aspartic acid (500 μL; pH 7.4) was added to a 100 μL sample and incubated at 37°C for 1 h. Further, 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 solutions were

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added to the above solution. The colour development was read at 505 nm using spectrophotometer within 15 min. Sodium pyruvate (160 U/L) was used as a calibrator. The activity of GOT was expressed as U/L. For GPT analysis, Buffered L-Alanine, 2-Oxoglutarate substrate (500 μL; pH 7.4) was added to a 100 μL sample and incubated at 37°C for 20 min. With this, 500 μL of 2, 4dinitrophenyl hydrazine was added and allowed to stand at room temperature for 30 min followed by the addition of 3 mL of freshly prepared 4N sodium hydroxide solution. The color development was read at 505 nm using a spectro photometer within 15 min. Sodium pyruvate (170 U/L) was used as a calibrator. The activity of GPT was expressed as U/L. 2.12. Statistical analysis The data were analyzed 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< 0.05) were observed. Data were expressed as mean ± SD. 3. Results 3.1. SEM and EDS Spectra of Mn-oxide nanoparticles The synthesis of Mn3O4 nanoparticles was monitored from the colour change after the reaction of KMnO4 solution with A. comosus peel extract. The complete reduction of Mn ions was perceived after 4-5 h incubation. The appearance of dark brown colour in reaction mixture during the incubation period, this colour change clearly indicated the formation of Mn3O4 NPs. The dark brown solution was then with ethanol several times to remove impurities. The formation of Mn3O4 NPs was further conformed by SEM and EDS. The surface morphology of Mn-oxide NPs was investigated using FE-SEM. As shown in Fig. 1a, Mn-oxide NPs nanoparticles were spherical with size ranging between 40-50 nm. The EDS spectrum of Mn-oxide showed only the peaks of Mn and oxygen elements. No trace amounts

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of other elements were seen, which confirmed that Mn-oxide NPs, were essentially free from impurities (Fig. 1b). 3.2. DLS measurements and surface charge of Mn-oxide nanoparticles The DLS instrument is known to measure the shell thickness of a capping or stabilizing agent enveloping the metallic particles along with the actual size of the metallic core. The DLS analysis results (Fig. 2a) revealed that the biosynthesized Mn3O4 nanoparticles had an average particle size of 50 nm. The particles size was larger and more polydisperse compared to the SEM result. The results of zeta potential value for Mn3O4 nanoparticles obtained from A. comosus peel extract was around -31.36 mV (Fig. 2b) indicating the surface charge of the synthesized nanoparticles. 3.3. FT-IR and XPS Spectrum of Mn-oxide nanoparticles FT-IR and XPS were used to further characterize the Mn3O4 nanoparticles. Fig. 3a displays the FT-IR spectrum of the NPs. The spectrum clearly shows Mn-O absorption b and near at 626 cm-1. The peaks at 421 cm-1 indicate that the presence of vibration of manganese (Mn3+) in an octahedral site. The amide linkages between the amino acid residues in the proteins give rise to peaks in the infrared region between 2900–3700 cm−1. The bands observed at 3450 cm−1 and 2335 cm−1 can be assigned to the stretching vibrations of the primary and secondary amines, O–H stretching of alcohols and C–H stretching of alkanes. The peaks around 1638 cm−1 and 1486 cm−1 are due to the amide I and amide II regions that are characteristic of proteins/enzymes. The intense bands observed at 1384 cm−1were indicative of C–N stretching vibrations of aliphatic and aromatic amines respectively. The Mn 2p spectrum is shown in Fig. 3b, the binding energy value of Mn 2p 3/2 was 642.6 eV and the observed spin-spitting between the Mn 2p3/2 and Mn 2p1/2 was 11.6 eV. Furthermore, the multiple splitting of the Mn 3s was 5.23 eV (Fig. 3c), which exactly matches with the reported values of Mn-oxide compound. Furthermore, the multiple splitting

