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Publications

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LIST OF PUBLICATIONS

1. Muralisankar, T., Bhavan, P.S., Radhakrishnan, S., Seenivasan, C., Manickam, N.,

Srinivasan, V., 2014. Dietary supplementation of zinc nanoparticles and its

influence on biology, physiology and immune responses of the freshwater prawn,

Macrbrachium rosenbergii. Biol. Trace Elem. Res. DOI 10.1007/s12011-014-0026-

4 (Impact factor 1.307).

2. Muralisankar, T., Bhavan, P.S., 2013. Chicken waste meal as an alternative for

fishmeal for better survival and growth of the freshwater prawn Macrobrachium

rosenbergii post larvae. Res. J. Biotech, 8(2), 61-66 (Impact factor 0.294).

3. Seenivasan, C., Radhakrishnan, S., Muralisankar, T., Bhavan, P.S., 2012.

Influence of combined probiotics Lactobacillus sporogenes and Bacillus subtilis on

survival, growth, biochemical changes and energy utilization performance of

Macrobrachium rosenbergii (De Man 1879) post larvae. J. Ecobiotech., 4(1), 29-34.

4. Seenivasan, C., Bhavan, P.S., Radhakrishnan, S., Muralisankar, T., 2012. Effects

of probiotics on survival, growth and biochemical constituents of freshwater prawn

Macrobrachium rosenbergii post larvae. Turk. J. Fish. Aquat. Sci., 12, 331-338

(Impact factor 1.07).

5. Seenivasan, C., Radhakrishnan, S., Muralisankar T., Bhavan, P.S., 2012. Efficacy

of probiotics on survival, growth, biochemical changes and energy utilization

performance of Macrobrachium rosenbergii (De Man 1879) post-larvae. J. Sci.

Res., 4 (3), 729-740.

6. Poongodi, R., Bhavan, P.S., Muralisankar, T., Radhakrishnan, S., 2012. Growth

promoting potential of garlic, ginger, turmeric and fenugreek on the freshwater

Prawn Macrobrachium rosenbergii. Int. J. Pharm. Bio Sci., 3(4), 914 – 926.

7. Seenivasan, C., Radhakrishnan, S., Muralisankar, T., Bhavan, P.S., 2013. Bacillus

subtilis on survival, growth, biochemical constituents and energy utilization of the

freshwater prawn Macrobrachium rosenbergii post larvae. Egyptian J. Aquatic

Res., 38, 195-203.


8. Aarumugam, P., Bhavan, P.S., Muralisankar, T., Manickam, N., Srinevasan, V.,

Radhakrishnan, S., 2013. Growth of Macrobrachium rosenbergii fed with Mango

seed kernel, Banana peel and Papaya peel incorporated feeds. Int. J. Applied Biol.

Phar. Technol., 4 (2), 12-25.

9. Bhavan, P.S., Saranya, C., Manickam, N., Muralisankar, T., Radhakrishnan, S.,

Srinivasan, V., 2013. Effects of Piper longum, Piper nigram and Zingiber officinale

on survival, growth, activities of digestive enzymes and contents of total protein,

vitamins and minerals in the freshwater prawn Macrobrachium rosenbergii. Elixir.

Bio. Tech., 58, 14824-14828.

10. Bhavan, P.S., Devi, N.N., Muralisankar, T., Manickam, N., Radhakrishnan, S.,

Srinivasan, V., 2013. Effects of Myristica fragrans, Glycyrrhiza glabra and

Quercus infectoria on growth promotion in the prawn Macrobrachium

rosenbergii. Int. J. Life. Sci. Biotechnol. Pharm. Res.2, 2250-3137.

11. Bhavan, P.S., Kavithamani, N., Radhakrishnan, S., Muralisankar, T., Srinivasan, V.,

Manickam, N., 2013. Comparison of nutritional quality of sunflower oil and cod

liver oil enriched with Artemia nauplii for assessing their efficacies on growth of

the prawn Macrobrachium rosenbergii post larvae. Int. J. Curr. Sci. 7, 67-79.

12. Seenivasan, C., Bhavan, P.S., Radhakrishna, S., Muralisankar, T., Immanuel, G.,

Srinivasan, V., Manickam, N., 2013. Effect of Saccharomyces cerevisiae on

survival, growth, biochemical constituents and energy utilization in the prawn

Macrobrachium rosenbergii. Int. J. Applied Biol. Phar. Technol., 4 39-47.

13. Bhavan, P.S., Ananthi, P., Satgurunathan, T., Sowdeswari, R., Muralisankar, T.,

Ponmathi, K., 2013. Effects of Quinalphos 25 EC and Dimethoate 30 EC on

Activities of AchE, Catalase, GOT and GPT in the Freshwater Prawn

Macrobrachium rosenbergii. Indian J. Appl. Res.,3, (8), 69-72.

14. Bhavan, P.S., Kirubhanandhini, V., Muralisankar, T., Manickam, N., Srinivasan, V.,

2013. Effects of fruits wastes (apple, grape and orange) incorporations on the

growth of the freshwater prawn Macrobrachium rosenbergii. Asian J. Sci. Technol.,

4, (10), 75-81.


15. Seenivasan, C., Radhakrishna, S., Shanthi, R., Muralisankar, T., Bhavan, P.S.,

2014. Influence of Probiotics on survival, growth, biochemical changes and energy

utilization performance of Macrobrachium rosenbergii post-larvae. Proc. Zool. Soc.

DOI 10.1007/s12595-014-0097-4.

16. Radhakrishnan, S., Bhavan, P.S., Seenivasan, C., Shanthi, R., Muralisankar, T.,

2014. Replacement of fishmeal with Spirulina platensis, Chlorella vulgaris and

Azolla pinnataon non-enzymatic and enzymatic antioxidant activities of

Macrobrachium rosenbergii. J. Basic Appl Zool. doi.org/10.101016/j.jobaz.2013.12.003

17. Seenivasan, C., Radhakrishna, S., Shanthi, R., Muralisankar, T., Bhavan, P.S.,

2014. Effect ofLactobacillus sporogeneson survival, growth, biochemical

constituents and energy utilization of freshwater prawn Macrobrachium rosenbergii

post larvae. J. Basic Appl Zool. (In press). http://dx.doi.org/10.1016/

j.jobaz.2013.12.002.

18. Radhakrishnan, S., Bhavan, P.S., Seenivasan, C., Muralisankar, T., Shanthi, R.,

2014. Effedts of native medicinal herbs (Alteranthera sessilis, Eclipta alba, Cissus

quadrangularis) on growth performance, digestive enzymes and biochemical

constituents of the monsoon river prawn Macrobrachium malcolmsonii. Aquacult.

Nutr. (In press).doi; 10.1111/anu.12180 (Impact factor 1.68).

19. P. Saravana Bhavan, T.C. Anisha, V. Srinivasan, T. Muralisankar, N. Manickam,

2014. Effects of spices, Papaver somniferum, Elettaria cardamomum, Foeniculum

vulgare and Syzygium aromaticum on growth promotion in Macrobrachium

malcolmsonii early juveniles. Indian J. Sci. Technol., (under review).


http://dx.doi.org/10.101016/j.jobaz.2013.12.003

http://dx.doi.org/10.1016/

CONFERENCES/ SEMINARS/ SYMPOSIUM AND WORKSHOPS

Paper presented

1. Muralisankar, T., Rekha, J., Shanthi, R., Bhavan, P.S. “Growth performance and

nutritional indices of macrobrachium malcolmsonii post larvae fed with feed

formulated from Horse gram, Ragi, Ground nut oil cake and Soy meal”. National

Level Symposium Emerging Trends in Life Sciences held at Department of

Zoology, Rajah Serfoji Govt. College, Thanjavur.

2. Muralisankar, T., Bhavan, P.S., Seenivasan, C., Aarumugam, P. “Effects of herbal

supplementation of growth and production of the freshwater prawn

macrobracchium rosenbergii post larvae. National Level Seminar on Biodiversity

Conservation and Management of Aquatic Resources (NSBCMA). Organized

by Directorate of Research and Extension (Fisheries), TANUVAS, Fisheries

College and Research Institute Campus, Thoothukudi.

3. Muralisankar, T., Bhavan, P.S., Aarumugam, P. “Growth promotion effects of

Tulasi (Holy basil), Thoothuvalai (Agnidamani) and Keelanelli (Niruri)

incorporated feeds on the giant freshwater prawn Macrobrachium rosenbergii post

larvae”. Emerging trends in Biological Research (NCEBR’11). Organized by

Department of Zoology, University of Madras, Maraimalai Campus, Guindy,

Chennai.

4. Muralisankar, T., Bhavan, P.S., Radhakrishnan, S., Seenivasan, C. Replacement

of fishmeal with chicken waste meal and soya bean meal on growth, contents of

biochemical constituents and energy utilization of the freshwater prawn

Macrobrachium rosenbergii post larvae. National Conference on Biotechnological

Approaces in Aquaculture (Live Aqua 2012). Organized by Dept.of Zoology,

Bharathiar University, Coimbatore during 1-13 Febuary 2012.


5. Bhavan, P.S., Saranya, C., Manickam, N., Muralisankar, T., Radhakrishnan, S.

“Effects of medicinal herbs, Piper longum, Piper nigram and Zinger officinale on

nutritional indices, activities of digestive enzymes and contents of biochemical

constituents in the freshwater prawn Macrobrachium rosenbergii". Regional

Science Congress held at Kongunadu Arts and Science Collage, Coimbatore, from

December 15th

to 16th

, 2012.

6. Bhavan, P.S., Parimala, P., Muralisankar, T., Manickam, N., Radhakrishnan,

S.Potency of medicinal herbs, Solanum xanthocarpum, Solanum torvum and

Phyllanthus emblica on nutritional indices, activities of digestive enzymes and

contents of biochemical constituents in the freshwater prawn Macrobrachium

rosenbergii. National Biodiversity Congress held at Thiruvanandapuram, from

27th

to 30th

December 2012.

7. Muralisankar, T., Bhavan, P.S., Radhakrishnan, S., Manickam, N. “Replacement

of fish oil with three vegetable oils for better survival and growth of the freshwater

prawn Macrobrachium rosenbergii post larvae”. 100th

Indian Science Congress

held at Kolkatta University, Kolkattar, from January 3rd

to 7th

, 2013.

8. Muralisankar, T., Bhavan, P.S., Radhakrishnan, S., Manickam, N. “Replacement

of live Artemia nauplii with boiled egg albumin and identification of a suitable

feeding schedule for nursery maintenance of Macrobrachium rosenbergii".

National level seminar on Opportunities in Aquaculture Biotechnology held at

SRTM University, NANDED, from January 27 and 28, 2013.

9. Muralisanakr, T., Bhavan, P.S., Radhakrishnan, S., Seenivasan, C., Srinivasan, V.,

Manickam, N. “Influence of herbs (Ocimum sanctum, Phyllanthus amarus, and

Solanum trilobatum) on survival, growth, amino acids, fatty acids and proximate

composition of freshwater prawn Macrobrachium rosenbergii". National

Biological Conference 2013, held at Calicut University, Calicut, and from11th

to13th

March 2013.


10. Muralisankar, T., Bhavan, P.S., Manickam, N., Srinivasan, V. Influence of dietary

supplementation of zinc nano particles on survival, growth, activity of digestive

enzymes, biochemical constituents and enzymatic antioxidants in the freshwater

prawn Macrobrachium rosenbergii“. Evolutionary Trends in Biological and

Pharmaceutical Chemistry (NCETBP)” organized by Dept Chemistry, Holy

Cross College, Tirucharappalli in Collabration with Dept. of Animal Science,

Bharathidasan University on 3oth to 31st January 2014.

Workshops attended

1. Workshop on Scientific Validation of Traditional Herbal Medicine held during

16th

and 17th

December 2009 at Department of Botany, Bharathiar University

Coimbatore.

2. National workshop on Freshwater Fish Culture Using Bio-encapsulated Live

feeds Towards Technological Empowerment of Fisherman and Self –Help

Groups held during 18th

t0 20th

of August 2010 organized by Department of

Zoology, Bharathiar University Coimbatore.

3. 3rd

Asian Lepidoptera Conservation Symposium and Training Program held at

Department of Zoology, Bharathiar University Coimbatore during 25th

to 29th

October, 2010.

4. National Training cum Workshop on Analytical and Technical Advancement

in AAS and GC (WATA-AG) organized by Department of Marine Science,

Bharathidasan University, Trichy during 24 and 25 January, 2012.

5. National workshop on Advanced Instruments for Characterization organized

by Department of Nano science and Technology, Bharathiar University,

Coimbatore on 27th

March 2012.

6. Workshop cum Hands on Training to Govt. College Teachers on “Basic

Molecular Techniques”, 21st to 27

th July, 2012 held at Department of Zoology,

Bharathiar University.


7. Workshop on “Image Processing and its Applications” held on 17th

September

2012 organized by the Department of Electronics and Instrumentation, Bharathiar

University, Coimbatore.

8. National workshop on Industry- Academia interaction meet on “Drugs and

Pharmaceuticals Research and Development at 12th

to13th

, April 2013 Organized

by Dept. of Biotechnology, Pondicherry University.

9. “One Day National Workshop on Recent Advances in aquatic Research” held

at 12th

December 2013 Organized by PG Research Dept. of Zoology, Kongunadu

Arts and Science College, Coimbatore.

10. “National Workshop on Researcher Competency: Mapping, Management and

Disaster Management” held on 13th

to 14th

March 2014 in Acedamic Staff

College, Pondicherry University, Pondicherry.

Awards received

University Research Fellow (URF) award from Bhrathiar University during 2013-2014.

Member in Professional Body

International Society of Zoologcal Sciences (ISZS), China.


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Biological Trace Element Research ISSN 0163-4984 Biol Trace Elem ResDOI 10.1007/s12011-014-0026-4

Dietary Supplementation of ZincNanoparticles and Its Influence on Biology,Physiology and Immune Responses ofthe Freshwater Prawn, MacrobrachiumrosenbergiiThirunavukkarasu Muralisankar,Periyakali Saravana Bhavan,Subramanian Radhakrishnan,Chandirasekar Seenivasan, et al.


1 23

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Dietary Supplementation of Zinc Nanoparticles and Its Influenceon Biology, Physiology and Immune Responses of the FreshwaterPrawn, Macrobrachium rosenbergii

Thirunavukkarasu Muralisankar & Periyakali Saravana Bhavan &

Subramanian Radhakrishnan & Chandirasekar Seenivasan &

Narasimman Manickam & Veeran Srinivasan

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

Abstract The present study was conducted to assess theinfluence of dietary zinc nanoparticles (size 50 nm) on thegrowth, biochemical constituents, enzymatic antioxidantlevels and the nonspecific immune response of the freshwaterprawn, Macrobrachium rosenbergii post larvae (PL). Theconcentrations of dietary supplement zinc nanoparticles(ZnNPs) were 0, 10, 20, 40, 60 and 80 mg kg−1 with the basaldiet, and the level of Zn in ZnNP-supplemented diets were0.71, 10.61, 20.73, 40.73, 60.61 and 80.60 mg kg−1, respec-tively. ZnNP-incorporated diets were fed toM. rosenbergii PL(initial body weight, 0.18±0.02 g) in a triplicate experimentalsetup for a period of 90 days. ZnNP supplemented feed fed PLup to 60 mg kg−1 showed significantly (P<0.05) improvedperformance in survival, growth and activities of digestiveenzymes (protease, amylase and lipase). The concentrationsof biochemical constituents (total protein, total amino acid,total carbohydrate and total lipid), total haemocyte count anddifferential haemocyte count were elevated in 10–60 mg kg−1

ZnNP supplemented feed fed PL. However, the PL fed with80 mg ZnNPs kg−1 showed negative results. Activities ofenzymatic antioxidants [superoxide dismutase (SOD) andcatalase (CAT)], metabolic enzymes [glutamate–oxaloacetatetransaminase (GOT) and glutamate–pyruvate transaminase(GPT)] and the process of lipid peroxidation (LPO) in thehepatopancreas and muscle showed no significant alterationsin 10–60 mg kg−1 ZnNP supplemented feed fed PL. Whereas,80 mg ZnNPs kg−1 supplemented feed fed PL showed signif-icant elevations in SOD, CAT, LPO, GOT and GPT.

Therefore, 80 mg ZnNPs kg−1 was found to be toxic toM. rosenbergii PL. Thus, the study suggests that up to60 mg ZnNPs kg−1 can be supplemented for regulating sur-vival, growth and immunity of M. rosenbergii.

Keywords Macrobrachium rosenbergii . ZnNPs . Growth .

Survival . Biochemical constituents . Antioxidant status .

