July | August 2010

Feature title: Effect of Orego-Stim on the growth performance and intestinal bacterial populations of channel catfish (Ictalurus punctatus)

The International magazine for the aquaculture feed industry

International Aquafeed is published five times a year by Perendale Publishers Ltd of the United Kingdom.All data is published in good faith, based on information received, and while every care is taken to prevent inaccuracies, the publishers accept no liability for any errors or omissions or for the consequences of action taken on the basis of information published. ©Copyright 2009 Perendale Publishers Ltd. All rights reserved. No part of this publication may be reproduced in any form or by any means without prior permission of the copyright owner. Printed by Perendale Publishers Ltd. ISSN: 1464-0058

China is the world’s largest producer of aquaculture, currently producing more than ten times more than

India, and over 30 different species of aquaculture products. Now that finan-cial incomes in developing nations such as China have increased, the demand for meat and dairy has also risen as more people can afford to consume these higher protein foods.

Between 1995 and 2009 soybean imports increased from 150 thousand tonnes to 41 million tonnes to help sustain this increased demand in animal feed.

During the same time period broiler production doubled to over 12 million tonnes and pork production increased by over 50 percent.

China is undeniably the world’s most powerful nation in its appetite for agricul-ture and aquaculture commodity use and has consistently consumed over 20 percent of the world’s production of fishmeal in the past 10 years to help support its expanding aquaculture industry.

Channel Catfish are an important aquaculture species originally from USA but intro-

duced into China in the mid 1980s and now widely cultured in China.

Last year Chinese channel catfish produc-tion was estimated at over 500 thousand MT in both small scale and larger commercial monoculture and poly-culture systems.

Channel catfish is tolerant to poorer water quality conditions with dissolved oxygen (DO) of less than 1ppm, though when produced in aquaculture DO is maintained at or above 4ppm in order to opti-

mise production efficiency.

Effect of Orego-Stim on the growth performance and

intestinal bacterial populations of channel catfish (Ictalurus punctatus)

Channel Catfish are an important aquaculture species originally from USA but introduced into China in the mid 1980s and now widely cultured in China. Last year Chinese channel catfish production

was estimated at over 500 thousand MT in both small scale and larger commercial monoculture and polyculture systems.

by Z. L. Zheng of China and Matt Pearce of the UK

Table 1: Formulation (g kg-1) and proximate composition of the diet

Ingredients Chemical composition

Soybean meal 300.00 DE (kJ kg-1) 12.51

Rapeseed meal 260.00 CP 345.00

Corn gluten meal 50.00 EE 38.50

Fish meal 100.00 ASH 62.40

Wheat shorts 220.00 Ca 8.50

Rapeseed oil 20.00 TP 12.20

Ca(H2PO4)2 16.00 AP 8.00

Vitamin premixa 1.70 Lys 17.50

Choline chloride 2.00 Met + Cys 13.20

Sodium chloride 1.00

Mineral premixb 5.00

Bentonite 24.30

Note: Crude protein, crude lipid, ash, crude fibre are expressed on a dry matter basis and given as means (n=2) a Vitamin mix provided the following vitamins (mg kg-1 diet unless otherwise stated): vitamin A, 4000 IU; vitamin D3, 2000 IU; vitamin K, 10; vitamin E, 50; thiamine, 10; riboflavin, 12; pyridoxine, 10; panthothenic acid, 32; nicotinic acid, 80; folic acid, 2; biotin, 0.2; vitamin B12, 0.01; L-ascorbyl-2-polyphosphate (250 g kg-1 vitamin C activity), 60b Trace mineral mix provided the following minerals (mg kg-1 diet): zinc (as ZnSO4·7H2O), 150; iron (as FeSO4·7H2O), 40; manganese (as MnSO4·H2O), 25; copper (as CuCl2), 3; iodine (as KI), 5; cobalt (as CoCl2·6H2O), 0.05; selenium (as Na2SeO3), 0.09

During the same time period broiler production doubled to over 12 million tonnes and pork production increased by

China is undeniably the world’s most powerful nation in its appetite for agricul-ture and aquaculture commodity use and has consistently consumed over 20 percent of the world’s production of fishmeal in the past 10 years to help support its expanding aquaculture industry.

Channel Catfish are an important aquaculture species originally from USA but intro-

though when produced in aquaculture DO is maintained at or above 4ppm in order to opti-

mise production efficiency.

