July | August 2010

Feature title: Effect of Orego-Stim on the growth performance and

intestinal bacterial populations of channel catfish (Ictalurus punctatus)
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,
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The International magazine for the aquaculture feed industry

 F: Growth performance F: Growth Aqua
performance
News
AwF launch
Lallemand range
Optimisation of feed intake of
and science-based, field-approved
nutritional composition of the diet redesigned website
solutions for
helps to ensure that a sustainable
channel catfish aquaculture
Effect of Orego-Stim on the A
are extracting the maximum possible quaculture without
growth from what they consume. Frontiers (AwF) is

L
Ingestion of aquatic
allemand Animal Nutrition feed inevita-
Further applications being under guarantying its purity and con- pleased to announce
the launch of its completely

growth performance and

bly iscauses
dedicateddrinking
to theofdevelop-
the localised
investigation to cover other major sistent quality. Agrimos contains
aquatic
ment,environment
production, and putting
mar-the aquaculture
gut species (tilapia, carp, one of the highest guaran- redesigned website.
interface
keting of catfishnatural
of profitable, into direct
and chal-
etc). Performance trials have teed levels of MOS on the This fulfills the commitment
to raise the profile of AwF and

intestinal bacterial populations

lenge with the
differentiated indigenous
yeast and bacteria-pathogenic
proven beneficial effects in terms market.
microbial
based community.
solutions for animal nutri- of growth enhancement, feed uti- Independent trials in various improve fundraising activities
Thehealth.
tion and gastrointestinal tract lisation, is as well as improved resist- salmonid and tropical fish species that Dr Barry Costa-Pierce and
responsive
The companyand hassensitive
developed to aa wide
ance under specific challenging have shown the benefits of Dr Mudnakudu Nandeesha,
international co-chairs, made at
of channel catfish (Ictalurus punctatus)
range
range ofofmicrobial-based
stressors. nutri- conditions. yeast mannan-oligosaccharides
tional Various
solutionsphytogenic
to support ingredients
per- on gut health, intestinal micro- the AwF Board of Directors
have been
formance andshown
health to of facilitate
aquatic ben- Overcoming intestinal flora and global performances meeting on September
eficialin effects
species a naturalonway. gut environment disturbance under various challenging condi- 26, 2009 during the World
Channel Catfish are an important aquaculture species originally from USA but introduced into China and microflora,
Lallemand’s though inno-
aquaculture some plant Levucell® SB, a probiotic yeast tions. Aquaculture Society confer-
in the mid 1980s and now widely cultured in China. Last year Chinese channel catfish production species
vative have a stronger
solutions are based impact
on thanstrain (Saccharomyces cerevi- Laltide® is a combination of ence in Veracruz, Mexico.
was estimated at over 500 thousand MT in both small scale and larger commercial monoculture and others.
specific yeast and bacteria strains siae var. boulardii CNCM 1079). available nucleotide specifically The AwF Board of Directors
polyculture systems. grownThe in theantimicrobial
company’s effectsown S.ofboulardii Figures
yeast is1-4.
well-known formulated for aquaculture appli- hopes visitors will find the new
Growth performance measured by total weight gain, specific growth
phenolic plants.
compounds which are in thehuman and cations. It protein
is recommended website informative and provide
production Each product’s animal
ratio, feedhealth for ratio
conversion and efficiencyasratio.
a
activesafety
origin, ingredient of phytogenic feed
and concentration its positive effects against intes- well-balanced source of nucle- a clear and comprehensive view
by Z. L. Zheng of China and Matt Pearce of the UK
areadditives,
guaranteed. target pathogenic bacte- tinal disturbance. Its modes of otides adapted to the needs of of work to date.
Visit the were website
used foratthe