Nonspecific immune response

Introduction

The giant freshwater prawn, Macrobrachium rosenbergii isone of the edible organisms among crustaceans. It is a mainprawn species for small- and large-scale freshwater farmingdue to its better environment tolerance, rapid growth, largersize, better meat quality, omnivorous feeding habit,established domestic and export markets in Asian countriesespecially in China, India, Bangladesh, Thailand, Malaysia,Vietnam, etc.

Zinc (Zn) is an essential micronutrient for stabilizing cel-lular membranes and component of all organs, tissues andfluids of an organism. It is involved in several metabolicpathways as cofactors in many enzyme systems and compo-nent of a large number of metalloenzymes (carbonicanhydrase, carboxypeptidase, alcohol dehydrogenase,glutamic dehydrogenase, D-superoxide dismutase etc.) [1]. Itregulates protein synthesis and acts as a regulator of energy,vitamin A and lipid metabolism. Zn-dependent physiologicalfunctions are influenced by its transport and storage. It isstored and metabolized via hepatic metallothionein proteinswhich are cytosolic proteins with high-cysteine content. Asingle metalloprotein like metallothionein-1 is capable ofbinding seven atoms of Zn [2–5]. The dietary Zn requirements

T. Muralisankar : P. S. Bhavan (*) : S. Radhakrishnan :C. Seenivasan :N. Manickam :V. SrinivasanCrustacean Biology Laboratory, Department of Zoology, School ofLife Sciences, Bharathiar University, Coimbatore 641046, TamilNadu, Indiae-mail: [email protected]

Biol Trace Elem ResDOI 10.1007/s12011-014-0026-4

Author's personal copy


as well the resultant effect of its deficiency or excess arespecies dependent. The role Zn in better survival, feed intake,growth, immune response, antimicrobial activities and stresstolerance has been reported in fishes supplemented with 40–120 mg Zn kg−1 [2–4] and crustaceans with 15–40 mg Znkg−1 [5, 6]. However, excess intake of Zn can be harmful tohuman beings and animals. The excess absorption of Znsuppresses the utilization of Cu and Fe which reduces thegrowth and reproductive performance in fishes and prawns[6, 7]. Similarly, the toxic effects of Zn-oxidized nanoparticles(ZnONPs) produce oxidative stress in the shrimp,Thamnocephalus platyurus and fish, Cyprinus carpio andDanio rerio [8–10].

The aquaculture industries can be revolutionized by usingnanotechnology with new tools to enhance the ability ofcultivable organisms to uptake drugs like hormones, vaccinesand nutrients [11]. The metal nanoparticles (NPs) such as Se,Al, Fe, FeO, and ZnO play a crucial role in aquacultureoperations [12]. The present study was focussed to understandthe influence of dietary Zn nanoparticles (ZnNPs) on survival,growth, activities of digestive enzymes (protease, amylase andlipase), contents of biochemical constituents (total protein,amino acid, carbohydrate and lipid), activities of antioxidantenzymes [superoxide dismutase (SOD) and catalase (CAT)],lipid peroxidation (LPO) activities of metabolic enzymes[glutamate–oxaloacetate transaminase (GOT) and gluta-mate–pyruvate transaminase (GPT)], total haemocyte count(THC) and differential haemocyte count (DHC) inM. rosenbergii PL.

Materials and Methods

Feed Formulation

Diets were prepared with locally available feed ingredients.Fishmeal (400 g kg−1) and soybean meal (200 g kg−1) wereused as protein sources; wheat flour (180 g kg−1) and tapiocaflour (150 g kg−1) were used as carbohydrate sources; codliver oil (20 ml kg−1) was used as lipid source; tapioca flourand egg albumin were served as binding agents; vitamin Bcomplex with vitamin C (thiamine mononitrate IP 10 mg,riboflavin IP 10 mg, pyridoxine hydrochloride IP 3 mg, vita-min B12 (as stablets 1:100) IP 15mcg, niacinamide IP 100mg,calcium pantothenate IP 50 mg, folic acid IP 1.5 mg, biotinUSP 100 mcg, ascorbic acid IP 150 mg) was also added. Zinc-free mineral mix (CuSO4·5H2O, 6 mg; CaCO3, 164 mg;NaH2PO4·2H2O, 148 mg; KH2PO4·2H2O, 337.6 mg; CaCl2,66.64 mg; MgSO4·7H2O, 80 mg; KCl, 22.40 mg;AlCl3·6H2O, 0.96 mg; MnSO4·H2O, 11.45 mg; FeSO4·7H2O,90 mg; COCl2·6H2O, 1.41 mg; KI, 1.81 mg; cellulose,69.74 mg) was also added. The graded concentrations of

dietary ZnNPs were designed according to dietary zinc (Zn)requirements in crustaceans [3].

In this study, 50-nm-sized ZnNPs (Sigma-Aldrich) weresupplemented with the basal diet at concentrations of 0, 10,20, 40, 60 and 80 mg kg−1. The content of Zn present in theseZnNP-supplemented diets was analysed [13] and found to be0.71, 10.61, 20.40, 40.73, 60.61 and 80.60 mg kg−1 respec-tively (Table 1). The diets were prepared by thoroughlymixing the ingredient powders in sterilized water. The doughwas then cooked in a closed aluminium container at 105 °C for20 min followed by cooling at room temperature. Cod liveroil, vitamins and egg albumin were added and thoroughlymixed until a stiff dough was obtained. The dough waspelletized by an indigenous hand pelletizer with mesh sizeof 0.1-mm diameter (Pigeon manufactures, Kolkata, India)and was cut into 3.0±0.97-mm-sized pieces. The pellets weredried at room temperature (27 °C) until constant weight wasreached. The prepared feed was stored individually in airtightplastic containers at −20 °C until used for the feeding trials.The formulated feed contains the proximate composition inthe following ratio with the energy value of 14.75 kJ g−1:protein, 41.78 %; carbohydrate, 29.37 %; lipid, 7.69 %; ash,11.46 %; moisture, 8.56 % [13].

Feeding Trial

M. rosenbergii PL (PL-5) were procured fromAqua Hatchery,Koovathur, Kanchipuram District, Tamilnadu, India. Theywere safely transported to the laboratory in plastic bags half-filled with hatchery water and well oxygenated. They wereacclimatized to ambient laboratory conditions for 3 weeks in alarge cement tank (1,000 L) with groundwater (pH, 7.10±0.20; total dissolved solids, 0.94±0.07 g L−1; dissolved oxy-gen, 7.20±0.36 mg L−1; BOD, 36.00±1.45 mg L−1; COD,125.0±7.00 mg L−1; ammonia, 0.018±0.004 mg L−1). Theprawns were provided aeration. During which they were fedwith boiled egg albumin, Artemia nauplii and control feedprepared with basal ingredients alternatively three times perday, and three fourths of aquarium water was changed daily.

Six groups of PL ranging from 1.42±0.35-cm length; 0.18±0.02-g weight was assigned in triplicate. A group was servedas control and fed with ‘0’ concentration of ZnNP-supplemented diet. The remaining five groups were fed with10, 20, 40, 60 and 80 mg kg−1 of ZnNP-supplemented dietsrespectively. Each group consisted of 40 PL in an aquariummaintained with 40 l of groundwater. The water medium wasrenewed every 24 hr by siphoning method without severedisturbance to the prawn and aerated adequately. The experi-mental prawns were fed with these feeds at 10 % of bodyweight two times per day (6.00 a.m. and 6.00 p.m.). Duringfeeding trial as well as acclimatization, the unfed feed, faecesand moult (if any) were removed on a daily basis while

Muralisankar et al.



renewing aquarium water. The experiment was performed fora period of 90 days.

Analysis of Survival, Growth and Nutritional Indices

At the end of the feeding trial, the survival rate and food indexparameters such as feed intake, length gain, weight gain,specific growth rate, feed conversion ratio and protein efficien-cy ratio were individually determined by the following equa-tions [14]:

Survival rate %ð Þ ¼ No:of live prawns=no:of prawns introduced� 100Length gain cmð Þ ¼ Final length cmð Þ − initial length cmð ÞWeight gain gð Þ ¼ Final weight gð Þ − initial weight gð ÞFeed intake g d−1

� � ¼ Feed eaten gð Þ = total number of daysSpecific growth rate %ð Þ¼logfinal weight gð Þ−loginitial weight gð Þ =

total number of days � 100Feed conversion ratio gð Þ ¼ feed intake gð Þ = weight gain gð ÞProtein efficiency ratio gð Þ ¼ weight gain gð Þ = protein intake gð Þ

Assay of Digestive Enzymes

Activities of digestive enzymes such as protease, amylase andlipase were assayed on initial and final days of feeding trial.The whole flesh except eyestalk, appendages and exoskeletonwas homogenized in ice-cold distilled water and centrifuged at9,300 g under 4 °C for 20 min. The supernatant was used as acrude enzyme source.

The activity of protease was estimated by the method ofFurne et al. [15]. One unit of enzyme activity represents the

amount of enzyme required to liberate 1 μg of tyrosine perminute under assay conditions. The reactionmixture consistedof 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. Thereaction was incubated for 1 h at 37 °C, then the reactionwas stopped by adding 0.6 ml 8 % (w/v) trichloroacetic acidsolution and kept for 1 h at 2 °C, then centrifuged at 1,800gfor 10 min, and the supernatant absorbance was measured at280 nm against blank. For the blank preparation, the enzymesource was added at the end of the incubation period, justbefore adding trichloroacetic acid. Tyrosine solution was usedas standard.

Amylase activity was assayed by starch hydrolysis methodof Bernfeld [16] in which the increase in reducing power ofbuffered starch solutions was measured. The specific activityof amylase was calculated as milligrams of maltose liberatedper gram of protein per hour (mg/g/h). The reaction mixtureconsisted of 0.125 ml of 2 % (w/v) starch solution, 0.125 ml of0.1 M citrate–phosphate buffer (pH 7.5) and 0.5 ml enzymesource. The reaction was incubated at 37 °C for 1 h, and theabsorbance was measured at 600 nm against a blank. For theblank, the enzyme source was added just after the incubationperiod. Maltose solution was used as standard.

The activity of lipase was assayed by the method of Furneet al. [15]. One unit of lipase activity was defined as theamount of free fatty acid released from triacylglycerol per unittime estimated by the amount of NaOH required to maintainpH constant and represented as mille equivalents of alkaliconsumed. A solution of 1 % polyvinyl alcohol (PVA) and5 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

Table 1 Composition of minerals in ZnNP-supplemented diets and carcass of M. rosenbergii fed with ZnNP-supplemented diets

Dietary ZnNP (mg kg−1) Cu Zn Fe Ca Mg Na K

0 Formulated diet (mg kg-1) 1.75±0.11a 0.71±0.04f 2.23±0.13a 1.84±0.10a 2.03±0.20a 1.49±0.21a 1.24±0.13a

10 1.74±0.15a 10.61±0.67e 2.11±0.21a 1.61±0.20a 2.05±0.23a 1.42±0.12a 1.32±0.17a

20 1.68±0.11a 20.73±1.59d 2.08±0.18a 1.73±0.16a 2.03±0.24a 1.48±0.10a 1.2±0.12a

40 1.76±0.14a 40.73±1.75c 2.06±0.21a 1.76±0.15a 2.1±0.11a 1.52±0.21a 1.26±0.16a

60 1.75±0.12a 60.61±1.24b 2.14±0.17a 1.56±0.16a 2.06±0.18a 1.38±0.11a 1.23±0.13a

80 1.75±0.15a 80.60±1.85a 2.03±0.12a 1.65±0.13a 2.06±0.16a 1.49±0.22a 1.29±0.18a

F value 0.077 1555.09 0.036 0.023 0.088 0.279 0.091

0 Carcass prawn (μg g−1) 37.05±1.28e 19.75±1.30f 26.25±2.01e 32.85±2.07f 78.50±3.11e 124.65±2.44f 146.28±3.01de

10 75.10±3.29d 69.05±4.01e 44.00±2.10d 117.35±5.04e 121.60±4.02d 144.65±2.84d 151.58±3.42d

20 81.15±4.30c 78.90±4.02d 50.45±2.35c 142.8±4.23d 133.60±4.65c 151.92±2.47c 162.27±3.21c

40 87.95±3.01b 138.60±4.07c 63.95±2.21b 156.1±5.32c 187.05±5.54b 174.49±3.14b 186.91±3.01b

60 95.25±3.19a 151.30±3.62b 68.4±2.35a 270.45±6.12a 196.65±5.76a 194.83±3.18a 203.78±4.61a

80 86.60±2.62bc 190.60±4.02a 45.95±1.83d 175.75±2.76b 137.60±3.54c 117.68±3.12e 156.92±3.91d

F value 136.405 892.317 149.263 894.295 277.667 321.743 195.752

Each value is mean ± SD of three individual observations; mean values within the same column sharing different alphabetical letter superscripts arestatistically significant at P<0.05 (one-way ANOVA and subsequent post hoc multiple comparisonwith 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

ZnNPs induced growth performance in prawn



NaOH. Virgin olive oil was added to an aliquot of this solutionfor obtaining 0.1 M substrate concentration. This mixture wasemulsified for 5 min. In addition, Mcllvaine buffer was pre-pared from 0.1 M citric acid and 0.2 M disodium phosphate.The reaction mixture consisted 1 ml of PVA-solution-emulsified substrate, 0.5 ml of Mcllvaine buffer (pH 8.0)and 0.5 ml enzyme source and was incubated at 37 °C for4 h. To stop the reaction and break the emulsion, 3 ml of 1:1ethanol-acetone solution was added. To the reaction mixture,phenolphthalein in ethanol 1 % (w/v) was added and titratedagainst 0.01 N NaOH. For the blanks, the same procedure wasfollowed but boiled enzyme was used.

Estimation of Biochemical Constituents and Carcass MineralContents

Analyses of total nitrogen, crude protein, moisture and ashcontents were performed according to standard AOAC proce-dures [13]. Dry matter was obtained by drying at 105 °C untila constant weight was achieved. Ash content was obtained byburning in a muffle furnace at 600 °C for 12 h. Total nitrogenand crude protein (N*6.25) were analysed after single aciddigestion using Kjeldhal apparatus (model: Kelplus DISTYL-BS, manufactured by Pelican Equipments Pvt. Ltd. Chennai,India).

Concentration of total protein was estimated by the methodof Lowry et al. [17], using ethanolic precipitated sample. Theblue colour was result of biuret reaction of protein with copperions in alkali solution and reduction of the phophomolybdic-phosphotungstic of Folin reagent by the tyrosine andtrphtophan present in the treated protein. Bovine serum albu-min (BSA) was used as a standard. This colour intensity wasmeasured at 650 nm against a blank which is devoid of proteinsample.

Concentration of total amino acid was estimated by themethod of Moore and Stein [18]. The total amino acid wasextracted with sodium tungstate and H2SO4. When aminoacids are heated with ninhydrine, they undergo deamination.The reaction of amino acid-hydratin complex with ninhydrinproduced purple colour, which was measured at 540 nm.Leusine was used as standard.

Concentration of total carbohydrate was estimated by themethod of Roe [19] using TCA extracted sample. Carbohy-drates are hydrolysed into simple sugars by diluted HCl in hotacidic medium. Glucose is dehydrated into hydroxyl-methylfurfural. This compound reacts with anthrone and produced agreen-coloured product, which was measured at 630 nm. Glu-cose was used as standard.

Total lipid was extracted with chloroform–methanol mix-ture following the method of Folch et al. [20] and estimated bythe method of Barnes and Blackstock [21]. Lipid reacts withvanillin in a medium of H2SO4 and phosphoric acid to form apink-coloured chromogen, which is proportional to the lipid

content of the sample and measured at 540 nm. Olive oil wasused as standard.

The carcass mineral contents including trace elements wereanalysed using the atomic absorption spectrophotometer(AAS) (PerkinElmer, model 2380) in air acetylene flame byadopting triple acid digestion method [13].

Total and Differential Haemocyte Counts

At the end of the experiment, 100 μl of haemolymph waswithdrawn from the ventral sinus in the first abdominal seg-ment 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 volumeup to 1 ml in the anticoagulated haemolymph. Further, avolume of 200 μl anticoagulated haemolymph was fixed withan equal volume of formalin (10 %) for 30 min. The fixedhaemolymph was used to THC and DHC determinations.

For THC, 100 μl of fixed haemolymph was diluted at 1:2ratio (v/v) with ice-cold phosphate-buffered saline (PBS,20 mM, pH 7·2). The diluted haemolymph was stained with20 μl of Rose Bengal strain (1.2 % Rose Bengal in 50 %ethanol) and incubated at room temperature for 20 min. THCwas determined by haemocytometer (Neubauer improved,Germany) under the light microscope at RP 1000× (Labomed,CXR2).