80; folic acid, 2; biotin, 0.2; vitamin B12, 0.01; L-ascorbyl-2-polyphosphate (250 g kg-1 vitamin C activity), 60b Trace mineral mix provided the following minerals (mg kg-1 b Trace mineral mix provided the following minerals (mg kg-1 bdiet): zinc (as ZnSO4·7H2O), 150; iron (as FeSO4·7H2O), 40; manganese (as MnSO4·H2O), 25; copper (as CuCl2), 3; iodine (as KI), 5; cobalt (as CoCl2·6H2O), 0.05; selenium (as Na2SeO3), 0.09

30 | InternatIonal AquAFeed | July-august 2010 July-august 2010 | InternatIonal AquAFeed | 31

F: Growth performance

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Optimisation of feed intake and nutritional composition of the diet helps to ensure that channel catfish are extracting the maximum possible growth from what they consume. Ingestion of aquatic feed inevita-bly causes drinking of the localised aquatic environment putting the gut interface of catfish into direct chal-lenge with the indigenous pathogenic microbial community.

The gastrointestinal tract is responsive and sensitive to a wide range of stressors.

Various phytogenic ingredients have been shown to facilitate ben-eficial effects on gut environment and microflora, though some plant species have a stronger impact than others.

The antimicrobial effects of phenolic compounds which are the active ingredient of phytogenic feed additives, target pathogenic bacte-rial cell walls by interaction with the cytoplasmic membrane and changing its permeability for cations, like H+ and K+.

Inhibitory effects on microorganisms

Orego-Stim® contains essential oils which include precise natural composi-tions of a-terpinene, p-cymene, carvacrol and thymol. These components are found to have inhibitory effects on micro-organisms especially in spore forming organisms.

It has been clearly demonstrated that chronic stress measured by prolonged elevated blood plasma cortisol concentra-tion in trout results in dose dependent increased mortality from common aquatic bacterial and fungal diseases.

This finding is not just confined to sal-monid species but is a normal physiological response in all fish species. The hypotha-lamic-pituitary-interrenal axis in fish which can be stimulated by both environmental and stress events affects the production of lymphocytes and antibody response as well as the reproductive capacity.

Gut Associated Lymphoid Tissue (GALT) is the interface between the diet, host physiology and gut microflora. GALT activ-ity can be modified through the diet by three principles – competitive bacterial exclusion, bacterial antagonism and immune modulation which in turn affect the health and commercial productive status of fish.

This latest experimental trial in Channel

catfish aimed to analyse the response of bacterial populations when exposed to Orego-Stim®.

Both aero-bic and anaero-bic beneficial and pathogenic p o p u l a t i o n s of bacteria were isolated and quantified from differ-ent regions of the intestine of channel catfish. Various other growth p a r a m e t e r s such as weight gain, FCR, SGR, Protein effi-ciency ratio and mortality were also quantified.

Fish and culture conditions

C h a n n e l catfish (Ictalurus p u n c t a t u s ) p r o p a g a t e d and reared by the Research Institute for F i s h e r i e s

(Chongqing, China) were used for the experiment. Experiments were conducted in a recirculation aquaculture system at

Figures 1-4. Growth performance measured by total weight gain, specific growth ratio, feed conversion ratio and protein efficiency ratio.

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The formulating method of FWA as fol-lows: MgSO4 (0.05g), beef extract (2.5g), NaCl (500g), K2HPO4 (0.2g), glucose (1g), peptone (5.0g), yeast extract (2.5g), agar powder (15.0g), add water to 1000 ml, pH 7.2-7.4, and was sterilised for 30 min at 121℃.

18 FWA plates were spread with homogenates of the intestinal sections in each group. Nine plates were incubated at 30℃ for 24-48 hours in an incubator, and nine plates were incubated at 30℃ for 48-72 hours in an anaerobic incubator. Plates with 20-200 bacterial colonies were counted and bacterial numbers in plates were converted to the number per unit weight of intestine.

Identification of bacteriaApproximately 10 colonies were ran-

domly picked from plates containing 20 to 200 bacteria. A total of 100 isolates were inoculated with a method of slope culture, respectively. 60 tubes were incubated at 30℃ for eight to 12 hours in an incubator, and placed at 4℃ after incubation.

Another 40 tubes were incubated at 30℃ for 24 to 36h in an anaerobic incuba-tor, and were identified after 4 hours.

The identification process of aerobic bac-teria was formulated according to Bergey’s Manual of Determinative Bacteriology and classified according to genera or groups on the basis of cell morphology, motility, gram reaction, catalase and oxidase and glucose fermentation, O/F test, salt tolerance for growth.