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of the Mn 3s is 5.23 eV which exactly matches with the reported values of Mn-oxide compound. Therefore, both FT-IR and XPS results confirm that the as obtained materials are Mn3O4 nanoparticles. 3.4. Growth, survival and food indices The results on growth performance, survival and nutritional directory of prawn diets supplemented with different concentrations of dietary Mn-oxide NPs are presented in Table 1. As seen in Fig. 4a, b, no significant difference was observed in the initial weight of prawn treated with manganese-oxide and control. In contrast, when the prawn were fed with diet supplemented with 0-18 mg Mn-oxide NPs/kg, it showed increasing level of (P < 0.05) growth performance, together with final weight and weight gain as compared to the control. Significant variations were observed (P < 0.05) in feed conversion ratio (FCR) and survival rate of prawn fed with different diets after 90 days (Table 1 and Fig 4c). The break point in the regression line, 16.53 and 16.42 manganese-oxide NPs/kg diets was considered to be the optimum dietary level for better response for weight gain and specific growth rate of M. rosenbergii respectively (Fig. 4d). 3.5. Activity of digestive enzymes Table 2 revealed that the enzymes activities such as amylase, lipase and protease functions were improved (P< 0.05) in prawns fed with diet supplemented with 0-18 mg Mnoxide NPs/kg when compared to the control. The protease activity was found to be significantly higher than control. Amylase and lipase activities were found to be lesser in prawns treated with 16 mg Mn-oxide NPs/kg diet. The results of Fig. 3 authentically proved that the all digestive enzymes were significantly increased in M. rosenbergii when fed with diet supplemented Mn-oxide NPs/kg.

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3.6. Mineral concentration and muscle biochemical compositions Effects of varying doses of dietary Mn-oxide NPs supplementation on mineral concentration and biochemical compositions are shown in Table 3. Prawn fed on diet supplemented with 16 mg nano manganese-oxide showed significantly (P < 0.05) higher total percentage of nitrogen and crude protein levels. Similarly, the muscle biochemical compositions of total protein, amino acids, carbohydrate, lipid and ash contents were also drastically increased (P < 0.05) in prawns fed on supplemented diet with 0-18 nano Mn-oxide NPs/kg (Table 3) compared to the control. The mineral salts contents and distribution of Cu, Fe, Ca, Mg, Na, Mn and K were significantly increased in prawns fed on supplemented diet with 0-18 nano Mn-oxide/kg compared with control. However, nano manganese-oxide content was considerably (P < 0.05) increased in prawns fed on supplemented diet with 0-18 nano Mn-oxide/kg (Table 3). 3.7. Antioxidant activities and metabolic enzymes activities Table 4 and 5 represents the results related to antioxidant response and metabolic enzyme activities of muscle and hepatopancreas of prawns treated with nano manganeseoxide respectively. No significant alterations were observed in antioxidant response and metabolic activities of muscle and hepatopancreas of prawns after 90 days compared to the control. 4. Discussion and Conclusion Current research in biosynthesis of non-metals using plant extracts has opened a new era in fast and nontoxic methods for the production of nanoparticles [39]. Plants contain a complex network of antioxidant metabolites and enzymes that work together to prevent oxidative damage to cellular components. Pineapple contains a number of essential nutrients, including vitamin C, manganese and fibre. It also contains beneficial phytochemicals which 15   

have antioxidant and anti-cancer activities [40]. This study has shown that pineapple peel extract otherwise considered as waste has transformed KMnO4 to nanoparticles with great stability. The appearance of dark brown colour is a clear indication of the formation of Mn3O4 nanoparticles. Previous studies suggested that sugars such as sucrose, glucose etc. could be responsible for synthesis of silver NPs using pineapple. It has also been reported that bio molecules with carbonyl, hydroxyl and amine functional groups have the potential for metal ion reduction and capping the newly formed particles during their growth processes [27]. Since amine was found to be the major functional group as characterized from FTIR spectra, it can be suggested that stabilization and reduction of the system may have resulted from binding of the amine group present in A. comosus extract. Many nanoparticles such as selenium, zinc, chitosan and iron supplemented with dietary feeds have shown to improve the growth several of fishes and prawns [41-46]. Previous study reported that metal oxides such as TiO2, ZnO, MgO, Mn2O3 and CaO are of particular interest in various fields as they are not only stable under harsh process conditions but also generally regarded as safe materials to human beings and animals [47]. However these metal oxides have not been used as animal feed so far. This is the first report to use a metal oxide based NPs like Mn3O4 nanoparticles as feeds for giant freshwater prawn M. rosenbergii development. Minerals such as cobalt, copper, iron, manganese, selenium, zinc, chromium and iodine etc. are required for the normal life processes, and all animals, including fish and crustacean, need these inorganic elements [48]. Of these minerals, manganese (Mn) an essential micronutrient is required for growth, reproduction and prevention of skeletal abnormalities in terrestrial animals and fish [49-50]. Different from terrestrial animals, fish can absorb Mn from aquatic environment as well as feed. Dietary supplementation of Mn is