THC �106cells ml−1� � ¼ Counted cells� depth of chamber � dilution factor

Number of 1−mm square

For DHC, fixed haemolymph was stained with Rose Ben-gal solution (10 %) for 10 min and smeared on a slide. Thenumbers of differential haemocytes, such as hyalinocytes,semigranulocytes and granulocytes, were characterised ac-cording to Tsing et al. [22], and 350–400 cells from eachsmear 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 individ-ually 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 thesupernatant was used to assay the enzyme activities. Solubleprotein concentration was determined by the method of Lowryet al. [17] using BSA as the standard.

Superoxide dismutase (SOD) activity was measured usingpyrogallol (10 mM) autoxidation in Tris buffer (50 mM, pH7.0) [23]. The assay mixture contained 1.2 ml of sodiumpyrophosphate buffer, 0.1 ml of phenazine methosulphate(PMS), 0.3 ml of nitro blue tetrazolium (NBT), 0.2 ml of theenzyme prepared and water accounting to a total volume of2.8 ml. The reaction was initiated by the addition of 0.2 ml

Muralisankar et al.



NADH. The mixture was incubated at 30 °C for 90 s andarrested by the addition of 1.0 ml glacial acetic acid. Thereaction mixture was then shaken with 4.0 ml n-butanol,allowed to stand for 10 min and centrifuged. The intensity ofthe chromogen in the butanol layer was measured at 560 nmusing spectrophotometer. The specific activity of the enzymewas expressed in U/mg protein.

Catalase (CAT) activity was measured using H2O2 as thesubstrate in phosphate buffer [24]. The reaction was initiatedby the addition of 1.0 ml of phosphate buffer (0.01M, pH 7.1),0.5 ml of H2O2 (0.2 M) and 0.4 ml of distilled water succes-sively to 0.5 ml of tissue homogenate. After 60 s, the reactionwas stopped by the addition of 2.0 ml of dichromate-aceticacid reagent. Further, the tubes were kept in a boiling waterbath for 10 min and cooled at room temperature. The absor-bance of the chromophore was read at 620 nm. A systemdevoid of enzyme served as the control. The activity ofcatalase was expressed as micromoles of hydrogen peroxideconsumed per minute per milligram of protein.

Lipid peroxidation (LPO) in the tissue homogenates wasmeasured by estimating the formation of thiobarbituric-acid-reactive substances (TBARS) [25]. The tubes containing 1 mlof 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 and1.5 ml of thiobarbituric acid (1.5%). The reactionmixture wasboiled for 15 min in the boiling water bath and cooled at roomtemperature. The content was centrifuged at 100g for 20 minand the supernatant was collected. The absorbance of thesupernatant was measured at 535 nm against the reagentblank. TBARS was expressed as nanomoles ofmalondialdehyde (MDA) per milligram of protein.

Activities of Metabolic Enzymes

The metabolic enzymes such as glutamic oxaloacetate trans-aminase (GOT) and glutamic pyruvate transaminase (GPT)were analysed according to the method of Reitman andFrankel [26] using a media source kit (Medsource OzoneBiomedicals Pvt. Ltd. Haryana, India). A total of 100 mg ofmuscle and hepatopancreas tissues was homogenized in0.25 M sucrose and centrifuged at 3,300g for 20 min in ahigh-speed cooling centrifuge at 4 °C. The supernatant wasused as the enzyme source.

GOT Analysis

The substrate solution, L-aspartic acid (500 μl, pH 7.4) wasadded with 100 μl sample and incubated at 37 °C for 1 h.Then, 500 μl of 2,4-dinitrophenyl hydrazine was added andallowed to stand for 20 min at room temperature, then 3 ml offreshly prepared 4 N sodium hydroxide solution was added tothe above solution. The colour development was read at

505 nm using spectrophotometer within 15 min. Sodiumpyruvate (160 U/l) was used as a calibrator. The activity ofGOTwas 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 for20 min. With this, 500 μl of 2,4-dinitrophenyl hydrazine wasadded and allowed to stand at room temperature for 30 min,then 3 ml of freshly prepared 4 N sodium hydroxide solutionwas added. The colour development was read at 505 nm usingspectrophotometer within 15 min. Sodium pyruvate (170 U/l)was used as a calibrator. The activity of GPTwas expressed asunits 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 multiplerange test to compare the differences among treatments wheresignificant differences (P<0.05) were observed. Data wereexpressed as mean ± SD.

Results

Content of Minerals

In formulated feeds, the levels of mineral salts, such as Cu, Fe,Ca, Mg, Na and K, were almost similar, whereas, in PL fedwith these diets, the levels of these mineral salts were signif-icantly increased (P<0.05) up to the level of 60 mg ZnNPskg−1 supplementation when compared with control (Table 1).

Survival, Growth, Nutritional Indices and Activitiesof Digestive Enzymes

In this study, the survival, growth, and other nutritional indicesparameters, such as feed intake, specific growth rate andprotein efficiency ratio, were significantly elevated (P<0.05)in PL fed with 10, 20, 40, 60 mg ZnNPs kg−1 supplementeddiets (Table 2) when compared with control. Among theseconcentrations, 60mg ZnNP kg−1 showed better performance.This was further confirmed through the observed lower feedconversion ratio in 60 mg ZnNP supplemented diet fed PLgroup when compared with control as well as other concen-trations of ZnNP supplemented diet fed PL.

In the present study, activities of protease, amylase andlipase were significantly increased (P<0.05) in PL fed with10–60 mg ZnNPs kg−1 incorporated diets when comparedwith control (Table 2). A maximum increase was recorded in




60 mg ZnNPs kg−1 incorporated diet fed PL. Whereas, the PLfed with 80 mg ZnNPs kg−1 incorporated diet acts as nega-tively influenced on activities of these enzymes. The activityof protease was found to be significantly higher when com-pared with control, but significantly lower when comparedwith 60 mg ZnNPs kg−1 incorporated diet fed PL. The activityof amylase was found to be lower in 80 mg ZnNPs kg−1

incorporated diet fed PL when compared with control, butthis was not statistically significant, whereas the decrease wasstatistically significant when compared with 60 mg ZnNPskg−1 incorporated diet fed PL. In the case of lipase activity,insignificant difference was seen between control and 80 mgZnNPs kg−1 incorporated diet fed PL, whereas the activity oflipase in 80 mg ZnNPs kg−1 incorporated diet fed PL showeda significant decrease when compared with 60 mg ZnNPskg−1 (Table 2).

Concentrations of Biochemical Constituents

In the present study, the percentage of total nitrogen and crudeprotein in ZnNP supplemented diet fed PL groups are present-ed in Table 3, and their levels were found to be graduallyelevated up to 60 mg ZnNPs kg−1 supplementation whencompared with control (P<0.05), whereas, their levels werefound to be declined in 80 mg ZnNPs kg−1 supplementationdiet fed PL. Similarly, the concentrations of total protein, total

amino acids, total carbohydrate, total lipid and ash weresignificantly increased (P<0.05) in PL fed with 20, 40 and60 mg ZnNPs kg−1 supplemented diets (Table 3) when com-pared with control, whereas their levels were found to bedeclined in 80 mg ZnNPs kg−1 supplementation diet fed PL.

Haemocytes Population

THC and DHC (hyalinocytes, semigranulocytes andgranulocytes) were also found to be elevated significantly(P<0.05) in PL fed with 20–60mg ZnNPs kg−1 supplementeddiets when compared with control (Table 3). In the case of80 mg ZnNPs kg−1 supplementation diet fed PL, the THC andDHC were found to be declined (Table 3).

Activities of Antioxidant and Metabolic Enzymes

In this study, no significant changes in activities of SOD andCAT were recorded in PL fed with 10–60 mg ZnNPs kg−1

supplemented diets throughout the sampling days (Tables 4and 5). Similarly, no significant changes in activities of GOTand GPT were recorded (Tables 4 and 5). The level of LPOalso showed no significant alteration up to 60mg ZnNPs kg−1.In the case of 80 mg ZnNPs kg−1 supplemented diet fed PL,the activities of SOD, CAT, GOT and GPT, along with the

Table 2 Nutritional indices and activities of digestive enzymes in M. rosenbergii fed with ZnNP-supplemented diets

Parameters Dietary ZnNP (mg kg−1)

0 10 20 40 60 80 F value

SR (%) 75.83±1.84c 76.66±3.81c 78.33±3.81bc 83.33±2.88ab 87.5±2.50a 68.33±3.81d 12.993

Length (cm) Time 0 1.42±0.35 1.42±0.35 1.42±0.35 1.42±0.35 1.42±0.35 1.42±0.35 –

Final 3.64±0.28c 5.02±0.55b 5.20±0.33b 5.62±0.65b 6.56±0.69a 5.76±0.80b 13.735

Weight (g) Time 0 0.18±0.02 0.18±0.02 0.18±0.02 0.18±0.02 0.18±0.02 0.18±0.02 –

Final 0.58±0.13c 1.06±0.43bc 1.14±0.27bc 1.40±0.51b 2.28±0.74a 1.60±0.60b 7.190

LG (cm) 3.29±0.32e 4.67±0.27d 4.85±0.24cd 5.27±0.31bc 6.21±0.29a 5.41±0.30b 33.958

WG (g) 0.4±0.01f 0.88±0.02e 0.96±0.02d 1.22±0.03c 2.10±0.08a 1.42±0.04b 570.59

FR (g d−1) 0.39±0.01d 0.45±0.02c 0.47±0.01c 0.50±0.01b 0.66±0.02a 0.49±0.02b 69.641

SGR (%) 0.55±0.03d 0.84±0.07c 0.87±0.08bc 0.97±0.06b 1.21±0.07a 0.94±0.06bc 30.011

FCR (g) 2.69±0.35a 1.15±0.13b 1.10±0.21b 0.92±0.09bc 0.70±0.03d 0.78±0.07d 50.945

PER (%) 1.22±0.06c 3.14±0.15b 3.2±0.18b 3.58±0.32b 4.88±0.38a 4.58±0.27a 80.504

Digestive enzymes(U/mg protein)

Protease Time 0 0.27±0.06 0.27±0.06 0.27±0.06 0.27±0.06 0.27±0.06 0.27±0.06 –

Final 1.00±0.12c 1.37±0.16b 1.44±0.15b 1.59±0.19b 2.07±0.21a 1.34±0.20b 12.674

Amylase Time 0 0.16±0.04 0.16±0.04 0.16±0.04 0.16±0.04 0.16±0.04 0.16±0.04 –

Final 0.75±0.11c 0.81±0.10bc 0.96±0.12b 0.97±0.14b 1.47±0.21a 0.67±0.08c 16.693

Lipase × 102 Time 0 0.74±0.05 0.74±0.05 0.74±0.05 0.74±0.05 0.74±0.05 0.74±0.05 –

Final 0.22±0.02e 0.31±0.04d 0.37±0.02c 0.48±0.01b 0.55±0.02a 0.23±0.04e 71.680

Each value is mean ± SD of three individual observations; mean values within the same row sharing the different alphabetical letter superscripts arestatistically significant at P<0.05 (one-way ANOVA and subsequent post hoc multiple comparisonwith 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; PER, protein efficiency ratio

Muralisankar et al.



level of LPO, showed significant elevations (P<0.05) whencompared with control and other diets (Tables 4 and 5).

Discussion

Zinc has a generally stable association with macromolecules,like protein, nucleic acid, carbohydrate and lipid and enzymes,to carry out diverse biological functions. Zinc acts as anelectron acceptor, which contributes to its catalytic activityin metalloenzymes, such as DNA and RNA polymerases,alcohol dehydrogenase, carbonic anhydrase, and alkalinephosphatase. Zn plays an important role in the functions oftranscription factor, antioxidant defense system and DNArepair. Dietary deficiencies of zinc can contribute to single-and double-strand DNA breaks and oxidative modifications toDNA that increase risk for cancer development [27]. Zndeficiency increases the levels of lipid peroxidation in mito-chondrial and microsomal membranes and the osmotic fragil-ity of erythrocyte membranes, while the presence of Zn

prevents lipid peroxidation and thus plays an important rolein protecting the cells from oxidative stress [28]. According toHedemann et al. [29], Zn promotes the processes of tissuerepair in the small intestine and stimulates the synthesis ofdigestive enzymes, resulting in a better digestion and absorp-tion of nutrients and potentially improving growth perfor-mance in pig.

The results of mineral contents clearly indicated thatthe supplementation of ZnNPs promotes mineral absorp-tion based on its level of supplementation. Increased ironand phosphorus utilization have been reported in Haliotisdiscus hannai and Oncorhynchus mykiss due to Zn sup-plementation [3, 30]. The supplementation of Cu, Zn andK in the diet of Gadus morhua showed maximum utiliza-tion of Cu, Zn, K, Mg and Ca [5]. In the present study,80 mg ZnNPs kg−1 supplemented diet fed PL showedpoor utilization of minerals (Cu, Fe, Ca, Mg, Na and K)when compared with 40–60 mg ZnNPs kg−1. This clearlyindicates the fact that mineral utilization by prawn wasbased on ZnNP concentration. A study by Rani et al. [8]indicated that Zn supplemented feed fed Carassius

Table 3 Concentrations of biochemical constituents and population of haemocyte (THC and DHC) in M. rosenbergii fed with ZnNP-supplementeddiets

Parameters Dietary ZnNP (mg kg−1)

0 10 20 40 60 80 F value

Total Nitrogen (%) Time 0 4.10±0.18 4.10±0.18 4.10±0.18 4.10±0.18 4.10±0.18 4.10±0.18 –

Final 8.17±0.36e 8.49±0.32de 8.82±0.28cd 9.45±0.19b 10.89±0.14a 9.05±0.22bc 39.121

Crude protein (%) Time 0 25.66±1.16 25.66±1.16 25.66±1.16 25.66±1.16 25.66±1.16 25.66±1.16 –

Final 51.10±2.29e 53.08±2.02de 55.12±1.75cd 59.09±1.24b 68.10±0.87a 56.58±1.42bc 38.988

Protein(mg/g wet wt.)

Time 0 46.64±2.14 46.64±2.14 46.64±2.14 46.64±2.14 46.64±2.14 46.64±2.14 –

Final 148.79±2.41e 169.6±3.76d 185.01±4.62c 199.0±3.10b 256.0±4.49a 174.02±4.43d 183.28

Amino acid(mg/g wet wt.)

Time 0 25.33±2.30 25.33±2.30 25.33±2.30 25.33±2.30 25.33±2.30 25.33±2.30 –

Final 90.00±3.46e 103.40±3.07d 116.66±3.05c 125.40±4.13b 155.40±2.94a 107.33±3.05d 102.39

Carbohydrate(mg/g wet wt.)

Time 0 18.07±1.07 18.07±1.07 18.07±1.07 18.07±1.07 18.07±1.07 18.07±1.07 –

Final 32.07±1.88e 40.61±2.34d 44.36±1.82c 50.11±1.42b 58.56±2.99a 41.31±2.26d 51.600

Lipid(mg/g wet wt.)

Time 0 10.68±0.74 10.68±0.74 10.68±0.74 10.68±0.74 10.68±0.74 10.68±0.74 –

Final 18.03±2.69c 21.70±2.18bc 25.98±2.94ab 27.09±1.26b 29.23±1.42a 23.84±2.85ab 7.673

Ash (%) Time 0 10.40±0.69 10.40±0.69 10.40±0.69 10.40±0.69 10.40±0.69 10.40±0.69 –

Final 15.50±1.37b 17.00±1.04a 17.43±0.87a 17.44±0.92a 17.93±2.10a 15.73±1.85b 1.432

Moisture (%) Time 0 78.33±1.52 78.33±1.52 78.33±1.52 78.33±1.52 78.33±1.52 78.33±1.52 –

Final 75.00±4.00a 72.00±3.60a 70.23±2.35a 68.00±4.35a 63.33±2.51a 74.33±2.08a 3.415

THC (×106 cells ml−1 ) 4.49±0.13d 4.84±0.21cd 5.75±0.11bc 6.42±0.15b 8.61±0.32a 4.69±0.15d 18.760

DHC(×106 cells ml−1 )

Hyalinocytes 2.10±0.11d 2.30±0.13d 2.71±0.12d 3.11±0.17b 4.10±0.16a 2.26±0.10d 74.862

Semigranulocytes 1.40±0.10c 1.50±0.12c 1.75±0.11b 1.82±0.13b 2.50±0.17a 1.35±0.12c 33.251

Granulocytes 0.08±0.08c 1.01±0.11c 1.15±0.13b 1.23±0.12b 1.54±0.11a 0.94±0.10c 21.577

Each value is mean ± SD of three individual observations; mean values within the same row sharing the different alphabetical letter superscripts arestatistically significant atP<0.05 (one-wayANOVA and subsequent post hocmultiple comparison with DMRT); a–e, order of performance (a > b > c > d> e); some of the mean sharing two superscripts means that it falls into two ranks/columns

THC total haemocyte count, DHC differential haemocyte count




auratus showed significant elevation in Fe and Cu con-tents only up to 60 mg Zn kg−1.