Anaerobic bacteria were identified with an automatic microorganism identifica-tion instrument (VITEK-�) by the French BioMerieux Company and identification plate (VITEK-ANA) for anaerobic bacteria.

Results - Growth performance

Survival rate of the experi-ment was high (>95%) and unre-lated to dietary treatment (see Table 2). Data on the growth performance of channel catfish, including weight gain (WG), spe-cific growth ratio (SGR), feed conversion ratio (FCR) and pro-tein efficiency ratio (PER) are shown in Table 2.

WG of fish fed the OS diet was significantly higher than those of fish fed the Con diet (P<0.05).

SGR of fish fed the OS diet

At each feeding, an excess amount of feed was fed to the fish and uneaten feed was collected one hour after feeding, dried at 70°C and reweighed. Leaching of uneaten feed was estimated by placing weighed samples of each diet into a tank without fish for one hour and then recovered, dried and

reweighed. The average leaching value

was used to correct the amount of uneaten feed.

Intestine bacteriological sampling

Six channel catfish were sampled from each diet group after one-day starvation.

The fish were killed by physical destruc-tion of the brain, and the number of inci-dental organisms was reduced by washing the fish skin with 700 ml L-1 ethanol before opening the ventral surface with sterile scissors to expose the body cavity.

The ventral belly surface of the fish was opened to expose the peritoneal cav-ity, and then the spleen, gallbladder and liver removed. The intestinal sections were emptied and thoroughly rinsed three times in 2ml sterile 9g/kg-1 saline to remove non-adherent bacteria.

Anterior intestine (AI), mid intestine (MI), and posterior intestine (PI) of the cor-responding sections were treated separately, and were then transferred to sterile plastic bags and homogenised in a Stomacher (Seward Laboratory, London, UK).

Bacterial plates spreading and count

Homogenates of the intestinal sections were diluted in sterile 9g/kg-1 saline and appropriate dilutions were spread on the surface of fresh water agar (FWA) plates.

Sou thwes t Un ivers i t y, Chongqing, China. Channel catfish with an average initial weight of 50 g were held in 200 L fibreglass tanks. Fish in randomly assigned duplicate aquaria (40 fish per aquaria) were fed one of the two experimental diets to apparent satiation six times daily. Water temperature and pH were constant (23-24°C; pH 8.5) during the experimental period. Water flow was maintained at 4.5 L/minute. Orego-Stim® (50g kg-1) was a commercial product of Meriden Animal Health Ltd., UK.

The dietary formulation of each of the diets was as follows: Control: without feed additives Orego-Stim®: 50g kg-1 Orego-Stim®

Experimental design and diets

Both diets were formulated to be isoca-loric (12.51kJ kg-1 diet) and isonitrogenous (3450g/kg-1 crude protein). All ingredients were finely ground, mixed in a Hobart mixer and pelleted through a 2.4mm diameter die in a Hobart meat grinder. The pellets were air-dried at room temperature, fragmented and stored in a freezer until use.

Ingredients and proximate composition of the experimental diets are presented in Table 1.

The feeding trial lasted eight weeks.

Table 2: WG, SGR, FCR, PER and survival of Channel catfish fed different diets with feed additives

Diets WG1 (g) SGR2 (%/d) FCR3 PER4 Survival

Control 102.63±2.22a 1.95±0.04a 1.94±0.092a 1.49±0.08a 95.83±2.89a

Car+Thy 116.77±2.49b 2.15±0.05b 1.84±0.03a 1.58±0.02a 95.00±2.50a

OS 127.53±6.40c 2.24±0.12b 1.64±0.04b 1.77±0.07b 98.33±1.44a

Data were presented as mean±SD (n=2). Value in the same column having different superscripts is significant (P<0.05).1 WG (Weight gain, g) = final body weight − initial bodyweight2 SGR (Specific growth ratio, �/d) = (loge average final weight - loge average initial weight) / no days) x

1003 FCR (Feed conversion ratio) = feed consumed (g, dry weight) / weight gain (g)4 PER (Protein efficiency ratio) = weight gain (g) / protein intake (g)

At each feeding, an excess amount of feed was fed to the fish and uneaten feed was collected one hour after feeding, dried at 70°C and reweighed. Leaching of uneaten feed was estimated by placing weighed samples of each diet into a tank without fish for one hour and then recovered, dried and

reweighed.

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order to maximise growth performance and immune defence.