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often required because Mn absorbed from water is not sufficient [51]. Hence as previously reported prawn probably requires supplemental Mn for growth [52]. In this present result, Mn3O4 NPs supplemented diet can significantly increase the survival, growth (length and weight gain), specific growth rate and protein efficiency ratio. About 0–18 mg/kg of Mn-oxide NPs had the ability to promote the survival, feed intake and growth of M. rosenbergii. However, the broken-line model indicated that the optimum dietary Mn-oxide NPs concentration for better responses in weight gain and specific growth rate for prawn species were 16.53 mg/kg and 16.42 mg/kg respectively. A previous report showed similar positive relationship between growth and dietary manganese supplementation necessary for the growth of fresh and marine water fishes, such as channel catfish [53], rainbow trout and common carp [54], grass carp [55], Atlantic salmon [56], gibel carp [57], juvenile tilapia [58], juvenile grouper [59], yellow catfish [60], juvenile cobia [61] and turbot [62]. Consistently it might be concluded that dietary Mn is an essential mineral for normal growth of freshwater prawn M. rosenbergii. As previously reported zinc, selenium, iron nanoparticles supplemented diet fed fishes showed an increase in muscle biochemical compositions, such as total nitrogen, protein, amino acid, carbohydrate, lipid and ash [42-44, 46]. Similarly our results suggest that Mn3O4 nanoparticles supplemented diet had significant influence on nutrient absorption and enhanced the synthesis and storage of protein, amino acids, carbohydrate and lipid in M. rosenbergii. Manganese deficiency (lack of mineral) includes increased mortality, retarded growth and skeletal abnormalities in fishes and crustaceans [63]. In the present investigation, we evaluated the contents of minerals in M. rosenbergii. Here the results evidently proved that 0–18 mg/kg of dietary Mn-oxide NPs endorse mineral utilization. About 16 mg nano Mnoxide/kg supplemented diet fed prawns showed higher absorption of minerals when 17   

compared to control. These results authorized that higher concentration of Mn3O4 nanoparticles could be the compensatory response for Mn-deficiency. The significance of Mn retention in tilapia fed Mn-deficient diet further supports the hypothesis [57]. The digestive enzyme in crustaceans plays an essential role in nutritional physiology and regulates the growth and molt cycle [64]. In this study, the important digestive enzyme such as protease, amylase and lipase activity shows that supplemented Mn3O4 nanoparticles had improved digestive enzyme secretion in M. rosenbergii. Similarly, other nanoparticles like Zinc supplemented feed fed M. rosenbergii produced significant improvements in digestive enzyme secretion [44]. In the present study, supplementations of Mn-oxide NPs significantly increased the activity of antioxidant defence system and metabolic activities such as SOD, CAT, GOT and GPT. Previous studies reported that Mn-SOD activity was regulated by dietary Mn contents in fish. For model, suppressed hepatic Mn-SOD activity has been observed in Mn-deficient yellow croaker, tilapia, juvenile cobia, rainbow trout [51, 58, 61, 65]. The results indicated that prawn fed the basal diet confronted with oxidative stress, which consisted with suppressed antioxidant responses in prawn fed the basal diet. In conclusion, simple and green bio synthesis route has been effectively developed to prepare Mn3O4 nanoparticles by using A. comosus peel extract as both reducing and capping agent. The as-prepared Mn3O4 nanoparticles had spherical morphology with size ranging between 40-60 nm. We further demonstrate that these Mn-based NPs are safe and effective dietary supplementation for fresh water prawn M. rosenbergii. Herein Mn3O4 nanoparticles supplemented in the diet could improve the performance in survival, growth, final weight and antioxidant activities of prawns, M. rosenbergii (Scheme. 1). This novel method has many advantages including low cost effect, simplicity and potential for large-scale production, green and eco-friendly method without using any toxic chemicals. Apart from these 18   

promising results, prospect studies are required for a complete evaluation of its wide application in various species of prawns. These findings break through the bottleneck in the application of Mn3O4 nanoparticles for feed technology and pave the way for the dietary supplementation of manganese-oxide nanoparticles as safe investigate for aquatic animals.

