Immunomodulatry and growth responses of Nile Tilapia fingerlings to dietary extract of Moringa flower

Esayas Welday Tekle1,2 & N. P. Sahu1

1 Fish Nutrition, Biochemistry and Physiology Division, Central Institute ofFisheries Education, Panch Marg, Off Yari Road, 400061 - Mumbai, India

2 College of Marine Sciences and Technology, Massawa, EritreaE-mail: e_welday@yahoo.com

Aquaculture Europe 2015European Aquaculture Society20 -23 October, 2015, Rotterdam, Netherlands

Outline

Bottomline Aquaculture Aquafeeds

Challenges Alternatives Dietary Moringa extract Effect on

Growth Enzyme activities Immunological

Tilapia Aquaculture

Aquaculture

Tilapia aquaculture Most widely distributed

(>135 countries) 2nd in production

Species of choice Hardy Low trophic level Flesh quality (fillet or

whole fried)

Farming and Production of O. niloticus (FAO, 2010)

Asia

Africa

Americas Others

Aquaculture production of tilapia by countryin million tonnes 1950–2009 (FAO, 2012)

Aquaculture production of tilapia by speciesin million tonnes as reported by the FAO, 1950–2009[1]

Challenges to industry sustainability

Intensification of farming systemsNutrient loadWater quality – stress

Feed and feed relatedEconomicEnvironmentalHuman health

ROS (FR)Oxidative stressInfectious diseases

Pathogens (opportunistic)

Stressful environment

Host (fish)

Overcrowding

Husbandry

Physicochemical (Poor WQ)

Parasite infestation

?

Modified from:Source: http://www.thefishsite.com/articles/1570/an-introduction-to-fish-health-management/ accessed on 11/10/2015.

Synthetic antioxidants BHT BHA Ethoxiquine

Chemoteraupitics Various antibiotics Disinfectants

Risks Bioacumulation Residues Development of resistant microbial strains

Environmental and human health concern

Restrictions

Cont. …

Alternatives

Plant/herbal extracts Natural Minimal or no negative impact

Antioxidant properties Mitigate oxidative stress – scavenge ROS Prevent lipid peroxidation - rancidity

Antimicrobial effect Enhance immune system Combat microbes

Moringa

Moringa?

Deciduous plant species Moringa spp. (Moringacea

family) Moringa oleifera – most

widely distributed worldwide “drumstick tree” “horse radish tree” “ben oil tree”

Native to Himalayan region of India Distributed worldwide

(resistant to drought)

Source: http://miracletrees.org/moringa_powder_content.html

Medicinal use of Moringa

Ayurveda (more than 300 diseases) The roots, leaves, gum, flowers, barks and

seeds/pods have active chemical constituents:

Nitrile mustard oil glycosides thiocarbamate glycosides (Anwar et al., 2007).

• Diuretic lowering• cholesterol lowering• Antiulcer• Hepatoprotective• cardiovascular

protective property

Cont. …

Other Phytochemicals with powerful antioxidant & antimicrobial attributes pterygospermin Kaempferol Quercetin Rutin caffeoylquinic acids antioxidant vitamins - C, E, and A carotenoids - lutein, alpha-carotene and beta-

carotene, xanthins, and chlorophyll

Objectives

Immunomodulatory

Growth performance and nutrient utilization

1. Extraction procedure

CentrifugingConcentrating Extract (lyophilized)

Pulverized Filtered Shaking (24 hrs) Filtering

2. Experimental design and methodology

60-days growth period 4 diets (isonitrogenous, isocaloric) basal diet (control) – without extract Three ext. inclusion levels (0.25%, o.5% and 1%)

Diet Denotations Extract level (%)

C (control) 0

EF.25 0.25

EF.5 0.5

EF1 1

EF = ethanolic flower exract (70% ethanol)

Cont. …

Experimental fish

60-day growth study 120 fish (8.78±1.36g ) in 12 plastic tanks (80 L)

Challenge experiment

Fish were exposed to A. hydrophila for 10 days after the 60-days growth period

20 fish per treatment in duplicate

Cont. …

After 60-days growth period and 10-days challenge:

Growth parameters

Metabolic and antioxidative stress enzymes

Immunological parameters

Results and discussion

Diets

Growth parameters Control EF.25 EF.5 EF1

Weight gain 44.90±0.48a 75.90±1.60c 68.29±1.23b 45.92±0.38a

FCR 2.13±0.02c 1.32±0.03a 1.42±0.02a 1.68±0.09b

SGR 0.68±0.03a 1.06±0.03c 0.92±0.02b 0.67±0.05a

Survival (%) 60.00±0.00a 90.00±0.00c 76.67±5.77bc 73.33±5.77ab

PER (%) 1.58±0.03a 2.53±0.05c 2.35±0.03c 1.99±0.10b

Energy retention (%) 12.93±1.08a 24.06±0.58d 20.53±0.18c 17.57±0.34b

Protein retention (%) 24.74±0.33a 51.69±1.22g 44.19±0.03e 43.68±0.52e

Survival(postchallenge) 29.63±6.41a 59.26±6.41c 51.85±6.42b 33.33±11.11a

Table 2 Growth and nutrient utilization

Some pictures taken after challenge with A. hydrophila

2. Metabolic enzymes activitiesMDH & LDH

d

c

ab

c

ab

c

a a

b

b

c

c

dd

LDH – anaerobic energy production; conversion of pyruvate (glycolysis) to lactateMDH – energy production from non-carbohydrate sources (TCA cycle); retarded growth