In the present study, the increased survival, weight gain,feed intake, specific growth rate and protein efficiency ratioindicate that ZnNPs have the ability to promote the survival,feed intake and growth of M. rosenbergii PL. The lower feedconversion ratio recorded in 60 mg ZnNPs kg−1 indicates theenhanced quality of the feed. The poor growth performance incontrol diet fed PL suggests deficiency of Zn. Zn deficiencyleads to cause slow growth and increased mortality in fish[31]. Poor survival, feed intake and growth recorded in 80 mgZnNPs kg−1 supplemented feed fed M. rosenbergii PL mightbe due to its toxicity associated with excess amount of ZnNPsin the diet. Concentration-dependent positive and negativeeffects of Zn-supplemented diets have been reported in fishes,Oreochromis niloticus, G. morhua, C. auratus and Cirrhinusmrigala, the shrimpPenaeus monodon and the Chinese mittencrab Eriochei sinensis [4–8, 32, 33].

The increased activity of digestive enzymes recorded indi-cates that supplemented ZnNPs have influence on digestiveenzyme secretion in M. rosenbergii PL. In support of thisobservation, increased activities of protease, amylase andlipase have been reported in Zn supplemented feed fed tilapia,O. niloticus and Oreochromis aureus [34]. It has also been

reported in rats and weaning pigs that dietary Zn has elevatedthe activity of maltase, pepsin, amylase and protease [29, 35].It is important to mention here that the digestive enzyme incrustacean plays an essential role in nutritional physiology andregulates the growth and moult cycle directly or indirectly[36].

The elevation recorded in biochemical constituents, such asprotein, amino acid, carbohydrate, lipid and ash, suggests thatdietary ZnNPs have influenced the absorption of nutrients andincrease the synthesis of these basic biochemical constituentsin M. rosenbergii PL. Concentration-based increase and de-crease in protein, lipid, fatty acid and ash contents have beenreported in Zn supplemented diet fed fishes G. morhua andO. niloticus [4, 5], the crab E. sinensis [33] and the gastropodH. discus hannai [3]. It is worthy tomention here that Zn playsa vital role in lipid, protein, carbohydrate and nucleic acidmetabolism [37].

The health status of an organism is established by itsimmunological responses. In aquatic organisms, the immunestatus can be determined by studying the haematologicalparameters like blood cell counts. Few researchers have prov-en the immune stimulating ability of minerals in aquaticorganisms [38, 39]. In the present study, the elevated THCand DHC (hyalinocytes, semigranulocytes and granulocytes)

Table 4 Activities of antioxidant and metabolic enzymes in the muscle of M. rosenbergii fed with ZnNP-supplemented diets

Enzymes and LPO Dietary ZnNP (mg/100 g)

0 1 2 4 6 8 F value

Initial SOD (μmol/min/mg protein) 4.81±1.01 4.81±1.01 4.81±1.01 4.81±1.01 4.81±1.01 4.81±1.01 –

CAT (U/mg protein) 11.20±1.01 11.20±1.01 11.20±1.01 11.20±1.01 11.20±1.01 11.20±1.01 –

GOT (U/l) 6.32±0.67 6.32±0.67 6.32±0.67 6.32±0.67 6.32±0.67 6.32±0.67 –

GPT (U/l) 7.95±1.01 7.95±1.01 7.95±1.01 7.95±1.01 7.95±1.01 7.95±1.01 –

LPO (nmol MDA/mg protein) 0.13±0.010 0.13±0.01 0.13±0.01 0.13±0.01 0.13±0.01 0.13±0.01 –

30 days SOD (μmol/min/mg protein) 8.28±1.17b 8.29±1.43b 8.29±1.52b 8.31±1.06b 8.33±1.44b 12.72±1.48a 5.275

CAT (U/mg protein) 20.90±1.23b 20.92±1.32b 21.36±2.01b 21.36±2.00b 21.45±1.87b 26.37±2.31a 4.037

GOT (U/l) 8.53±1.11b 8.53±0.52b 8.53±0.60b 8.54±0.79b 8.50±1.08b 12.19±1.42a 7.032

GPT (U/l) 9.21±1.08b 9.25±1.28b 9.26±1.33b 9.25±1.17b 9.26±1.16b 12.23±0.79a 3.339

LPO (nmol MDA/mg protein) 0.63±0.02b 0.63±0.01b 0.64±0.01b 0.65±0.02b 0.67±0.02b 1.68±0.01a 989.11

60 Days SOD (μmol/min/mg protein) 8.27±1.24b 8.33±1.19b 8.40±1.23b 8.44±1.16b 8.52±1.06b 14.55±1.45a 12.602


GOT (U/l) 8.53±1.12b 8.52±0.49b 8.51±0.48b 8.53±0.74b 8.53±1.10b 14.51±2.28a 12.322

GPT (U/l) 9.22±0.83b 9.26±1.27b 9.26±1.08b 9.25±1.32b 9.26±1.26b 13.44±2.61a 3.867




GOT (U/l) 8.54±1.13b 8.54±0.56b 8.54±0.61b 8.52±0.75b 8.53±1.07b 15.66±3.88a 8.112

GPT (U/l) 9.23±1.13b 9.25±1.30b 9.26±1.39b 9.26±1.26b 9.27±1.33b 16.62±2.93a 9.596


Each value is mean ± SD of three individual observations; mean values within the same row sharing the different alphabetical letter superscripts arestatistically significant at P<0.05 (one-way ANOVA and subsequent post hoc multiple comparison with DMRT); a–b, order of performance (a > b)

Muralisankar et al.



of the supplemented animals with 10–60 mg ZnNPs kg−1

suggest that it has influenced the production of haemocytesin M. rosenbergii PL. It has been reported that Zn promotedthe haemocytes production in the shrimp P. monodon, and, atthe same time, its excess reduced THC production [7]. Thedecreased THC and DHC observed in control and 10 mg kg−1

ZnNP supplemented diet fed PL were probably associatedwith Zn deficiency, whereas overdose can be recorded in thesupplemented prawns with 80mg ZnNPs kg−1. Therefore, it issuggested that either deficits or excess of ZnNPs affects theproduction of haemocytes in M. rosenbergii PL which is inturn reflected on its immune performance.

The antioxidant enzymes, SOD and CAT are responsiblefor scavenging superoxide radicals and are involved in pro-tective mechanisms within tissue injury following oxidativeprocess and phagocytosis. In this study, 80 mg ZnNP kg−1

supplementation has produced some toxic effects onM. rosenbergii PL, and, therefore, activities of SOD andCAT were increased, which leads to lower performance ingrowth, nutritional indices, activities of digestive enzymes,and haemocyte count of M. rosenbergii PL. The elevationrecorded in the activities of GOT and GPT confirms that80 mg ZnNPs kg−1 is toxic to M. rosenbergii PL. The eleva-tion of LPO level recorded in M. rosenbergii PL fed with

80 mg ZnNPs kg−1 supplemented diet further confirms itstoxicity. In the present study, 10–60 mg ZnNPs kg−1 was notfound to be toxic to M. rosenbergii PL, since no significantalterations in the activities of SOD, CAT, GOTandGPT, alongwith the level of LPO, were recorded. Thus, these concentra-tions of ZnNPs (10–60 mg kg−1) can be taken as a safe dietarylevel. Zn-concentration-dependent elevation and decline inliver SOD level in C. auratus has been reported [8]. It hasalso been reported that higher concentrations of ZnONPsproduced cellular oxidative stress in the brine shrimp Artemiasalina and in the zebrafish D. rerio [10, 40].

The primary success of aquaculture is determined by thesurvival, growth and health of the cultivable species. In thepresent study, effective host defence of the test prawnsprevailed due to the elevation of haemocytes, and good healthdue to unaltered SOD, CAT, GOT, GPT and LPO leads togood digestive physiology by increasing the activity of diges-tive enzymes, better survival and growth with enhanced basicbiochemical constituents (protein, amino acid, carbohydrate,lipid and minerals) in M. rosenbergii PL fed with ZnNP (10–60 mg kg−1) supplemented diets.

Acknowledgments Bharathiar University, Coimbatore, Tamil Nadu,India, is gratefully acknowledged for the financial support provided in

Table 5 Activities of antioxidant and metabolic enzymes in the hepatopancreas of M. rosenbergii fed with ZnNP-supplemented diets

Enzymes and LPO Dietary ZnNP (mg/100 g)

0 1 2 4 6 8 F value

Initial SOD (μmol/min/mg protein) 5.81±1.01 5.81±1.01 5.81±1.01 5.81±1.01 5.81±1.01 5.81±1.01 -

CAT (U/mg protein) 14.20±1.01 14.20±1.01 14.20±1.01 14.20±1.01 14.20±1.01 14.20±1.01 -

GOT (U/l) 8.32±0.67 8.32±0.67 8.32±0.67 8.32±0.67 8.32±0.67 8.32±0.67 -

GPT (U/l) 9.15±1.01 9.15±1.01 9.15±1.01 9.15±1.01 9.15±1.01 9.15±1.01 -

LPO (nmol MDA/mg protein) 0.14±0.010 0.13±0.010 0.13±0.010 0.13±0.010 0.13±0.010 0.13±0.01 -

30 days SOD (μmol/min/mg protein) 14.49±1.43b 14.49±2.00b 14.72±1.34b 14.87±1.44b 15.03±1.21b 20.01±2.14a 5.327


GOT (U/l) 13.85±1.12b 13.84±1.11b 13.85±2.16b 13.85±1.39b 13.85±1.24b 15.70±2.00a 3.871

GPT (U/l) 14.32±0.86b 14.31±0.93b 14.32±0.79b 14.35±0.97b 14.35±0.70b 18.44±3.06a 2.844




GOT (U/l) 13.86±1.31b 13.85±1.46b 13.85±1.51b 13.86±1.47b 13.86±1.30b 16.25±1.55a 34.498

GPT (U/l) 14.33±0.88b 14.32±0.88b 14.32±0.78b 14.33±0.85b 14.31±0.59b 20.91±0.69a 2.496




GOT (U/l) 13.86±1.92b 13.84±1.18b 13.84±1.26b 13.85±1.29b 13.86±1.24b 20.06±2.60a 35.192

GPT (U/l) 14.32±0.93b 14.33±0.90b 14.32±0.73b 14.33±0.98b 14.34±0.69b 22.61±1.51a 9.170


Each value is mean ± SD of three individual observations; mean values within the same row sharing the different alphabetical superscripts are statisticallysignificant at P<0.05 (one-way ANOVA and subsequent post hoc multiple comparison with DMRT); a–b, order of performance (a > b)




the form of University Research Fellowship to the first author. TheUniversity Grants Commission, Government of India, New Delhi, is alsogratefully acknowledged for the acquired laboratory facility by the secondauthor through aMajor Research Project operated (2009–2012) on prawnnutrition.

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Research Journal of Biotechnology Vol. 8(2) February (2013)

Res. J. Biotech

(61)

Chicken waste meal as an alternative for fishmeal for better survival and growth of the freshwater prawn

Macrobrachium rosenbergii post larvae Muralisankar T. and Saravana Bhavan P.*

Department of Zoology, Bharathiar University, Coimbatore–641046, Tamilnadu, INDIA

*[email protected]

Abstract The present study was conducted to assess the suitability of chicken waste meal as an alternative protein source for fishmeal in sustainable nursery maintenance of Macrobrachium rosenbergii post larvae (PL). Three types of feeds with 40% optimum protein were formulated by using fishmeal (FM), chicken waste meal (CWM) and soya bean meal (SBM) independently with other basal ingredients, such as green gram and ground nut oilcake. Cod liver oil was used as lipid source. Egg albumin and tapioca flour were used as binding agents. Vitamin B-complex with vitamin-C and a pinch of salt were also mixed. M. rosenbergii PL-20 (0.9-1.14 cm length and 0.12-0.16 g weight) was subjected to feeding trail with these feeds for a period of 60 days. The overall growth performance was found to be in the following order: FM > CWM > SBM. The differences recoded11 in survival rate, weight gain and food conversion ratio; colorific energy utilized through feeding, absorption, conversion, ammonia excretion and metabolism; concentrations of total protein, amino acid, carbohydrate, lipid, ash and moisture between FM and CWM including feeds fed PL groups were not statistically significant. However, the differences obtained for these parameters between FM and SBM, and between CWM and SBM were found to be significant (P<0.05). The staining intensity of polypeptide bands resolved between 116-14 kDa as well was found to be appreciable in PL groups fed with FM and CWM when compared with SBM. Therefore, CWM was equally good and nutritious as FM and the superior quality of these two meals are attributed to their protein and lipid contents. Hence, FM can be replaced by CWM as an ideal protein source in low cost feed formulation for sustainable nursery maintenance of M. rosenbergii PL.

Keywords: Fish meal, Chicken waste meal, Soya bean

meal, M. rosenbergii, growth, Protein. Introduction Aquaculture is one of the fastest growing industries of the

world food sector. Crustaceans, such as prawns, shrimps,

lobsters and crabs have vital role in aquaculture due to their nutritious delicacy for human consumption. The freshwater

prawn, Macrobrachium rosenbergii is one of the important

cultivable prawn species. It earns valuable foreign

exchange. In culture of this species, artificial feed

constitutes a major operating cost1. Animal and plant by-

products have been important contributors to the growth

and extension of the world aquaculture food production and

have supplied the chief source of proteins, essential amino

acids, carbohydrate, fats, minerals and vitamins. Fish meal

(FM) is the major protein source, which has worldwide

demand2. According to Amaya et al,

3 FM (25-50%) is the

primary and most expensive ingredient in commercial feed

formulations. The increasing price of feed is considered

one of the most important factors limiting profitability in

aquaculture. Therefore, there is necessity for replacing FM

with low cost animal protein sources4-5.

PBM and SBM have been widely studied as two important

alternative protein sources for FM in aqua feed6-10

. From

the plant source side, soya bean meal (SBM) is the most

promising and most studied protein source11-12

. SBMs are

widely used as the most cost-effective alternative for high-

quality FM in feeds due to its high protein content

(approximately 48%), excellent amino acid profile, lower

cost and consistent availability13-16

.

Yet, sometimes, the presence of various anti-nutritional

factors, such as protease inhibitors, lectins, antigenic or

estrogenic factors, oligosaccharides, limits its use in

crustacean diets. These factors can be nullified while

preparing the feeds12

. The other plant ingredients used as

protein source are cotton seed meal, various legumes like

cowpea, horse gram and black gram etc. due to their low

price and consistent quality13,19-22

.

Animal by-products, like meat, bone meal and poultry by

product meal (PBM) have supplied the majority of the

proteins (45–65%), fats and minerals, vitamins and

indispensable amino acids23-24

. There are few reports ava-

ilable with animal by-product as meals in aqua feeds23-25

.

Material and Methods Experimental animals: M. rosenbergii post larvae were

procured from Government Prawn Hatchery at Thrissur in

Kerala state, India. They were acclimatized to the

laboratory condition for two weeks before the

commencement of experiments (water temperature,

25.3±0.41°C; dissolved oxygen, 30±0.86 mg/L; pH,

7.2±0.21; salinity, 0.533±0.02 ppt). During acclimatization,

PL were fed with boiled egg albumin, live Artemia nauplii and commercially available scampi feed alternatively thrice

a day. Water was routinely changed every day in order to

maintain a healthy environment apart from providing


mailto:[email protected]


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artificial aeration. This ensures sufficient oxygen supply for

the prawn and an environment devoid of accumulated

metabolic wastes.

Ingredients and feed preparation: The feed ingredients,

such as FM, SBM, green gram, groundnut oilcake and

tapioca flour were purchased from local merchants. These

ingredients were shadow dried and stored at room

temperature in laboratory. Vitamin B-complex with

vitamin-C capsules and cod liver oil were purchased from

local medical shop. Chicken hen eggs were procured from

local shop. Chicken intestine were collected from local

chicken meat markets at free of cost. Chicken intestine

were thoroughly cleaned in running water and boiled at

above 100°C in pressure cooker and then sun dried. Three

different feeds were formulated by using three different

major protein sources, the FM, SBM and CWM). Along

with each, green gram and groundnut oilcake were used as

common basal ingredients.

Tapioca flour was used as carbohydrate source as well as

binding agent (Table 1). The feeds were prepared by

mixing dry ingredients for 15 min. The mixture was then

steams cooked at 90-100ºC for 5 minutes and allowed to

cool at room temperature. Cod liver oil (as lipid source)

and egg albumin (as binding agent) were added. Vitamin B-

complex and a pinch of salt were also mixed. The prepared

mix was pelletized in a manual pelletizer fixed with 3 mm

diameter mesh. The pellets were dried at room temperature

under fan until they reached constant weight.