Conflict of interest The authors declare no conflicts of interest in this work.

Acknowledgements We would like to acknowledge University Grant Commission (UGC), New Delhi for supporting financial assistance in the form of Rajiv Gandhi National Fellowship (RGNF) (Ref No: RGNF-SC-TAM-39192 (SA-III)). We also thank Department of Zoology, Bharathiar University, Coimbatore for supporting and providing essential infrastructure facility needed for this study. We thank the CeNSE Department, Indian Institute of Science (IISc) Bangalore 560 012 India for help with all characterizations.

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Reference [1] FAO. The State of World Fisheries and Aquaculture Opportunities and challenges. Food and Agriculture Organization of the United Nations. Rome, 2014. Available at: http://www.fao.org/3/a-i3720e.pdf [2] Johnson SK. Diseases of Macrobrachium. In: New, M.B. (Ed.), In: Giant Prawn Farming Developments in Aquaculture and Fisheries Science, 10. Elsevier, Amsterdam 1982; 10: 269– 277. [3] New M. Status of freshwater prawn farming: a review. Aquac. Res 1995; 26: 1–54. [4] Roustaian P, Kamarudin MS, Omar HB, Saad CR, Ahmad MH. Biochemical changes in freshwater prawn Macrobrachium rosenbergii during larval development. J. World Soc. Aquacul 2001; 32: 52–59. [5] FAO, Macrobrachium rosenbergii (de Man) Statistics. Cultured Aquatic Species Information Programme, Fisheries and Aquaculture Department, Food and Agriculture Organization

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27   

Figure Legends

Fig 1. (a). High Resolution-Scanning Electron Microscopy analysis (HR-SEM) of Mnoxide NPs confirms spherical in shape and inter-particle interactions are quite large. (b) EDAX spectrum shows manganese (Mn) and oxide (O) peaks present in the Mn3O4 NPs.

28   

Fig 2. (a) DLS measurement to determine the size distribution of manganese-oxide nanoparticles. (b). Particle size distribution pattern of manganese-oxide nanoparticles (Mn).

29   

Fig 3. (a). FT-IR spectra for manganese oxide nanoparticles (Mn).(b) Mn 2p and (c) Mn 3s of XPS spectra for manganese-oxide nanoparticles. 30   

31   

Fig 4. Growth performance of M. rosenbergii fed with Mn-oxide NPs supplemented diets. (a). (Length, Length gain (LG); (b). weight; weight gain (WG); (c). specific growth rate (SGR) compared with control. (d). Relationship between weight gain (WG) specific growth rate (SGR) on dietary Mn levels based on a broken-line regression analysis, where

32   

X represents dietary Mn requirement of M. rosenbergii and containing graded levels of Mn for 90 days. The term “Criteria values” represents the values of a selected parameter, such as WG and SGR.

33   

Scheme 1. Schematic illustration shows the bio synthesized and characterization of Mn-oxide NPs used to diet supplementation for M. rosenbergii. Mn-oxide NPs enhanced the growth performance, antioxidant and digestive enzymes activities and improved survival of the M. rosenbergii.

34   

Table Legends Table 1. Survival, growth and nutritional indices of M. rosenbergii and fed with different concentrations of Mn-oxide nanoparticles supplemented diets. Table 2. Activities of digestive enzymes (U/mg protein) in M. rosenbergii and fed with different concentrations of Mn-oxide nanoparticles supplemented diets. Table 3. Concentrations of biochemical constituents in M. rosenbergii fed with Mn-oxide NPs supplemented diets. Table 4. Activities of antioxidant and metabolic enzymes in the muscle of M. rosenbergii fed with different concentrations of Mn-oxide nanoparticles supplemented diets. Table 5. Activities of antioxidant and metabolic enzymes in the hepatopancreas of M. rosenbergii fed with different concentrations of Mn-oxide nanoparticles supplemented diets.