This experimental trial isolated the bac-terial species, compositional proportion and the region of the intestine that different bacteria species colonised.

Orego-Stim® demonstrated the ability to selectively reduce or increase different species of bacteria. Numbers of key patho-genic bacteria such as Ent. Aer. and Vib were reduced in the Orego-Stim® group of catfish, where as beneficial bacteria which helped to promote gut health such as Lac. and Bif had higher numbers of bacteria.

This study shows that Orego-Stim® acts in a similar way as probiotics using competitive exclusion, but unlike probiot-ics Orego-Stim® has much more potent antimicrobial properties.

Orego-Stim® is a phytogenic feed additive.

This specific discipline of aquaculture feed science is still an innovative and pio-neering technology.

At the time of writing the exact bio-chemical mechanism by which beneficial bacteria are allowed to grow in the intes-tines and pathogenic bacteria are killed, is still not fully understood. The theory is that there must be a structural difference in the cell wall of the two classes of bacteria possibly relating to epitopes and virulence factors which selectively promotes or pre-vents cell lysis upon exposure to Orego-Stim®, but this needs further research to get the required scientific evidence.

What is known is that both the holistic and generic effects of Orego-Stim® work in a way that alters intestinal bacterial populations and benefits the host fish by increasing aquaculture productivity, both experimentally and commercially.

References are available on request.

Numbers of Aer. also declined with the Orego-Stim® diet and this result was significantly different from the control (P<0.05). Conversely, Bac. numbers increased signifi-cantly upon addition of OS (P<0.05). Addition of Orego-Stim® had no effect on the numbers of Staphylococcus (Stap).

In MI, the num-bers of Ent. sig-nificantly decreased upon addition of OS (P<0.05). The feed additives had no effect on the numbers of Aer., Stap. and Vib.. The numbers of Bac. also increased significantly in the Orego-Stim® diets compared to con-trol (P<0.05).

In PI, the numbers of Ent., Aer., Stap. and Vib. decreased with the Orego-Stim® diet.

A n a e r o b i c bacteria

The three intes-tinal anaerobic bacteria identified with the identification plate (VITEK-ANA) of anaerobic bacteria were Bifidobacterium (Bif.), Lactobacillus (Lac.) and Bacteroides (Bact.), respectively. The predominant intestinal anaerobic bacteria were Bact. Bif. and Lac. (Figure 7).

In the AI, MI and PI numbers of Bif. and Lac in the Orego-Stim® group significantly increased compared to that of the control (P<0.05). Numbers of Bact. significantly decreased in the Orego-Stim® group com-pared to that of control (P<0.05).

Discussions and conclusionAs there is evidence that intestinal com-

petitive bacterial exclusion and bacterial antagonism are the unique modes of action that help the immune response of fish, it is pertinent to understand how Orego-Stim® can beneficially balance the gut flora in

were significantly higher than those of fish fed the control diet. FCR and PER were also affected by OS.

Lowest FCR and highest PER values were observed for catfish fed the OS diet compared to catfish fed the control diet (P<0.05).

Composition of bacteria flora - Aerobic Bacteria

Bacterial numbers are shown in tables 5-7.

Intestinal aerobic bacteria of 4 families (genera) of channel catfish were identi-fied. The intestinal predominant aerobic bacteria were Enterobacteriaceae Ent.）, Aeromonas (Aer.), Bacillus (Bac.) and Vibrio (Vib.) (Figure 5-6).

In AI, the numbers of Enterobacteriaceae in fish fed Orego-Stim® decreased sig-nificantly compared to control (P<0.05).

About the authors:Z. L. Zheng of China - School

of Veterinary Medicine of Sichuan Agriculture University, Sichuan 625014, PR China and the Fisheries Breeding and Healthy Cultivation Research Centre, South-west University, Chongqing 402460, PR China

Matt Pearce - Meriden Animal Health Limited, Cranfield Innovation Centre, University Way, Cranfield Technology Park MK43 0BT, United Kingdom

Figures 5-7: Composition of Aerobic and Anaerobic bacteria species in the Anterior, Mid and posterior intestine of Channel Catfish

34 | InternatIonal AquAFeed | July-august 2010

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VOLUME 13 I S SUE 4 2 010

Inclusion of a mineral premix for low-salinity culture

of Litopenaeus vannamei

Evaluation of Vibrio control

- with a multi-species probiotic in shrimp aquaculture

Recent advances in the use of diformates in fish

IAF10-04.indd 1 30/06/2010 09:13

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