Experimental procedure: M. rosenbergii post larvae (PL-

15) ranging from 0.9-1.14 cm in length and 0.12-0.18 g in

weight was used in this experiment. The PL was divided

into three groups. Each group consisted of 30 animals and

kept in an aquarium (plastic trough) with 40L water. Each

group was fed with each feed formulated (10% of body

weight) for period of 60 days. 80% of the water medium

was renewed daily by siphoning method without severe

disturbance to the prawn. The feeding schedule was

maintained two times a day at 9.00 am and 9.00 pm. The

unfed feed, faeces and exuvia if any were removed and

separately dried at 65ºC in a hot air oven. The experiment

was conducted in triplicate.

Calculation of nutritional indices: Parameters of the

nutritional indices such as survival rate (SR), specific

growth rate (SGR) and weight gain (WG) were calculated 26

, and also the feed conversion ratio (FCR) and protein

efficiency ratio (PER) were calculated27

:

SR (%) = Final number of prawns / Initial number of

prawns × 100

WG (g) = Final weight – Initial weight

SGR (%) = log Final weight – log Initial weight / No. of total

experimental days × 100 FCR (g) = Feed consumed (g) / Live weight gain (g)

PER (%) = Weight gain (g) / Protein intake (mg) × 100

Calculation of energy utilization: Energy utilization

parameters such as feeding rate (FR), absorption (AR),

food conversion rate (FCR) and metabolic rate (MR) were

calculated. The energy content of whole prawn, feeds,

faeces and exuvia was measured using Parr 1281 Oxygen

Bomb Calorimeter. The energy budget was calculated using

the equation28

:

C = (P+E) + R + F +U

where C is the energy consumed in food; P, is the

conversion or growth; R, the material lost as heat due to

metabolism; F, the energy lost through faeces; U, the

energy lost in ammonia excretion and E, the energy lost

through exuvia.

The daily excretion of ammonia by the prawn (after

feeding) was estimated as per the phenol hypochloride

method 29

. The energy lost through ammonia excretion was

calculated using the ammonia calorific quotient, 1 mg NH3:

5.9 cal 30

. The food energy consumed was measured as the

difference between the energy content of food offered and

that of the uneaten food. The quantity of absorbed food

energy was calculated by subtracting F from C. Conversion

or growth is the sum of energy channeled to somatic

growth (P) and exuvia (E). Following the estimations of C,

F, U, and P, the metabolism (R=Respiration) was

calculated by dividing the respective amount of energy by

initial live weight of the prawn per unit time in days.

Feeding Rate = Mean Food Consumption (k.cal/day) /

Initial live weight of the prawn (g)

Absorption Rate = Mean Absorption (k.cal/day) / Initial

live weight of the prawn (g)

Conversion Rate (P) = Mean Conversion (k.cal/day) /


Excretion Rate (U) = Mean NH315

Excretion (k.cal/day) /


Metabolic rate (R) = Absorption Rate (k.cal/g/day) –

Conversion Rate (k.cal/g/day)+NH316

excretion Rate

(k.cal/g/day).

Estimations of Biochemical constituents: The prawns

were sacrificed and muscle tissues were sampled. The

samples were immediately processed for analyzing the

concentration of total protein, amino acids, carbohydrate

and lipid. The content of total protein was estimated by

following the method of Lowry et al31

using alcoholic

precipitated sample.

Amino acids were extracted using Sodium tungstate and

H2SO4. The content of total amino acid was assayed by the method of Moore and Stein

32. Total carbohydrate was

estimated by the method of Roe33

using TCA extracted

sample. Total lipid was extracted with chloroform-



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methanol mixture following the method of Barnes and

Black-Stock34

and estimated by the method of Folch et al.35

The pre weighed wet tissue samples were dried under 40oC

to measure the moisture content. The dried tissue sample

was subjected to 600oC under a Muffle Furnace to measure

the ash content.

Protein profile analysis : The muscle tissue samples were

first defrosted in phosphate buffer (137 mM NaCl, 2.7 mM

KCl, 10 mM Na2HPO4 and 2 mM KH2PO4, pH- 7.4),

homogenized under ice cooled condition and centrifuged at

1500 rpm at 4°C for 5 min. The soluble protein content in

supernatant was determined.31

SDS-PAGE was performed on vertical slab gel with 4%

stacking gels and 10% separating gels 36

. Protein marker

consisting of six different molecular weights (Medox-Bio

Pvt. Ltd., India), such as β-galactosidase (116 kDa), bovine

serum albumin (66 kDa), ovalbumin (45kDa), carbonic

anhydrase (29 kDa), soyabean trypsin inhibitor (20 kDa)

and lysozyme (14 kDa) was also run. The patterns were

compared by using information on apparent molecular

masses of bands and their intensity.

Statistical analysis: The data obtained from all the

parameters were expressed as mean ±SD. Significance of

variations between groups was determined by two-tailed

paired sample t-test. One way analysis of variance

(ANOVA), which was followed by Duncan’s Multiple

Range Test was also performed using SPSS software (11.5

version).

Results and Discussion The parameters of nutritional indices such as SR, WG,

SGR and PER in PL fed with feeds prepared from different

meals were found to be in the order of FM > CWM > SBM

(Table 2) whereas the FCR was naturally higher in SBM

followed by CWM and FM. The lowest FCR recorded in

FM including diet fed PL indicates its superior quality.

However, the value of FCR recorded indicates the fact that

the feeds formulated with FM and CWM were of equal

quality.

Thus, CWM included feed produced growth performance

closely similar to that of FM included feed in M. rosenbergii PL. Similar higher nutritional indices were

reported in the following studies: in fish fed with FM and

CWM included diets due to higher digestibility37

; in

Macrobrachium nipponense fed with 15% of FM replaced

by PBM 25

; in Pacific white shrimp 11

and sunshine bass38

fed with diets of 80 % and 100 % replacement of FM with

PBM respectively. Appreciable PER was reported in

Litopenaeus vannami fed with diet of 50% FM replaced by

PBM39

.

Animals utilize energy from oxidation of the complex

molecules (protein, carbohydrate and lipids) which have

been eaten. These complex molecules are broken down in

to simpler molecules by digestion. The dietary protein

supply is one of the major factors that influence growth and

energy budget in aquaculture. In this study, the parameters

of energy utilization, such as FR, AR, CR, NH3 excretion

rate and MR were all found to be higher in FM included

feed fed PL followed by CWM and SBM (Table 2).

Therefore, the poor energy utilization observed in PL fed

with SBM included feed might be due to presence of anti-

nutritional factors in SBM or due to the inferior feed

quality than that of FM and CWM included feeds.

Concurrently, the contents of biochemical constituents,

such as total protein, amino acid, carbohydrate, lipid and

ash were found to be higher in FM included feed fed PL

followed by CWM and SBM (Table 2). An increase in ash

content with increase in size has been reported in juveniles

of P. monodon 40

. Prawn with increase of ash represents

attaining good proportion of minerals. Most studies

suggested that growth increases with increasing protein

level41

. The protein sparing effect on growth can be

increased by optimized concentrations of carbohydrate and/

or lipid in the diet42

.The protein-sparing effect was more

obvious when the dietary protein level was reduced from

40 to 30% by increasing the dietary corn starch level from

20 to 30% in P. monodon diet43

. In the present study, the

contents of total protein, carbohydrate and lipid were

optimum as no negative effect was seen. However, the

source of protein influences the growth performance of M. rosenbergii PL.

In this study, polypeptide bands of molecular weight

between 116-14 were observed in the muscle of prawn fed

with these feeds prepared by inclusion of FM, SBM and

CWM (Fig.1).

Figure 1: SDS- PAGE protein profile of prawns fed

with different meal incorporated

There were five Coomassie blue stained protein bands

(116, 57, 43, 19 and 15kDa) calculated against the standard

markers of β-galactosidase, Bovine serum albumin, ovalbumin, carbonic anhydrase, soyabean trypsin inhibitor

and lysozyme which represent 116, 66, 45, 29, 20, 14 kDa

respectively. Generally, all the five bands resolved were



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very visible. However, the staining intensity of these bands

was found to be very obviously higher in FM incorporated

feed fed PL when compared with SBM. However, the

differences in the staining intensity of these polypeptides

between FM and CWM incorporated feed fed PL were

invisible (Fig. 1). Therefore, the protein quality of both FM

and CWM is almost equal.

Conclusion CWM is a readily available protein source at very low price

or even at free of cost. The results of the present study

suggest that CWM is an excellent alternative protein source

to FM as far as the survival and growth of M. rosenbergii PL is concerned. Therefore, sustainable aquaculture of

Macrobrachium can be promoted by utilizing the low cost

CWM. Furthermore, utilization of CWM in aquaculture

would also aid to avoid the solid waste management

problem arising due to its dumping. Therefore, wealth out

of waste can be generated.

Acknowledgement The University Grants Commission, Government of India,

New Delhi is gratefully acknowledged for the financial

support provided.

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Table 1

Composition of ingredients in different experimental diets (g/ 100g)

Nutrients sources Ingredients FM SBM CWM

Protein

FM 40 0 0

SBM 0 40 0

CWM 0 0 40

Protein and carbohydrate Green gram 24 24 24

Ground nut oil cake 24 24 24

Binding agents Tapioca flour 6 6 6

Egg albumin 3 3 3

Lipid Cod liver oil 2 2 2

Vitamin B- complex with vitamin C Vitamin mix* 1 1 1

Total 100 100 100

Concentration of biochemical constituents (dry weight basis)

Protein (%) 41.76 40.00 41.83

Carbohydrate (%) 25.19 25.35 25.19

Lipid (%) 7.09 5.0 9.09

Moisture (%) 9.0 9.6 7.6

Ash (%) 12.3 11.6 12

*BECOSULES CAPSULES (Manufactured by PFIZER)

Each capsule contains, Total mg = 438.5mg; Thiamine Mononitrate IP, 10mg; Riboflavin IP, 10mg; Pyridoxine

Hydrochloride IP, 3mg; Vitamin B12 (as tablets 1:100) IP, 15mcg; Niacinamide IP, 100mg; Calcium

pantothenate IP, 50mg; Folic acid IP, 1.5mg; Biotin USP, 100mcg; Ascorbic acid IP, 150mg.

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Table 2

The morphometric data, nutritional indices, bioenergetics performance and biochemical composition of M.

rosenbergii17

fed with diets prepared from different meal sources

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Litopenaeus vannamei, and digestibility of the tested ingredients

and diets, Aquaculture, 272, 466–476 (2007).

(Received 23rd

April 2012, revised 26th September 2012

accepted 15th December 2012)

Parameters FM SBM CWM

t- value/ significant

F-

value

FM

vs

SBM

FM

vs

CWM

SBM

vs

CWM

Morphometry Length (cm)* 4.30±0.68

a 4.00±0.15

a 4.22±0.57

a 0.84/0.44 0.24/0.81 -0.77/0.48 <1

Weight (g)* 0.88±0.21

a 0.73±0.08

a 0.82±0.14

a 1.20/0.28 0.70/0.52 -1.14/0.31 <1

Survival rate

(%)

80.0±3.2a 70.33±2.4

c 78.0±3.5

b 36.09/0.01 57.73/0.00 -

10.50/0.09

22.43

Nutritional

indices

Weight gain (g) 0.72±0.02a 0.57±0.07

b 0.66±0.04

ab 5.19/0.35 5.19/0.35 5.19/0.35 48.82

Feed conversion

ratio (g)

1.70±0.08a 1.84±0.14

a 1.73±0.12

a 4.04/0.05 9.52/0.01 -1.29/0.32 1.21

NS

Specific growth

rate (%)

1.16±0.13a 1.11±0.12

a 1.15±0.10

a 8.66/0.01 0.57/0.62 -3.46/0.07 <1

Protein

efficiency ratio

(%)

0.52±0.04a 0.49±0.02

a 0.51±0.05

a 1.73/0.225 5.19/0.07 1.73/0.22 <1

Bioenergetics

(k.cal/g/day)

Feeding rate 0.31±0.010a 0.22±0.017

c 0.27±0.015

b 22.26/0.08 29.44/0.01 -9.39/0.03 25.80

Absorption rate 0.26±0.011a 0.20±0.015

b 0.23±0.020

ab 25.98/0.01 5.77/0.02 -

10.39/0.00

10.86

Conversion rate 0.15±0.017a 0.10±0.019

b 0.13±0.011

a 40.70/0.00 4.90/0.03 6.49/0.02 9.60

NH3 excretion

rate

0.026±0.003a 0.023±0.005

a 0.025±0.004

a 2.59/0.12 1.73/0.22 -3.46/0.00 <1

Metabolic rate 0.13±0.004a 0.11±0.015

a 0.12±0.006

a 10.39/0.00 12.12/0.00 -9.52/0.01 <1

Biochemical

constituents

(mg/g wet

wt.)

Protein 163.03±5.4a 133.55±3.43

c 151.46±2.4

b 6.74/0.21 6.33/0.24 -6.85/0.35 41.78

Amino acid 102.66±3.0a 85.33±4.16

b 91.33±5.03

b 4.91/0.03 4.25/0.05 -2.51/0.12 13.41

Carbohydrate 43.02±4.57a 42.84±3.39

a 42.96±3.53

a 0.47/0.61 0.01/0.99 -0.03/0.97 <1

Lipid 16.15±1.84a 14.01±1.45

a 14.87±2.47

a 1.29/0.32 1.98/0.18 -0.39/0.73 <1

Moisture (%) 63.66±2.08a 69.00±4.58

a 67.00±4.58

a 0.756/0.52 0.37/0.74 -0.25/0.82 2.47

NS

Ash (%) 17.33±1.52a 16.66±1.52

a 17.00±1.00

a -3.46/0.07 -0.958/0.43 1.51/0.27 <1


Journal of Ecobiotechnology 2012, 4(1): 29-34 ISSN: 2077-0464 Available Online: http://journal-ecobiotechnology.com/

Influence of combined probiotics Lactobacillus sporogenes and Bacillus subtilis on survival, growth, biochemical changes and energy utilization performance of Macrobrachium rosenbergii (De Man 1879) post larvae C. Seenivasan*, S. Radhakrishnan, T. Muralisankar and P. Saravana Bhavan Crustacean Biology Laboratory, Department of Zoology, Bharathiar University, Coimbatore-641046, Tamilnadu, India

Abstract A 90-day feeding experiment was studied to determine the influence of the bacterial combination Lactobacillus sporogenes and Bacillus subtilis (LS+BS) on survival, growth, biochemical constituents and energy utilization performance of the freshwater prawn Macrobrachium rosenbergii post larvae (PL). Experimental diets were the same, except for the variation in probiotic levels. The probiotics LS+BS (4:3) were used at 0%, 1%, 2%, 3% and 4% inclusion rates in the experimental diets. After the feeding trail, the growth parameters of the PLs, such as survival, weight gain, specific growth rate, feed conversion efficiency and protein efficiency rate were significantly (P<0.05) higher in 4% LS+BS incorporated diet as compared with control. The food conversion ratio was significantly (P<0.05) lower in 4% LS+BS incorporated diet. The biochemical composition of the total protein, amino acid, carbohydrate and lipid ash content were significantly (P<0.05) higher in 4% LS+BS incorporated diet. However, insignificant difference was recorded in moisture content between control and experimental groups. The energy utilization parameters, such as feeding rate, absorption rate, conversion rate and metabolic rate were significantly (P<0.05) higher in 4% LS+BS incorporated diet fed PL. These results revealed the benefits of the incorporation of the probiotic (LS+BS) in aqua feed for M. rosenbergii PL. Keywords: Biochemical composition, B. subtilis, energy utilization, growth, L. sporogenes

INTRODUCTION The culture of freshwater prawn offers tremendous scope to meet the awe some challenge of providing adequate levels of nutritious food to the growing human population [1]. The giant freshwater prawn (Macrobrachium rosenbergii) is a species, which plays an important role in the aquaculture and fisheries industry. Parker [2] coined the term probiotic and defined the term as “organisms and substances which contribute to intestinal microbial balance”. Probiotics can also be considered as microbes to improve the nutritive value of an animal feed [31]. A number of studies have shown that a single probiotic ingredient can improve the growth performance of the freshwater prawns and shrimps [4-14]. The present investigation was conducted to determine the effects of combined probiotics, L. sporogenes and B. subtilis (4:3) on survival, growth, biochemical constituents and energy utilization of the freshwater prawn M. rosenbergii post larvae (PL). MATERIALS AND METHODS The post larvae of freshwater prawn, M. rosenbergii (PL 15)

were purchased from a Happy Bay Annexe, Kanchipuram, Tamilnadu, India and were stocked in a cement tank (1000 L) filled with freshwater. The PL were acclimatised at ambient laboratory conditions for 15 days (up to PL 30) and starved for 24 h before the commencement of the feeding experiment. The experimental water had these physicochemical parameters: pH 7.00±0.30; total dissolved solids 0.90±0.08 g/L-1; dissolved oxygen 7.10±0.10 mg/L-1; BOD 4.10±2.60 mg/L-1; COD 12.00±10.00 mg/L-1 and ammonia 0.098±0.018 mg/L-1. Diet preparation The composition of the experimental diets is given in Table 1. The probiotics, L. sporogenes (Uni-Sankyo Ltd., Maharashtra, India) and B. subtilis (Tablets, India Ltd), one gram of lyophilized powders contains 15x107 and 10x107cfu cells respectively. The probiotics, LS+BS (4:3) were incorporated in to the test diets at five different concentrations individually 0% (control), 1%, 2%, 3% and 4% respectively. Diet formulation was done basically by ‘‘Pearson’s square-method’’ using determined values of 40% protein content (Table 1). The proportion of each ingredient was calculated precisely providing allowance for the premix. The dough was steam cooked and cooled to room temperature. After that different concentration of LS+BS (4:3) was mixed with the dough and the diets were pelletized separately with a locally made (Kolkata, India) hand pelletizer. The pellets were dried in a thermostatic oven (M/s Modern Industrial, Mumbai, India) at 400 C until it reached constant weight and stored in airtight jars at room temperature.