35   

Table 1. Survival, growth and nutritional indices of M. rosenbergii and fed with different concentrations of manganese-oxide nanoparticles supplemented diets.

Control a

Parameters

Mn concentrations (mg/kg)

3.0 SR (%)

6.0

9.0

12

15

18

76.58±1.71d

77.83±1.16c

79.62±1.84bc

81.81±2.22b

83.43±2.37ab

85.67±2.89a

72.15±1.15c

Length

Initial

1.64±5.11

1.64±5.11

1.64±5.11

1.64±5.11

1.64±5.11

1.64±5.11

1.64±5.11

(cm)

Final

3.831±0.43c

4.53±0.21b

5.87±0.53b

5.97±0.62ab

6.33±1.12ab

9.64±1.41a

8.97±0.16ab

Weight (g)

Initial

0.23±0.06

0.23±0.06

0.23±0.06

0.23±0.06

0.23±0.06

0.23±0.06

0.23±0.06

Final

0.66±0.11c

0.82±0.26bc

1.33±0.38bc

1.74±0.44ab

1.97±0.72ab

2.45±1.21a

2.11±0.22ab

LG (cm)

3.38±0.41f

4.39±0.17e

4.72±0.46d

5.39±0.29b

5.85±0.48db

6.74±0.44a

6.01±0.11ab

WG (g)

0.52±0.02e

0.71±0.04d

1.02±0.05c

1.32±0.05b

1.62±0.06a

2.11±0.05a

1.95±0.05b

SGR (%)

0.64±0.03d

0.88±0.04c

1.04±0.05c

1.34±0.05c

1.83±0.04b

2.11±0.05a

1.96±0.06b

FCR (g)

2.25±0.12a

1.33±0.13b

0.92±0.19b

0.79±0.16c

0.61±0.14c

0.54±0.11c

0.62±0.31c

Each value is mean ± SD of three individual observations; mean values within the same row sharing the different alphabetical letter superscripts are statistically significant at P< 0.05 (one-way ANOVA and subsequent post hoc multiple comparison with DMRT); a–f, order of performance (a > b > c > d > e > 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. a Mn free diet; n = 3 (three samples from each treatment), mean ± SD.

36   

Table 2. Activities of digestive enzymes (U/mg protein) in M. rosenbergii and fed with different concentrations of manganese-oxide  nanoparticles supplemented diets. Concentrations of Supplemented diets Enzymes

Control

(mg/kg) 3.0

Protease

Amylase

Lipase*

6.0

9.0

12

15

18

Initial

0.28±0.0554

0.28±0.0554

0.28±0.0554

0.28±0.0554

0.28±0.0554

0.28±0.0554

0.28±0.0554

Final

1.16±0.22c

1.27±0.24b

1.41±0.33b

1.56±0.49ab

1.72 ±0.58ab

1.91±0.82a

1.51±0.91b

Initial

0.17±0.03

0.17±0.03

0.17±0.03

0.17±0.03

0.17±0.03

0.17±0.03

0.17±0.03

Final

0.79±0.22c

0.84±0.27b

0.90±0.32b

0.95±0.52b

1.23±0.66b

1.61±0.77a

1.01±0.82b

Initial

0.76±0.04

0.76±0.04

0.76±0.04

0.76±0.04

0.76±0.04

0.76±0.04

0.76±0.04

Final

0.25±0.03d

0.27±0.03d

0.31±0.04c

0.44±0.04b

0.51±0.05b

0.59±0.11a

0.26±0.17d

Each value is mean ± SD of three individual observations; mean values within the same row sharing the different alphabetical letter superscripts are statistically significant at P< 0.05 (one-way ANOVA and subsequent post hoc multiple comparison with DMRT.*, ×102; n=3 (three samples from each treatment), mean ±SD. a

Mn free diet.

b

x 102; n = 3 (three samples from each treatment), mean ± SD.