Transaminases

a

ab

c c

b

c

d

aa

ab b

b

c

d

ALT AST

ALT & AST • Synthesis and deamination of AA.; gluconeogenesis• Higher levels - energy utilization from non-carbohydrate sources – slow down increase in

body mass

Antioxidative stress enzymes

Treatments

Liver GillPre-

challengePost-

challengePre-

challengePost-

challengeControl 30.41±0.96dA 34.36±1.53cB 28.20±1.70dA 32.03±0.26dB

EF.25 7.01±0.37aA 10.86±1.01aB 15.95±0.80a 13.10±0.74a

EF.5 20.52±0.33c 21.03±0.56b 22.72±1.79bA 26.45±0.71bB

EF1 15.82±1.80b 20.79±0.95b 25.83±1.39c 28.95±0.15c

SOD (U/min/mg protein)

Control 1.94±0.06aB 1.31±0.03aA 1.45±0.1aA 2.12±0.01aB

EF.25 5.28±0.19dB 2.47±0.46cA 2.86±0.3dA 4.21±0.08cB

EF.5 4.44±0.19cB 2.05±0.10cA 1.92±0.2bA 2.92±0.02bB

EF1 3.57±0.15bB 2.00±0.05bA 2.34±0.14cA 2.24±0.00aA

CAT (mmols/min/mg protein)

SOD – cleaving superoxide radicals into elemental O2 and H2O2CAT – decomposes H2O2 into O2 and H2O

Lowercase superscripts in a column; uppercases in a row = significant difference (P<0.05)

Cont. …

a

abb

c

a

a

cbc

b

c

cba

c

a

GST – catalyzes conjugation of glutathione to toxic cpds. (epoxides, aliphatic, aromatic and heterocyclic radicals, etc.) – measured as µM of CDNB released

3. Immunological

Treatme

nts

NBT Lysozyme activity

Prechallenge Postchallenge Prechallenge Postchallenge

Control 0.721±0.013aB 0.604±0.009aA 0.367±0.025aA 0.490±0.030aB

EF.25 1.526±0.018cB 0.877±0.040cA 1.983±0.0.085dA 2.243±0.045cB

EF.5 1.093±0.025bB 0.684±0.031bA 1.080±0.076cA 1.533±0.015bB

EF1 0.759±0.013aB 0.677±0.033bA 0.790±0.060bA 1.450±0.120bB

NBT = Nitroblue tetrazolium (absorbance 620nm) – respiratory burst activityLysozyme activity = A450 – a measure of bacterial lysis by blood cells.

Table 4

Cont. …

Treatments

Phagocytic activity (%) Antiprotease activity (%)

Prechallenge Postchallenge Prechallenge Postchallenge

Control 46.170±1.830aB 34.627±1.375aA 85.633±0.575aB 77.070±0.520aA

EF.25 94.547±.815dB 70.000±2.082dA 94.253±0.385cB 89.540±0.360cA

EF.5 86.670±0.000cB 63.750±2.165cA 90.997±0.575bB 81.897±0.515bA

EF1 78.887±0.964bB 59.167±0.722bA 84.673±0.765aB 76.210±0.690aA

Table 5 Serum phagocytic and antiprotease activities (%) of the various experimental groups

Conclusion

Growth parameters, enzyme activities and immunlologicalanalysis revealed the EF.25 found to be most effective Recommended as antioxidant and immunostimulant Replace synthetic antioxidants and drugs

As the extract inclusion level increases, performance decreased In some instances less than the control May be due to the toxic effect of the extracts at higher doses Cytotoxicity, histopathological study should be carried out

Mechanism of action, at molecular level

References Anwar F., Ashraf M. & Bhanger M.I. (2005) Inter-provenance variation in the composition

of Moringa oleifera oilseeds from Pakistan. Journal of the American Oil Chemists' Society82(1), 45-51.

Asaolu, V. O. & Okewoye, A. T. (2013) Moringa multinutrient block supplementation effects on feed Utilization by West African dwarf goats fed a basal diet of Cassava peels. Science Focus 18 (1), 63-72.

Bauer A.K., Dwyer-Nield L.D., Hankin J.A., Murphy R.C. & Malkinson A.M. (2001) The lung tumor promoter, butylated hydroxytoluene (BHT), causes chronic inflammation in promotion-sensitive BALB/cByJ mice but not in promotion-resistant CXB4 mice. Toxicology169(1), 1-15.

Fawole F.J., Sahu N.P., Pal A.K. & Ravindran A. (2015) Haemato-immunological response of Labeo rohita (Hamilton) fingerlings fed leaf extracts and challenged by Aeromonas hydrophila. Aquaculture Research June 25 DIO:10.1111/are.12829.

Moringa oleifera extract against waterborne Pb stress (Sirimongkolvorakul et al., 2012) Siddhuraju P. & Becker K. (2003) Antioxidant properties of various solvent extracts of

total phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa oleifera Lam.) leaves. Journal of agricultural and food chemistry 51(8), 2144-2155.

Sirimongkolvorakul et al., 2012Moringa oleifera extract against waterborne Pb stress Tekle E.W., Sahu N.P. & Makesh M. (2015) Antioxidative and antimicrobial activities of

different solvent extracts of Moringa oleifera: An in vitro evaluation. International Journal of Scientific and Research Publications 5(5), 255-266.