Received: Nov 17, 2011; Revised: Dec 22, 2011; Accepted: Jan 18, 2012.

*Corresponding Author C. Seenivasan Crustacean Biology Laboratory, Department of Zoology, Bharathiar University, Coimbatore-641046, Tamilnadu, India

Tel:+91-9488175470; Email: [email protected]


Turkish Journal of Fisheries and Aquatic Sciences 12: 331-338 (2012)

www.trjfas.org ISSN 1303-2712

DOI: 10.4194/1303-2712-v12_2_19

© Published by Central Fisheries Research Institute (CFRI) Trabzon, Turkey in cooperation with Japan International Cooperation Agency (JICA), Japan

Effects of Probiotics on Survival, Growth and Biochemical Constituents of

Freshwater Prawn Macrobrachium rosenbergii Post Larvae

Introduction

Macrobrachium rosenbergii was fast growing,

being able to grow in freshwater and low brackish

water conditions. This species has many biological

advantages for commercial culture including attaining

maturation in captivity, a relatively large size, rapid

growth rate. Other advantages include omnivorous

feeding habits (Ling, 1969).

The term “probiotic” which literally means “for

life” has since been employed to describe these

health-promoting bacteria. The World Health

Organization has defined probiotic bacteria as “live

microorganisms which when administrated in

adequate amounts confer a health benefit on the host”

(FAO/WHO, 2001). The use of probiotics in the

culture of aquatic organisms is increasing with

demand for more environment friendly aquaculture

practices (Gatesoupe, 1999). The microorganisms

used as probiotics, including Lactobacillus, Bacillius

and yeasts, have been reported in penaeids and fish

(Rengpipat et al., 2000; Hong et al., 2005; Li et al.,

2005; El-Haroun et al., 2006; Balcázar et al., 2007a,

2007b; Bagheri et al., 2008; Ghosh et al., 2008;

Capkin and Altinok, 2009; Al-Dohail et al., 2009;

Pooramini et al., 2009; Boonthai et al., 2011). The

effects of probiotics for freshwater prawn, M.

rosenbergii have been reported by Seenivasan et al.

(2012); Seenivasan et al. (2011); Saad et al. (2009);

Shinde et al. (2008); Keysami et al. (2007); Venkat et

al. (2004) and Suralikar and Sahu, (2001). The

present study was attempted to examine the effect of

combined probiotics, Lactobacillus sporogenes,

Bacillus subtilis and yeast, Saccharomyces cervisiae

on survival, growth, biochemical changes and energy

utilization performance of the freshwater prawn M.

rosenbergii PL.

Materials and Methods

The post larvae of freshwater prawn, M.

rosenbergii (PL 15) were purchased from a Happy

C. Seenivasan1,*, P. Saravana Bhavan

1, S. Radhakrishnan

1, T. Muralisankar

1

1 Bharathiar University, Department of Zoology, Crustacean Biology Laboratory, Coimbatore-641046, Tamilnadu, India.

* Corresponding Author: Tel.: +91.94881-75470; Fax: +88.176 4692554;

E-mail: [email protected] Received 06 January 2012

Accepted 26 April 2012

Abstract

The present study was attempted to examine the combined effects of probiotics, Lactobacillus sporogenes (LS), Bacillus

subtilis (BS) and yeast, Saccharomyces cerevisiae (SC) on survival, growth, biochemical changes and energy utilization

performance of the freshwater prawn M. rosenbergii post larvae. The probiotics, L. sporogenes (4), B. subtilis (3) and S.

cerevisiae (4) were taken and mixed. 1%, 2%, 3% and 4% of LS+BS+SC (4+3+4) was incorporated with basal diet. Diet

without probiotics served as control. PL-30 of M. rosenbergii was fed with LS+BS+SC (4+3+4) incorporated diet for a period

of 90 days. The growth parameters, such as survival, weight gain, specific growth rate, feed conversion efficiency and protein

efficiency rate were significantly (P<0.05) higher in 3% LS+BS+SC incorporated diet fed post larvae (PL) followed by other

experimental groups when compared with control. Whereas, the feed conversion ratio was significantly (P<0.05) lower in 3%

LS+BS+SC incorporated diet fed PL. Similarly the tissues biochemical composition such as protein, amino acid, carbohydrate

and lipid content were significantly (P<0.05) higher in 3% LS+BS+SC incorporated diet fed PL. However, insignificant

difference was recorded in moisture content between control and experimental groups. The energy utilization parameters, such

as feeding rate, absorption rate, conversion rate, NH3 excretory rate and metabolic rate were significantly (P<0.05) higher in

3% LS+BS+SC incorporated diet fed PL. Therefore, the probiotics 3% LS+BS+SC incorporated diet fed PL was produced

better growth performance.

Keywords: M. rosenbergii, L. sporogenes, B. subtilis, S. cerevisiae, growth performance, biochemical constituents, energy

utilization.


Available Online

Publications

J. Sci. Res. 4 (3), 729-740 (2012)

JOURNAL OF SCIENTIFIC RESEARCH

www.banglajol.info/index.php/JSR

Efficacy of Probiotics on Survival, Growth, Biochemical Changes and

Energy Utilization Performance of Macrobrachium rosenbergii (De Man 1879) Post-larvae

C. Seenivasan*

doi:

, S. Radhakrishnan, T. Muralisankar, and P. S. Bhavan

Crustacean Biology Laboratory, Department of Zoology, Bharathiar University, Coimbatore-641046, Tamilnadu, India

Received 26 March 2012, accepted in final revised form 18 August 2012

Abstract

The efficacy of combined probiotics, Lactobacillus sporogenes and Saccharomyces cerevisiae (LS+SC) on survival, growth, biochemical changes and energy utilization of Macrobrachium rosenbergii post larvae (PL) was examined. Each probiotic organism was individually tested at four different concentrations (1-4%) separately. The best concentration in each probiotic species was combined and tested for its suitability in aquaculture usage. The basal diet was incorporated with probiotics, LS+SC (4:4) at five different concentrations 0% (control), 1%, 2%, 3% and 4%. These diets were fed to M. rosenbergii PL for a period of 90 days. After the feeding trail, 2% LS+SC incorporated diet had significantly (P<0.05) higher survival, WG, SGR, FCE and PER compared with other experimental groups than the control. Whereas, the FCR was significantly (P<0.05) lower in 2% LS+SC incorporated diet fed PL. Similarly the proximate composition of the protein, amino acid, carbohydrate, lipid and ash content were significantly (P<0.05) higher in 2% LS+SC incorporated diet fed PL than the control. The energy utilization parameters were significantly (P<0.05) higher in 2% LS+SC incorporated diet fed PL than the control. This study indicated that combined probiotics, LS+SC incorporated diets were beneficial for M. rosenbergii in terms of increasing growth and enhancing energy utilization performances. Keywords: M. rosenbergii; Growth; Biochemical composition; Energy utilization; L. sporogenes; S. cerevisiae. © 2012 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved.

http://dx.doi.org/10.3329/jsr.v4i3.10193 J. Sci. Res. 4 (3), 729-740 (2012) 1. Introduction The giant river prawn, Macrobrachium rosenbergii is one among crustaceans, native to Southeast Asia, South Pacific countries, northern Oceania, and Western Pacific islands. M. rosenbergii has become the main freshwater prawn species for small-scale and large-scale farming because of its fast growth, large size, better meat quality, omnivorous feeding habit and established domestic and export markets in Asia [1]. A probiotic is

*Corresponding author: [email protected], [email protected]


http://dx.doi.org/10.3329/jsr.v4i3.10193�

mailto:[email protected]�

Int J Pharm Bio Sci 2012 Oct; 3(4): (B) 914 - 926

This article can be downloaded from www.ijpbs.net

B - 914

Research Article Bio Chemistry

International Journal of Pharma and Bio Sciences ISSN

0975-6299

GROWTH PROMOTING POTENTIAL OF GARLIC, GINGER,

TURMERIC AND FENUGREEK ON THE FRESHWATER

PRAWN MACROBRACHIUM ROSENBERGII

R. POONGODI, P. SARAVANA BHAVAN*, T. MURALISANKAR

AND S. RADHAKRISHNAN

Department of Zoology, Bharathiar University, Coimbatore- 641046, Tamilnadu, India

ABSTRACT

A 60 day feeding trial was conducted to determine the growth performance of Macrobrachium rosenbergii post larvae (PL) on Allium sativum (Garlic), Zingiber officinale (Ginger), Curcuma longa (Turmeric) and Trigonella foenum-graecum (Fenugreek) incorporated diets. Survival rate (SR) ranged from 69.0-80.0% in experimental diets fed PL groups were recorded against 66.0% in control. The elevation in SR was in the order of garlic > ginger > turmeric > fenugreek. Similar trend in elevation of FR was recorded, which in turn increases the WG in experimental PL over control. The same patterns of improvements were observed in SGR, FCE, PCE, ME and GE. Concurrently, the values obtained for CF and FCR were in decreasing pattern (garlic < ginger < turmeric < fenugreek). This indicated the fact that the experimental feeds were superior in quality than that of control. Concentrations of total protein, amino acid, RNA, DNA, RNA/DNA and carbohydrate as well were found to be elevated in experimental diets fed PL when compared with control. Instead, herbal dependent utilization of lipid was noted in PL, and thus, the elevation of total lipid was recorded in just the reverse order, garlic < ginger < turmeric < fenugreek. All the data were significant at P<0.05. The herbal growth promoter induced transcription rate, which in turn increased RNA and total amino acid, which ultimately enhances proteins synthesis in M. rosenbergii PL. This ability was higher with garlic included feed followed by ginger, turmeric and fenugreek. Therefore, these herbs could be taken as supplementary materials in aqua feed formulation. KEY WORDS: M. rosenbergii, Garlic, Ginger, Turmeric, Fenugreek, Survival, Growth, Protein, RNA/DNA.

*Corresponding author

P. SARAVANA BHAVAN Department of Zoology, Bharathiar University, Coimbatore- 641046, Tamilnadu, India

[email protected], [email protected]


FULL LENGTH ARTICLE

Bacillus subtilis on survival, growth,

biochemical constituents and energy utilization of

the freshwater prawn Macrobrachium rosenbergii postlarvae

C. Seenivasan *, S. Radhakrishnan, T. Muralisankar, P. Saravana Bhavan

Crustacean Biology Laboratory, Department of Zoology, Bharathiar University, Coimbatore 641 046, Tamilnadu, India

Available online 29 January 2013

KEYWORDS

Biochemical constituents;

B. subtilis;

Energy utilization;

Growth;

M. rosenbergii

Abstract This experiment was carried out to evaluate the effects of the probiotic Bacillus subtilis

on survival, growth, biochemical constituents and energy utilization in Macrobrachium rosenbergii

PL. Experimental diets were the same in all, except for the variation in probiotic levels. The probi-

otic B. subtilis was used at 0% (diet 1), 1% (diet 2), 2.0% (diet 3), 3.0% (diet 4) and 4.0% (diet 5)

inclusion in the experimental diets. These diets were fed to M. rosenbergii PL for a period of

90 days. The growth parameters, such as survival, weight gain, specific growth rate, feed conversion

efficiency and protein efficiency rate were significantly (P < 0.05) higher in 3% B. subtilis incorpo-

rated diet fed PL followed by other experimental groups than the control. Similarly the biochemical

constituents of the protein, amino acid, carbohydrate, lipid and ash content were significantly

(P < 0.05) higher in 3% B. subtilis incorporated diet fed PL. The energy utilization parameters,

such as feeding rate, absorption rate, conversion rate and metabolic rate were significantly

(P < 0.05) higher in 3% B. subtilis incorporated diet fed PL. From the results of this experiment,

we conclude that B. subtilis can be used as a probiotic in M. rosenbergii culture, to enhance the sur-

vival, growth and improved energy utilization performance.ª 2013 National Institute of Oceanography and Fisheries. Production and hosting by Elsevier B.V. All

rights reserved.

Introduction

The animal protein for human consumption is rapidly in-creased, which is mainly depend upon the terrestrial and aqua-

tic farm animals. Macrobrachium rosenbergii, known as thegiant river prawn, Malaysian prawn, freshwater scampi (espe-cially in India) is a species of freshwater shrimp native to the

Indo-Pacific and northern Australia. The feeding habits ofM. rosenbergii were studied by Ling (1969) who reported thatit is omnivorous. Aquaculture, however, is an increasinglyimportant option in animal protein production. This activity

* Corresponding author. Tel.: +919488175470.E-mail address: [email protected] (C. Seenivasan).

q Peer review under responsibility of National Institute of Oceanog-

raphy and Fisheries

Production and hosting by Elsevier

Egyptian Journal of Aquatic Research (2012) 38, 195–203

National Institute of Oceanography and Fisheries

Egyptian Journal of Aquatic Research

http://ees.elsevier.com/ejarwww.sciencedirect.com

1687-4285 ª 2013 National Institute of Oceanography and Fisheries. Production and hosting by Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.ejar.2012.12.009





http://www.sciencedirect.com/science/journal/16874285


Received: 22nd Jan-2013 Revised: 31st Jan-2013 Accepted: 2nd Feb-2013 Research article

GROWTH OF MACROBRACHIUM ROSENBERGII FED WITH MANGO SEED KERNEL, BANANA PEEL AND PAPAYA PEEL INCORPORATED FEEDS

P. Aarumugam, P. Saravana Bhavan*, T. Muralisankar, N. Manickam,

V. Srinevasan and S. Radhakrishnan

Department of Zoology, Bharathiar University, Coimbatore – 641046, Tamilnadu, India *Corresponding Author E-mail: [email protected], [email protected]

ABSTRACT: The growth promoting potential of fruits wastes, mango seed kernel, banana peel and papaya peel on the freshwater prawn, Macrobrachium rosenbergii post larvae (PL) was evaluated. Basal diet equated to 35% protein was prepared by using soybean meal, groundnut oilcake, horse gram and wheat flour. Each fruit waste powder was separately incorporated with basal diet at a proportion of 10%. Sunflower oil was used as lipid source. Egg albumin and tapioca flour were used as binding agents. Vitamin B-complex with Vitamin-C was also mixed. Feed without any fruit waste was served as control. M. rosenbergii PL (length: 1.2-1.4 cm; weight: 0.09-0.13 g) was fed with these feeds for a period of 90 days. Significant improvements in the nutritional indices (survival rate, weight gain, biomass index, specific growth rate and condition factor), concentrations of biochemical constituents (total protein, carbohydrate and lipid), levels of non-enzymatic antioxidants (vitamin-C and E), content of minerals (Na+ and K+), activities of digestive enzymes (protease, amylase and lipase), and profiles of essential amino acids and fatty acids were recorded in fruits wastes incorporated feeds fed PL when compared with control (P< 0.003 – 0.878). The overall results indicated the fact that mango seed kernel incorporated feed was produced the best performance, followed by better performance of banana peel and good performance of papaya peel. These fruits wastes incorporated feeds enhance digestive enzymes activities and act as appetizer, which in turn enhances food utilization and ultimately yielded better survival and growth of M. rosenbergii PL. Therefore, these fruits wastes have considerable potentials in sustainable development of Macrobrachium culture. Key words: Prawn, Mango seed kernel, Banana peel, Papaya peel, Growth, Protein, Vitamins, Minerals, Digestive enzymes, Amino acids, Fatty acids. INTRODUCTION This study was conducted to evaluate the growth promoting potential of fruits wastes, such as mango seed kernel, banana peel and papaya peel on Macrobrachium rosenbergii post larvae (PL) and to recommend the feasibility of incorporation of these fruits wastes in aqua feed formulation. The crustaceans are one of the most numerous and diverse group of non-insect arthropods, comprising approximately 52,000 described species (Martin and Davis, 2001) including prawns, crabs, crayfish, lobster etc. The commercial culture of various shrimp and prawn species for human consumption is one of the fastest growing areas of aquaculture (Rosenberry, 2005). In this connection a large amount of production has taken place in China and there has been rapid expansion in India and Bangladesh (New, 2005). In India, the export of shrimps and prawns earn valuable foreign exchange, they comprise more than 60% of exports among the crustaceans and occupy a unique position in both capture and culture fisheries. After arising serious environmental threats due to shrimp farming and disease outbreak, the freshwater prawn farming got momentums. Among the freshwater prawns, M. rosenbergii and Macrobrachium malcolmsonii are the two members of the family, Palaemonidae potentially important for aquaculture. These prawn species are fetched for high price and have demand in both domestic and export markets, as it provides nutritious delicacy for human being (Radheshyam, 2009). Among these two, M. rosenbergii has become the main species for small-scale as well as large-scale commercial farming because of its fast growth, large abdominal size (compared to M. malcolmsonii), better meat quality, omnivorous feeding habit, and established domestic and export markets. It is transported to many parts of the world including South America and China (New, 2005).