37   

Table 3. Concentrations of biochemical constituents in M. rosenbergii fed with Mn-oxide NPs supplemented diets.

Parameters Total nitrogen (% dry wt.) Crude protein (% dry wt.) Total protein (mg/g wet wt.) Amino acid (mg/g wet wt.) Carbohydrate (mg/g wet wt.) Lipid (mg/g wet wt.) Ash (%) Moisture (%) Whole body minerals content (μg/g)

Concentrations of Supplemented diets (mg/kg)

Control Initial Final Initial Final Initial Final Initial Final Initial

4.22 ± 0.13 8.27 ± 0.04d 25.44 ± 1.214 54.01 ± 1.11d 47.53 ± 1.21 133.11 ± 2.11e 26.42 ± 3.21 83.17± 1.23d 19.14 ± 1.28

3.0. 4.22 ± 0.13 8.52± 0.11cd 25.44 ± 1.214 56.21 ± 1.22cd 47.53 ± 1.21 147.37 ± 4.22d 26.42 ± 3.21 94.25 ± 2.12cd 19.14 ± 1.28

6.0 4.22 ± 0.13 8.94 ± 0.23bc 25.44 ± 1.214 59.37± 1.31bc 47.53 ± 1.21 158.43 ± 2.17c 26.42 ± 3.21 100.21 ± 2.01c 19.14 ± 1.28

9.0 4.22 ± 0.13 9.41 ± 0.34b 25.44 ± 1.214 62.11 ± 1.13b 47.53 ± 1.21 172.51 ± 2.45b 26.42 ± 3.21 107.12 ± 2.61b 19.14 ± 1.28

12 4.22 ± 0.13 9.86 ± 0.51b 25.44 ± 1.214 66.28 ± 1.16b 47.53 ± 1.21 192.12 ± 3.23b 26.42 ± 3.21 121.23 ± 2.73b 19.14 ± 1.28

15 4.22 ± 0.13 10.10 ± 0.15a 25.44 ± 1.214 71..1 ± 1.03a 47.53 ± 1.21 224.32 ± 2.34a 26.42 ± 3.21 140.11 ± 2.91a 19.14 ± 1.28

18 4.22 ± 0.13 8.89 ± 0.31abc 25.44 ± 1.214 59.56 ± 1.57abc 47.53 ± 1.21 141.21 ± 2.11de 26.42 ± 3.21 91.14 ± 3.02cd 19.14 ± 1.28

Final Initial Final Initial Final Initial Final Ca Cu Fe Mn K Mg Na Zn

33.19 ± 1.11d 10.33 ± 1.39 19.11 ± 1.32c 10.13 ± 0.14 14.15 ± 2.13a 76.21 ± 1.61 70.01 ± 2.12a 33.11 ± 1.12f 36.18 ± 1.23d 25.22 ± 1.11d 1.9.23±1.43d 122.15 ± 2.10c 76.11 ± 2.11f 120.18 ± 1.32e 18.14 ± 1.31f

38.23 ± 2.12c 10.33 ± 1.39 22.12 ± 1.42bc 10.13 ± 0.14 15.32 ± 2.22 a 76.21 ± 1.61 69.23 ± 2.09a 73.15 ± 2.13d 51.11 ± 1.42c 30.17 ± 1.32c 11.38.±2.67c 129.42 ± 2.41b 92.32 ± 2.14e 127.33 ± 1.56d 33.16 ± 2.11e

43.32 ± 2.31bc 10.33 ± 1.39 25.34 ± 2.11ab 10.13 ± 0.14 16.55 ± 3.22 a 76.21 ± 1.61 67.32 ± 2.026a 84.33 ± 2.43c 66.17 ± 1.66b 38.13 ± 1.23b 26.44.±2.88c 137.11 ±1.12b 115.12 ± 2.13d 138.35 ± 1.64c 41.11 ± 2.11d

48.42 ± 2.44ab 10.33 ± 1.39 28.14 ± 3.01ab 10.13 ± 0.14 16.84 ± 1.10 a 76.21 ± 1.61 65.45 ± 1.84a 97.43± 2.64b 72.21 ± 2.12b 42.23 ± 1.32b 38.76.±1.75b 143.19 ±3.04b 131.14 ± 3.18c 146.18 ± 1.92c 49.17 ± 1.18c