International Journal of Applied Biology and Pharmaceutical Technology Page: 12 Available online at www.ijabpt.com


P. Saravana Bhavan et al./ Elixir Bio Tech. 58 (2013) 14824-14828

14824

Introduction

The giant river prawn, Macrobrachium rosenbergii is dominating in India, and it is one of the major contributors of national economy. It is good source for protein, essential amino acids and polyunsaturated fatty acids, it is very low in fat, and therefore, it can be used as a healthy choice of food for human (Bhavan et al. 2010). The successful prawn culture depends on quality live feeds and nutrient rich commercial feeds. Both are very costly and non-affordable to small farmers. Therefore, low cost artificial feeds are much needed for promoting inland aquaculture of freshwater prawns. Nowadays, herbals are widely used in veterinary and human medicine. The herbal active principles in the diets induce the secretion of the digestive enzymes. It would stimulate the appetite and ultimately increased the food consumption and efficiencies (Bhavan et al. 2011; Shanthi et al. 2012; Radhakrishnan et al. 2013). Herbal active principles induce transcription, which lead to high protein synthesis (Citrasu et al. 2002, 2009). Several plant products found to have potent antiviral activity against fish and shrimp viruses. For example, shrimp fed with ethanolic extract of Clinacanthus nutans showed 95% survival rate against yellow head virus (YHV), but

the control showed only 25% survival (Direkbusarakom et al. 1996). Herbs such as, Solanum trilobatum, Psoralea corylifolia and Clematis paniculata enriched Artemia fed Penaeus

monodon had reduced vibrio species (Citrasu et al. 2002, 2009). Many plants have antibacterial and antifungal properties. For instance, the extract of Ocimum basilicum has controlled infection of Aspergillus flavus and Fusarium oxyspoum (Adiguzel et al., 2005). Herbal compounds have the ability to inhibit the generation of oxygen anions and scavenge free radicals, hence reduces the effects due to stress. Piper longum (long pepper) is a component of medicines which is reported as good remedy for treating gonorrhea, menstrual pain, tuberculosis, sleeping problems, respiratory tract infections, chronic gut-related pain and arthritic conditions. It has antioxidant, antimicrobial, antipyretic, antiallergic, immunomodulatory and antitumour activities (Joy et al. 2010; Khushbu, 2011). Other beneficial effects of P. longum include analgesic and diuretic effects, relaxation of muscles tension and alleviation of anxiety. Alkaloids, piperine, piper longumine and pipernonaline are the active principles of P. longum. Piper nigrum (black pepper) has many medicinal uses, including the ability to control worm infestations, and to provide

Tele:

E-mail addresses: [email protected]

© 2013 Elixir All rights reserved

Effects of Piper longum, Piper nigram and Zingiber officinale on survival, growth, activities of digestive enzymes and contents of total protein, vitamins

and minerals in the freshwater prawn Macrobrachium rosenbergii P. Saravana Bhavan*, C. Saranya, N. Manickam, T. Muralisankar, S.Radhakrishnan and V. Srinivasan

Department of Zoology, Bharathiar University, Coimbatore – 641046, Tamilnadu, India.

ABST RA CT

In this study, Piper longum (long pepper), Piper nigram (black pepper) and Zingiber

officinale (dried zinger) were incorporated with artificial feeds formulated to understand their effects on survival, growth, activities of digestive enzymes and biochemical constituents in Macrobrachium rosenbergii post larvae (PL). These herbs were independently incorporated with basal ingredients at a concentration of 5% each, and fed to M. rosenbergii PL (1.56±0.08 cm; 0.074± 0.02 g) for a period of 60 days under laboratory condition with triplicates. A significant improvement (P<0.05) in survival and growth performance (weight gain, specific growth rate, food conversion ratio), elevation in activities of digestive enzymes (protease, amylase and lipase), increased concentrations of total protein, non-enzymatic antioxidants (vitamins C and E), and mineral salts (sodium and potassium) were recorded in P. longum incorporated feed fed PL followed by P. nigram and Z. officinale when compared with control. Polypeptide bands of molecular weight between 116-14 kDa were resolved in the muscle of PL fed with control as well as herbs incorporated feeds. Generally, there were eleven Coomassie blue stained protein bands (116, 58, 51, 45, 38, 33, 25, 18, 17, 16 and 14kDa) were calculated in herbal incorporated test samples. The general comparison between control and experimental groups revealed that there was more number of bands resolved in herbals incorporated feeds fed PL. Therefore, it is suggested that these medicinal herbs have their own influence in protein synthesis. In this study, the overall results indicated the fact that these herbs have acted as appetizers and hence, enhanced the secretion of digestive enzymes, which facilitated efficient digestion, absorption of nutrients and favoured for general health, which in turn ultimately produced better survival and growth of M. rosenbergii PL. Therefore, these herbs can be taken as feed additives in sustainable development of freshwater prawn culture. © 2013 Elixir All rights reserved.

ARTICLE INF O

Article h istory: Received: 6 April 2013; Received in revised form: 7 May 2013; Accepted: 9 May 2013;

Keywords

Prawn, Long pepper, Black pepper, Dried zinger, Growth, Survival.

Elixir Bio Tech. 58 (2013) 14824-14828

Bio Technology

Available online at www.elixirjournal.org


169

This article can be downloaded from http://www.ijlbpr.com/currentissue.php

Int. J. LifeSc. Bt & Pharm. Res. 2013 P Saravana Bhavan et al., 2013

EFFECTS OF MYRISTICA FRAGRANS,GLYCYRRHIZA GLABRA AND QUERCUS

INFECTORIA ON GROWTH PROMOTION INTHE PRAWN MACROBRACHIUM ROSENBERGII

P Saravana Bhavan1*, N Nandhini Devi1, T Muralisankar1,N Manickam1, S Radhakrishnan1 and V Srinivasan1

Research Paper

Objective: To understand whether the medicinal herbs, Myristica fragrans (nutmeg) Glycyrrhizaglabra (liquorice) and Quercus infectoria (gallnut) have effects on growth promotion in the postlarvae (PL) of the prawn, Macrobrachium rosenbergii. Methods: M. fragrans (seed powder), G.glabra (stem powder) and Q. infectoria (fruit powder) were incorporated with basal diet at threedifferent concentrations (1%, 3%, and 5%) individually and fed to the PL of M. rosenbergii(1.56±0.18 cm; 0.074± 0.02 g) for a period of 60 days under triplicate experimental set-up.Results: Significant elevations (P < 0.05) in weight gain, survival rate, activities of digestiveenzymes (protease, amylase and lipase), concentrations of total protein, non-enzymaticantioxidants (vitamins C and E) and minerals (Na+ and K+) were recorded in M. fragransincorporated feed fed PL followed by G. glabra and Q. infectoria when compared with control.Nine polypeptide bands of molecular weight between 116-14 kDa were resolved in the muscletissue of PL. These bands were stained more intensely in experimental PL when compared withcontrol. Conclusion: These herbs have the ability to induce secretion of protease, amylase andlipase in M. rosenbergii PL, which lead to increased food consumption and absorption of nutrients,and resulted in elevation of total protein, vitamins and minerals.

Keywords: Prawn, Nutmeg, Liquorice, Gallnut, Growth, Digestive enzymes, Vitamins, Protein

*Corresponding Author: P Saravana Bhavan, [email protected]

ISSN 2250-3137 www.ijlbpr.comVol. 2, No. 3, July 2013

© 2013 IJLBPR. All Rights Reserved

Int. J. LifeSc. Bt & Pharm. Res. 2013

1 Department of Zoology, Bharathiar University, Coimbatore - 641046, Tamil Nadu, India.

INTRODUCTIONAquaculture is the production of aquatic plants

and animals under the controlled or semi

controlled conditions (Stickney et al., 2000). It is

one of the fastest growing animal food sectors

and provides over 13% of the animal protein for

the human consumption (WHO, 2003; FAO,

2010). In addition to contributing to global food

production, aquaculture is a major economic

activity and an important source of foreign

exchange for several developing countries.

Currently aquaculture supplies about 50% of the

global demand for fish and fishery products with

about 90% of the products coming from the Asian

region (FAO, 2009). The production of carp and


INT J CURR SCI 2013, 7: E 67-79

RESEARCH ARTICLE ISSN 2250-1770

Comparison of nutritional quality of sunflower oil and cod liver oil enriched with

Artemia nauplii for assessing their efficacies on growth of the prawn

Macrobrachium rosenbergii post larvae

Saravana Bhavan P*, N. Kavithamani, S. Radhakrishnan, T. Muralisankar, V. Srinivasan and N. Manickam

Department of Zoology, Bharathiar University, Coimbatore-641 046, Tamil Nadu, India

*Corresponding author: [email protected]; [email protected]; Phone: +91-422-2428495; Fax: +91-422-2425706

Abstract

The quality of chiefly available sunflower oil and costlier cod liver oil enriched Artemia nauplii was compared to

understand whether the plant oil (sunflower oil) with low levels of highly unsaturated fatty acids (HUFA) or fish oil (cod

liver oil) with high levels of HUFA is good for improving the food value of Artemia nauplii for attaining better survival,

growth and biochemical constituents of M. rosenbergii early post larvae (PL-30). Sunflower oil enriched Artemia showed

significant (P<0.01) elevations in total protein, amino acid, carbohydrate and lipid than that of cod liver oil enriched Artemia

and un-enriched Artemia. After a feeding trial of 45 days, the weight gain, survival rate, concentrations of total protein,

amino acid, carbohydrate and lipid were found to be significantly (P<0.01) higher in PL fed with sunflower oil enriched

Artemia followed by cod liver oil enriched Artemia and un-enriched Artemia. Similarly, the comparison of fatty acid profiles

revealed that the sunflower oil enriched Artemia fed PL showed more quantity (P<0.1) of stearic and lignoceric acids

among saturated fatty acids (SFA), oleic acid in monounsaturated fatty acids (MUFA) category, and linoleic acid in the

category of poly unsaturated fatty acids (PUFA). However, the HUFA such as linolenic acid, eicosapentaenoic acid (EPA)

and docosahexaneoic acid (DHA) were found to be significantly declined (P<0.1). The overall results suggest that sunflower

oil enriched Artemia have more growth promotion potential in M. rosenbergii early PL than that of cod liver oil enriched

Artemia. This may be because of presence of high amount of linoleic acid (n-6), palmitoleic acid (n-7) and oleic acid (n-9) in

sunflower oil. Therefore, it is suggested that diet with rich HUFA is not required to M. rosenbergii early PL since it is

transferred and acclimatized to freshwater. Hence, the costlier cod liver oil can be replaced with low priced sunflower oil in

sustainable nursery maintenance of M. rosenbergii is concerned.

Keywords: Macrobrachium rosenbergii, growth, survival, sunflower oil, cod liver oil

Received: 20th February; Revised: 26thMarch; Accepted: 24thApril; © IJCS New Liberty Group 2013

Introduction

Prawns are well known for their nutritious delicacy

to human consumption with high protein and low fat

contents. The success in the hatchery production of prawn

larvae and early post larvae (PL) for stocking in grow out

production system is largely dependent on the availability

of suitable live food organisms for feeding. Live feeds are

filter feeders and have been used successfully as biological

carriers for transferring essential nutrients to predator

larvae (Citarasu et al., 1998). Live feeds such as rotifers,

Brachionus sp. and nauplii of the brine shrimp, Artemia

and Moina micrura satisfy both the dimensional and

nutritional requirements to M. rosenbergii (Sorgeloos

et al., 1991; Sorgeloos and Leger, 1992; Alam et al., 1993,


Received: 29th April-2013 Revised: 06th May-2013 Accepted: 17th May-2013 Research article

EFFECT OF SACCHAROMYCES CEREVISIAE ON SURVIVAL, GROWTH, BIOCHEMICAL CONSTITUENTS AND ENERGY UTILIZATION IN THE PRAWN MACROBRACHIUM

ROSENBERGII

C. Seenivasan1, P. Saravana Bhavan*1, S. Radhakrishnan1, T. Muralisankar1, G. Immanuel2, V. Srinivasan1 and N. Manickam1

1Department of Zoology, Bharathiar University, Coimbatore - 641046, Tamilnadu, India

2Centre for Marine Science and Technology, Manonmanium Sundaranar University, Rajakkamangalam - 629502, Tamilnadu, India

*Corresponding Author E-mail: [email protected], [email protected] ABSTRACT: A probiotic yeast, Saccharomyces cerevisiae was incorporated in basal diet prepared with fish meal, soybean meal, groundnut oil cake, corn flour, tapioca flour, egg albumin, cod liver oil and vitamin B-complex, at four different concentrations (10g, 20g, 30g and 40g kg-1) and fed to Macrobrachium rosenbergii post larvae (PL) for 90 days. The effect of this probiotic incorporation on the growth and survival performances, concentration of protein, amino acid, carbohydrate and lipid, and energy utilization was found to be significantly (P<0.05) higher at 40g kg-1 followed by 30g, 20g and 10g kg-1. 40g kg-1 S. cerevisiae incorporation was found to be established the highest rate of colony formation, 234x10-4 cfu (colony formation units). Actually, presence of Bacillus spp., Bacillus cereus, Pseudomonas spp., Escherichia coli, Streptococcus spp., and Klebsiella pneumoniae were deducted in water medium and the PL gut of control group. There is a general belief that Pseudomonas spp. and K. pneumonia are pathogenic to prawns. The establishment of S. cerevisiae colony in the gut of experimental PL has eradicated these pathogenic bacteria. Therefore, it is suggested that establishment of S. cerevisiae colony has led to better growth, survival and biochemical constituents in M. rosenbergii PL. Thus, S. cerevisiae could be taken as a useful probiotic in M. rosenbergii culture. Key words: M. rosenbergii, S. cerevisiae, Survival, Growth, Protein INTRODUCTION The freshwater prawn, Macrobrachium rosenbergii is a commercially important species in India and south Asian countries. M. rosenbergii farming has the potential to revolutionize the rural employment and economy (Radheyshyam 2009). Probiotic organisms are live microorganisms that are thought to be beneficial to the host organism. According to FAO/WHO (2001), probiotics are "live microorganisms which when administered in adequate amounts confer a health benefit on the host". Probiotics (antonym of antibiotics) include certain bacteria, yeasts and fungi involved in growth of useful microbes, and suppress the growth of harmful ones (Kollath 1953; Parker 1974). Probiotics have been used to improve the growth performance and disease resistance in prawns (Intriago et al. 1998; Scholz et al. 1999; Suralikar & Sahu 2001; Venkat et al. 2004; Keysami et al. 2007; Deeseenthum et al. 2007; Shinde et al. 2008; Saad et al. 2009). Probiotic yeast, Sacharomyces cerevisiae, Sacharomyces exiguous and Phaffia rhodozyma have been reported to increase β-glucan level in penaeid prawns infected with Vibriosis (Scholz et al. 1999). Recently, it is reported that in M. rosenbergii PL probiotics, BinifitTM, Lactobacillus sporogenes, Bacillus subtilis have enhanced the growth, survival, biochemical constituents and feed utilization performance (Seenivasan et al. 2011, 2012a, b, 2013). However, there is paucity of literature in relation to S. cerevisiae usage in this economically important species of prawn. Therefore, we aimed to study the effect of S. cerevisiae on growth, survival, biochemical constituents and energy utilization of M. rosenbergii PL. MATERIALS AND METHODS The post larvae of the freshwater prawn, M. rosenbergii (PL-15; 0.86±0.11cm; 0.014±0.005g) were purchased from a Happy Bay Annexe, Kanchipuram, Tamilnadu, India and acclimatised to ambient laboratory conditions for 15 days (up to PL 30) with ground water (pH, 7.10; total dissolved solids, 0.100 g l-1 dissolved oxygen, 7.0 mg l-1 BOD, 32.00 mg/L-1; COD, 120.00 mg l-1 and ammonia, 0.024 mg l-1 in a cement tank (1000 L).