53.16 ± 1.22ab 10.33 ± 1.39 31.33 ± 2.13b 10.13 ± 0.14 16.95 ± 2.11 a 76.21 ± 1.61 63.11 ± 1.68a 118.18±1.32ab 81.27 ± 2.19b 55.32 ± 2.44a 44.33.±2.08b 148.22 ± 3.32b 146.16 ± 2.11b 157.11 ± 2.23b 56.43 ± 1.28b

60.32 ± 0.17a 10.33 ± 1.39 34.25 ± 1.11a 10.13 ± 0.14 11.21 ± 3.44 a 76.21 ± 1.61 61.24 ± 1.43a 138.53 ± 1.59a 90.33 ± 2.46a 64.21 ± 2.62a 66.14.±2.87a 151.13 ± 2.01a 161.223± 3.11a 169.74 ± 2.93a 62.16 ± 1.42a

43.12 ± 2.11c 10.33 ± 1.39 21.21 ± 1.03bc 10.13 ± 0.14 14.65 ± 2.28 a 76.21 ± 1.61 69.10± 4.62a 56.18 ± 2.61e 87.03 ± 2.63a 23.11 ± 2.34a 46.18.±1.21b 1147.12 ± 1.09b 151.11 ± 2.21b 159.56 ± 1.23b 54.33 ± 1.15b

38   

Table 4. Activities of antioxidant and metabolic enzymes in the muscle of M. rosenbergii fed with different concentrations of Mn-oxide NPs supplemented diets.

Enzymes

Concentrations of Supplemented diets (mg/kg)

Control 3.0

Initial 30days

SOD(µmol/min/m g protein)

6.0

9.0

12

3.78± 1.07

3.78± 1.07

3.78± 1.07

3.78± 1.07

3.78± 1.07

3.78± 1.07

3.78± 1.07

b

b

b

b

b

b

8.54±1.63 a

7.96±1.11

7.97±1.18

8.05±1.21

8.26±1.28

8.35±1.42

8.44±1.52

8.10 ± 1.14b

8.10 ± 1.19b

8.13±1.22b

8.32±1.31b

8.43±1.42 b

8.52±1.49 b

8.82±1.61 a

90days

8.22± 1.17b

8.24± 1.14b

8.48± 1.17b

8.55± 1.22b

8.64±1.31 b

8.71±1.44 b

8.87±1.35 a

11.32± 1.05

11.32± 1.05

11.32± 1.05

11.32± 1.05

11.32± 1.05

11.32± 1.05

11.32± 1.05

b

b

b

b

21.69±1.45a

30days

CAT (U/mg protein)

b

20.82±1.11

21.13±1.23

21.35±1.24

21.43±1.34

21.60±1.37

21.27±1.19b

21.45±1.24b

21.57±1.29b

21.66±1.32b

21.70±1.51a

60days

20.88±1.09b

20.83±1.16b 20.90±1.17b

90days

20.93±1.16b

20.93±1.23b

21.34±1.11b

21.44±1.21b

21.52±1.32b

21.75±1.31 b

21.83±1.32a

6.28± 1.06

6.28± 1.06

6.28± 1.06

6.28± 1.06

6.28± 1.06

6.28± 1.06

6.28± 1.06

30days

8.22± 1.14b

8.22± 1.14b

8.20± 1.19b

8.22±1.21b

8.23±1.22b

8.24±1.35b

8.25±1.45 a

60days

8.21± 1.37b

8.21± 1.39b

8.22± 1.53b

8.23± 1.62b

8.23± 1.71 b

8.24± 1.44b

8.24± 1.96 a

90days

8.22±1.18b

8.22± 1.19b

8.23± 1.53b

8.23± 1.74b

8.23± 1.86 b

8.24± 1.46 b

8.24± 1.465a

7.67± 1.04

7.67± 1.04

7.67± 1.04

7.67± 1.04

7.67± 1.04

7.67± 1.04

7.67± 1.04

b

b

b

b

Initial

Initial

GOT (U/L)

GPT (U/L)

b

b

9.22± 1.56

9.22± 1.75 a

30days

9.11± 1.05

9.11± 1.07

9.16± 1.14

9.22± 1.23

9.22± 1.46

60days

9.15± 1.12b

9.15± 1.13b

9.18± 1.42b

9.21± 1.19b

9.23± 1.62 b

9.23± 1.72 b

9.223 1.84 a

90days

9.18± 1.15b

9.18± 1.16b

9.22± 1.21b

9.23± 1.12b

9.23± 1.87 b

9.23± 1.94 b

9.23± 1.99a

Mean values within the same row sharing the same superscript are not significantly different (P > 0.05). Mn free diet; n = 3 (three samples from each treatment), mean ± SD.

39   

18

60days Initial

a

15

Table 5. Activities of antioxidant and metabolic enzymes in the hepatopancreas of M. rosenbergii fed with different concentrations of Mn-oxide NPs supplemented diets.

Enzymes

Concentrations of Supplemented diets (mg/kg)

Control 3.0

6.0

9.0

12.0

15.0

18

4.93± 1.07

4.93± 1.07

4.93± 1.07

4.93± 1.07

4.93± 1.07

4.93± 1.07

4.93± 1.07

9.70±1.03b

9.70±1.08b

9.92±1.13b

10.28±1.22b

10.52±1.37 b

10.72±1.42 b

10.85±1.54 a

60days

9.91 ± 1.13b

9.91 ± 1.20b

10.12±1.21b

10.35±1.30b

10.63±1.45 b

10.84±1.62 b

11.04±1.07 a

90days

10.14± 1.04b

10.42± 1.15b

10.78± 1.21b

10.97± 1.36b

11.14±1.08 b

11.46±1.08 b

11.81±1.18 a

12.13± 1.07

12.13± 1.07

12.13± 1.07

12.13± 1.07

12.13± 1.07

12.13± 1.07

12.13± 1.07

30days

22.05±1.04b

22.45±1.16b

22.84±1.42b

23.06±1.17 b

23.36±1.39 b

23.55±1.44a

60days

22.42±1.02b

22.33±1.08b 22.52±1.18b

22.65±1.21b

22.73±1.31b

23.88±1.43 b

23.91±1.57 b

23.95±1.33 a

90days

22.57±1.16b

22.61±1.21b

22.84±1.34b

23.16±1.08b

23.44±1.19 b

23.58±1.26 b

23.97±1.37a

8.12± 1.03

8.12± 1.03

8.12± 1.03

8.12± 1.03

8.12± 1.03

8.12± 1.03

8.12± 1.03

b

b

b

b

b

b

9.32±1.18 a

Initial 30days

Initial

Initial

SOD(µmol/min/mg protein)

CAT (U/mg protein)

GOT (U/L)

30days

8.32± 1.05

8.36± 1.17

8.53± 1.21

8.86±1.33

60days

8.52± 1.07b

8.66± 1.15b

8.90± 1.23b

9.05± 1.04b

9.34± 1.18 b

9.66± 1.24 b

9.73± 1.43 a

90days

8.72±1.08b

8.94± 1.18b

9.09± 1.22b

9.35± 1.32b

9.56± 1.41 b

9.81± 1.49 b

9.93± 1.66 a

8.15± 1.06

8.15± 1.06

8.15± 1.06

8.15± 1.06

8.15± 1.06

8.15± 1.06

8.15± 1.06

b

b

b

b

Initial

GPT (U/L)

b

10.14± 1.14

10.32± 1.18 a

9.16± 1.12

9.35± 1.31

9.51± 1.45

9.75± 1.62

9.96± 1.69

60days

9.29± 1.13b

9.48± 1.26b

9.69± 1.36b

9.90± 1.49b

10.22± 1.12 b

10.35± 1.17 b

10.53± 1.22 a

90days

9.56± 1.21b

9.66± 1.37b

9.88± 1.48b

9.98± 1.59b

10.39± 1.07 b

10.61.± 1.17 b

10.75± 1.26a

Mn free diet; n = 3 (three samples from each treatment), mean ± SD.

40   

b

9.23±1.04

30days

Mean values within the same row sharing the same superscript are not significantly different (P > 0.05). a

8.95±1.44