International Journal of Applied Biology and Pharmaceutical Technology Page: 39 Available online at www.ijabpt.com


INDIAN JOURNAL OF APPLIED RESEARCH X 69

Volume : 3 | Issue : 8 | Aug 2013 | ISSN - 2249-555XReseaRch PaPeR Zoology

Effects of Quinalphos 25 EC and Dimethoate 30 EC on Activities of AchE, Catalase, GOT and GPT in the

Freshwater Prawn Macrobrachium rosenbergii

Saravana Bhavan P Ananthi P Satgurunathan TDepartment of Zoology, Bharathiar

University, Coimbatore-641046, India.

Department of Zoology, Bharathiar University, Coimbatore-641046,

India.


India.

Sowdeswari R Muralisankar T Ponmathi KDepartment of Zoology, Bharathiar

University, Coimbatore-641046, India.


India.


India.

Keywords Prawn, quinalphos, dimethoate, AChE, catalase, GOT, GPT

ABSTRACT The commercially important freshwater prawn, Macrobrachium rosenbergii post larvae (PL), 1.5±0.2 cm and 0.1±0.03 g were subjected to static renewal type acute toxicity bioassays against two orgnophophate insec-

ticides, quinalphos (Ekalux EC 25) and dimethoate 30% EC (TAFGOR). The 96 hr LC50 values were determined to be 0.774 µgl-1for quinalphos and 0.856 mgl-1for dimethoate. The PL were exposed to lethal (the 96 hr LC50) and sub-lethal (1/2nd and 1/4th of the 96 hr LC50) concentrations of these insecticides (quinalphos: 0.774, 0.384 and 0.193 µgl-1; dimethoate: 0.856, 0.428, 0.214 mgl-1) for a duration of 4, 8 and 12 days to study their acute and chronic impacts on whole body activities of enzymes, the neurotransmitter, acetyl cholinesterase (AChE), the antioxidant, catalase and metabolic enzymes, glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT). The activity levels of AChE and catalase were found to be significantly (P<0.05) decreased in test prawns when compared with control, whereas, there were sig-nificant elevations in GOT and GPT levels (P<0.05). Among GOT and GPT, the impact was more on GOT than GPT. The dosage and time dependent manner of inhibition or elevation in activities of these enzymes were recorded. Among these two insecticides, quinalphos showed more impacts than dimethoate on this non-target organism.

IntroductionPesticides have tremendous benefits to man by increasing crop protection and thereby increasing food production, and controlling the vectors of man and animal diseases. They are transported over long distances by global circulation, and through run-off, find their way into aquatic systems. At the same time the pollution of freshwater ecosystem by chemical pesticides has become one of the most critical environmental problems (Northoff and William, 2004). This causes extensive damage to the activities of the living resources of food-web due to their toxicity, persistency with half-lives of decades and tendency to accumulate in the organisms (Joseph and Raj, 2010; Joseph et al., 2010; Joseph and Raj, 2011).

Quinalphos (C12H15N2O3PS), O,O-diethyl O-quinoxalin-2-yl phosphorothioate, an ester of OP is used as insecticide and acaricide having a quick knock down effect through contact and stomach poisoning (David and Kumaraswami, 1988; Has-sal, 1990). It is frequently used in many countries and repre-sents a source of toxicity to humans and vertebrate animals (Kegley et al., 2010). Dimethoate (C5H12NO3PS2), O,O-di-methyl S-[2-(methylamino)-2-oxoethyl] dithiophosphate is a broad-spectrum OP insecticide and acaricide exhibit both contact and systemic activity (David and Kumaraswami, 1988; Hassal, 1990). Its degradation by esterases and amidases are very low in insects as compared with those of mammals (Rose and Hodgson, 2004).

The effects of pesticides, such as endosulfan, carbaryl, dichlo-rvos, lindane, chlorpyrifos, monocrotophos, carbofuran and methomyl have been studied on acute toxicity (Bhavan et al., 1997a, b, 2008; Key and Fulton, 2006; Satapornvanit et al., 2009) and biochemistry (Bhavan and Geraldine, 1997, 2000a, b; 2001, 2002, 2004, 2007, 2009; Geraldine et al., 1999; Bhavan et al., 2011) of freshwater prawns. However, no data is available pertaining to quinalphos and dimethoate toxic-

ity induced changes on the activities of the neurotransmit-ter, acetyl cholinesterase (AChE), the enzymatic antioxidant, catalase and metabolic enzymes, glutamate oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) in Macrobrachium. Therefore, it was necessitated to generate information on these pesticides in Macrobrachium rosenbergii to establish the potential of predictive biomark-ers for use in water pollution monitoring.

Materials and MethodsThe post larvae of freshwater prawn, M. rosenbergii were purchased from Happy Bay Aqua Nova Hatchery, Mugaiyur, Marakanam Taluk, and Kancheepuram District, Tamilnadu, In-dia. They were safely brought to the laboratory in polythene bags filled with hatchery water and well-oxygenated. They were stocked in large cement tank (6’ x 4’ x 3’) and acclimatized for 2 weeks in ground water. During which they were fed with boiled egg albumin, Artemia nauplii and commercially avail-able scampi crumble feed alternatively thrice a day. The excreta, unfed feed and exuvia if any were removed daily, three fourth of the water was renewed daily and adequately aerated.

Quinalphos (Ekalux EC 25) and dimethoate 30% EC (TAF-GOR) were purchased from local agro service centre. Ten concentrations of each quinalphos (0.250-1.375 µgl-1) and dimethoate (0.060-1.350 mgl-1) were prepared by mixing in distilled water afresh on every day. Post larvae (PL) of M. rosenbergii (1.5 ±0.2 cm and 0.1 ± 0.03g) were transferred to plastic aquaria of 10 l capacity (each with 10 PL) with ground water for 2 days. Out of eleven groups one PL group was served as control and others were exposed to ten dif-ferent known concentrations (quinalphos: 0.250-1.375 µgl-1; dimethoate: 0.060-1.350 mgl-1) of each insecticide for 96 hours to assess their LC50 value as per the guidelines pre-scribed by ASTM (1980). The experiment was conducted


RESEARCH ARTICLE

Influence of Probiotics on Survival, Growth, Biochemical Changesand Energy Utilization Performance of Macrobrachiumrosenbergii Post-larvae

C. Seenivasan • S. Radhakrishnan • R. Shanthi •

T. Muralisankar • P. Saravana Bhavan

Received: 19 August 2013 / Revised: 2 January 2014 / Accepted: 28 January 2014

� Zoological Society, Kolkata, India 2014

Abstract A 90 days feeding trial was carried out to

determine the effects of the combined probiotics Bacillus

subtilis (BS) and yeast Saccharomyces cerevisiae (SC) on

the survival, growth, biochemical changes and energy uti-

lization performance of the freshwater prawn Macrob-

rachium rosenbergii post larvae (PL). The probiotics, BS

(3) and SC (4) were taken and mixed, as 1, 2, 3 and 4 % of

BS ? SC (3:4) was incorporated with basal diet. Diet

without probiotics served as control. PL-30 of M. rosen-

bergii was fed with BS ? SC (3:4) incorporated diet for a

period of 90 days in triplicates. After the feeding experi-

ment, the growth parameters such as survival, weight gain,

specific growth rate, feed conversion efficiency and protein

efficiency rate were significantly (P \ 0.05) higher in 3 %

BS ? SC incorporated diet fed PL group. Similarly, the

biochemical composition of the total protein, amino acid,

carbohydrate, lipid and ash content were significantly

(P \ 0.05) higher in 3 % BS ? SC incorporated diet fed

PL group. The energy utilization parameters, such as

feeding rate, absorption rate, conversion rate, NH3 excre-

tory rate and metabolic rate were significantly (P \ 0.05)

higher in 3 % BS ? SC incorporated diet fed PL group.

However, BS ? SC incorporated diet fed PLs produced

better growth performance.

Keywords Bacillus subtilis � Biochemical composition �Energy utilization � Growth � Saccharomyces cerevisiae

Introduction

Macrobrachium rosenbergii popularly known as ‘scampi’ is

an economically important and farmed crustacean species.

The farming of giant river prawn, M. rosenbergii has gained

increased interest in recent years, due to its high economic

value and an annual production of over 30,000 t has been

achieved through the use of culture practices (FAO 2010).

Probiotics (antonym of antibiotics) include certain bacteria,

yeasts and fungi involved in growth of useful microbes, and

suppress the growth of harmful ones (Kollath 1953; Parker

1974). Probiotics in aquaculture have been shown to have

several modes of action, competitive exclusion of pathogenic

bacteria through the production of inhibitory compounds,

improvement of water quality, enhancement of immune

response of host species and enhancement of nutrition of host

species through the production of supplemental digestive

enzymes (Gomez-Gill et al. 2000; Verschuere et al. 2000).

The use of probiotics in the aquatic organisms is increasing

along with the demand for more environment-friendly aqua-

culture practices (Gatesoupe 1999).

Bacillus subtilis (BS) is a Gram positive, spore forming

bacterium, used commercially in probiotic products because its

beneficial effects have been documented and the pathogenic

potential is generally described as low or absent (Kim and

Austin 2006). Moreover, as a spore former, Bacillus prepara-

tions are resistant and have a long lasting shelf life and thus can

be stored in a state of dehydration (Boer and Diderichsen 1991).

BS can promote survival and growth (Wang et al. 2008;

Seenivasan et al. 2012d), by stimulating the immune system (Ai

et al. 2011) and by controlling pathogenic bacteria (Vaseeharan

and Ramasamy 2003). Saccharomyces cerevisiae, SC (baker’s

yeast) is unicellular fungus and it is very cost effective if used in

diets of fish (Tewary and Patra 2011). Oral administration or

injection of the SC has been shown to increase growth

C. Seenivasan (&) � S. Radhakrishnan � R. Shanthi �T. Muralisankar � P. S. Bhavan

Crustacean Biology Laboratory, Department of Zoology,

Bharathiar University, Coimbatore 641046, Tamil Nadu, India

e-mail: [email protected]

123

Proc Zool Soc

DOI 10.1007/s12595-014-0097-4

TH

EZ

O

OLOGICAL SOC

IET

YKO LK ATA


Replacement of fishmeal with Spirulinaplatensis, Chlorella vulgaris and Azolla pinnataon non-enzymatic and enzymatic antioxidant

activities of Macrobrachium rosenbergii

S. Radhakrishnan *, P. Saravana Bhavan, C. Seenivasan, R. Shanthi,

T. Muralisankar

Crustacean Biology Laboratory, Department of Zoology, Bharathiar University, Coimbatore, Tamil Nadu, India

Received 15 July 2013; revised 2 October 2013; accepted 18 December 2013

KEYWORDS

Fishmeal replacement;

Algae;

Azolla;

Antioxidants;

Macrobrachium rosenbergii

Abstract The fishmeal replaced with Spirulina platensis, Chlorella vulgaris and Azolla pinnata and

the formulated diet fed to Macrobrachium rosenbergii postlarvae to assess the enhancement ability

of non-enzymatic antioxidants (vitamin C and E), enzymatic antioxidants (superoxide dismutase

(SOD) and catalase (CAT)) and lipid peroxidation (LPx) were analysed. In the present study, the

S. platensis, C. vulgaris and A. pinnata inclusion diet fed groups had significant (P < 0.05)

improvement in the levels of vitamins C and E in the hepatopancreas and muscle tissue. Among

all the diets, the replacement materials in 50% incorporated feed fed groups showed better

performance when compared with the control group in non-enzymatic antioxidant activity. The

50% fishmeal replacement (best performance) diet fed groups taken for enzymatic antioxidant

study, in SOD, CAT and LPx showed no significant increases when compared with the control

group. Hence, the present results revealed that the formulated feed enhanced the vitamins C and

E, the result of decreased level of enzymatic antioxidants (SOD, CAT and LPx) revealed that these

feeds are non-toxic and do not produce any stress to postlarvae. These ingredients can be used as an

alternative protein source for sustainable Macrobrachium culture.ª 2014 The Egyptian German Society for Zoology. Production and hosting by Elsevier B.V. All rights

reserved.

Introduction

Microalgae are valuable in aquaculture and have been used aslive feeds for larval or juvenile crustaceans and finfish, for all

bivalve mollusks including oysters, scallops, clams andmussels, and as feed for zooplankton used in aquaculture.Microalgae are rich sources of vitamins, essential amino acids,

minerals, essential fatty acids, and carotenoid pigments for

* Corresponding author. Tel.: +91 9842289425.

E-mail addresses: [email protected], sjsrikrishna@

gmail.com (S. Radhakrishnan).

Peer review under responsibility of The Egyptian German Society for

Zoology.


The Journal of Basic & Applied Zoology (2014) xxx, xxx–xxx

The Egyptian German Society for Zoology

The Journal of Basic & Applied Zoology

www.egsz.orgwww.sciencedirect.com

2090-9896 ª 2014 The Egyptian German Society for Zoology. Production and hosting by Elsevier B.V. All rights reserved.


Please cite this article in press as: Radhakrishnan, S. et al., Replacement of fishmeal with Spirulina platensis, Chlorella vulgaris and Azolla pinnata on

non-enzymatic and enzymatic antioxidant activities of Macrobrachium rosenbergii. (2014), http://dx.doi.org/10.1016/j.jobaz.2013.12.003Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.









Effect of Lactobacillus sporogenes on survival,

growth, biochemical constituents and energy utilization

of freshwater prawn Macrobrachium rosenbergii post larvae

C. Seenivasan *, S. Radhakrishnan, R. Shanthi, T. Muralisankar,

P. Saravana Bhavan

Crustacean Biology Laboratory, Department of Zoology, Bharathiar University, Coimbatore 641046, Tamil Nadu, India

Received 29 June 2013; revised 19 December 2013; accepted 22 December 2013

KEYWORDS

Prawn;

L. sporogenes;

Growth;

Protein;

Carbohydrate;

Lipid

Abstract The present study was conducted to investigate the optimization of probiotic, Lactoba-

cillus sporogenes on survival, growth, biochemical constituents and energy utilization of the fresh-

water prawn Macrobrachium rosenbergii post larvae (PL). Experimental diets were the same in all,

except for the variation in probiotic levels. The probiotic L. sporogenes was used at 0%, 1%, 2%,

3% and 4% inclusion in the experimental diets. These diets were fed to M. rosenbergii PL for a per-

iod of 90 days. The food index parameters, such as SR, WG, SGR, FCE and PER were significantly

(P < 0.05) higher in 4% L. sporogenes incorporated diet fed PL, whereas the FCR was significantly

(P < 0.05) lower in 4% L. sporogenes incorporated diet fed PL. This indicates the fact that this feed

produced higher growth rate than that of other experimental diets. Similarly the proximate compo-

sition of the total protein, total free amino acid, total carbohydrate, and total lipid content was sig-

nificantly (P < 0.05) higher in 4% L. sporogenes incorporated diet fed PL. However, insignificant

differences were recorded in ash and moisture contents between control and experimental groups.

Energy utilization parameters, such as feeding rate, absorption rate, conversion rate and excretory

rate were significantly (P < 0.05) higher in 4% L. sporogenes incorporated diet fed PL. Statistically

insignificant differences were recorded in metabolic rate between control and experimental groups.

This indicates that there were no differences in energy loss between control and experimental

groups. However, L. sporogenes incorporated diet fed PL produced better growth performance.ª 2014 The Egyptian German Society for Zoology. Production and hosting by Elsevier B.V. All rights

reserved.

Introduction

Macrobrachium rosenbergii is a species of aquaculture impor-tance owing to its high fecundity, rapid growth, wide range

of salinity and temperature tolerance, disease resistance as wellas its superior taste and high commercial value (Johnson, 1982;New, 1995; Roustaian et al., 2001). M. rosenbergii is highly

* Corresponding author. Mobile: +91 9488175470.E-mail address: [email protected] (C. Seenivasan).

Peer review under responsibility of The Egyptian German Society for

Zoology.


The Journal of Basic & Applied Zoology (2014) xxx, xxx–xxx

The Egyptian German Society for Zoology

The Journal of Basic & Applied Zoology

www.egsz.orgwww.sciencedirect.com

2090-9896 ª 2014 The Egyptian German Society for Zoology. Production and hosting by Elsevier B.V. All rights reserved.


Please cite this article in press as: Seenivasan, C. et al., Effect of Lactobacillus sporogenes on survival, growth, biochemical con-stituents and energy utilization of freshwater prawn Macrobrachium rosenbergii post larvae (2014), http://dx.doi.org/10.1016/j.jobaz.2013.12.002









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