C
rial development
The cell walls by and interaction
valida- withaction in the gutcatfish aimed
are well to analyse
under- fish andthecrustacean
response species,
of (Chongqing,
par- China)
hina is the world’s largest duced into China in theof cytoplasmic
tion our ‘field led membrane
and scienceandstood, changing bacterial
creating a real populations whenduring
‘shield ticularly exposed to of intense
periods www.aquaculturewithout
experiment. Experiments were conducted
Table 1: Formulation (g kg-1) and proximate composition of frontiers.org
producer of aquaculture, the mid 1980s and now the diet
its permeability
supported’ products foriscations,
based on like H+ and K+.
effect’, Orego-Stim®.
reinforcing the microbial metabolism needs. in a recirculation aquaculture system at
currently producing more widely cultured in China. scientific research and perform- ecosystem and protecting Both aero- the
than ten times more than Last year Chinese Ingredients Chemical composition anceInhibitory effects on
trials conducted in par t- animal’s bic
digestive and
tract. anaero-
Levucell
India, and over 30 different species of channel catfish produc- microorganisms
nership with leading interna- SB is also a source bic ofbeneficial
beneficial
aquaculture products. Now that finan- tion was estimated tional Orego-Stim®
research centres, contains essential oils
universi- nutrients and
particularly pathogenic
useful to
cial incomes in developing nations
such as China have increased, the
at over 500 thousand
MT in both small scale
Soybean meal
Rapeseed meal
300.00
260.00
DE (kJ kg-1)
CP
12.51
345.00
tieswhich include precise
and commercial
thetions
world:
natural promote
firms around
of a-terpinene, p-cymene, aquaculture
composi-the growth
carvacrol species.
p o p uof
of
l a certain
tions
bacteria FIGAP/ VIV
demand for meat and dairy has also and larger commercial
Corn gluten meal 50.00 EE 38.50
and thymol.
Bactocell® These
(live components
bacteria strain are found a were
Alkosel®, isolated
yeast product América Latina 2010
risen as more people can afford to monoculture and poly- to have inhibitory
Pediococcus acidilactici CNCMeffects on micro- elevated
containing and quantified
levels of
consume these higher protein foods. culture systems. Fish meal 100.00 ASH 62.40 organismsthe
MA18/5M), especially in sporeselenomethionine,
first probiotic forming fromthe highest
differ- Your natural partner for aquaculture
Channel catfish is Wheat shorts 220.00 Ca 8.50 organisms.
strain authorised for use in aqua- bioavailable form ent ofregions
selenium. of
Between 1995 and 2009 soybean imports tolerant to poorer cultureIt inhas
thebeen clearly
European demonstrated
Union, is Alkoselthat the intestine
is recommended as a We offer a range of innovative microbial-based
Rapeseed oil 20.00 TP 12.20
increased from 150 thousand tonnes to 41 water quality conditions chronic stress
a guarantee measured
of its safety, qualityby prolonged
source of natural of seleniumchannel for solutions for sustainable aquaculture:
million tonnes to help sustain this increased with dissolved oxygen Ca(H2PO4)2 16.00 AP 8.00 andelevated
efficacy blood fish and shrimpcatfish.
plasma cortisol concentra-
in aquaculture. production.Various October 21-23, 2010
Vitamin premixa

Guadalajara
demand in animal feed. (DO) of less than 1ppm, 1.70 Lys 17.50 tion in istrout
Bactocell one ofresults in dose
the best A g r i m o s ® ,other
sci- dependent a c ogrowth
ncen-
During the same time period broiler though when produced increased mortality from
entifically-documented common
probiotic aquatic
trated sourcep aofr ayeast
m e t ecellrs Probiotics
Choline chloride 2.00 Met + Cys 13.20
production doubled to over 12 million in aquaculture DO is bacterial
strains and fungal
in aquatic diseases.
species such as weight
with wall mannan-oligosaccharides
Sodium chloride 1.00
tonnes and pork production increased by maintained at or above many This finding isinnot
applications just confined
salmonids, (MOS) to sal- gain, through
obtained FCR, SGR, a
over 50 percent. 4ppm in order to opti- Mineral premixb 5.00 monidand
shrimps species
otherbut is a normal
marine fishes. physiological
specific production Proteinprocess effi-
China is undeniably the world’s most mise production response in all fish species. The hypotha- ciency ratio and
Selenium Enriched Yeast
Bentonite 24.30
powerful nation in its appetite for agricul- efficiency. lamic-pituitary-interrenal axis in fish which mortality were Register now
Note: Crude protein, crude lipid, ash, crude fibre are expressed
ture and aquaculture commodity use and can be stimulated by both environmental also quantified. for free entr
has consistently consumed over
on a dry matter basis and given as means (n=2)
and stress events affects the production of ance
a Vitamin mix provided the following vitamins (mg kg-1 diet Yeast Mannan-Oligosaccharides (MOS)
20 percent of the world’s
unless otherwise stated): vitamin A, 4000 IU; vitamin D3, 2000
lymphocytes and antibody response as well Fish and www.viv.net
production of fishmeal IU; vitamin K, 10; vitamin E, 50; thiamine, 10; riboflavin, as the reproductive capacity.
FES Consultants (UK) Ltd culture
in the past 10 years 12; pyridoxine, 10; panthothenic acid, 32; nicotinic acid, Gut Associated Lymphoid Tissue parts for allconditions
• Wear(GALT) single and
to help support its 80; folic acid, 2; biotin, 0.2; vitamin B12, 0.01; L-ascorbyl-2- is the interface between thetwin diet, hostextrudersC h a n n e l
screw Purified
Guadalajara, Jalisco - MexicoNucleotides
expanding aquaculture polyphosphate (250 g kg-1 vitamin C activity), 60 physiology and gut microflora. • The
GALT supply and refurbishment
activ- catfish (Ictalurusof
b Trace mineral mix provided the following minerals (mg kg-1 used extruders for the pet, aqua
industry. ity can be modified through and the human
diet byfood industry
punctatus)
Channel Catfish are an diet): zinc (as ZnSO4·7H2O), 150; iron (as FeSO4·7H2O), three principles – competitive bacterial propagated It is time to explore a wide range of
important aquaculture 40; manganese (as MnSO4·H2O), 25; copper (as CuCl2), 3; exclusion, bacterial antagonism and www.fes-ltd.com
immune and reared by challenging opportunities
iodine (as KI), 5; cobalt (as CoCl2·6H2O), 0.05; selenium (as Unit 11, Barnwell Manor Estate, Lallemand Animal Nutrition- 19, rue des Briquetiers BP 59 31702 Blagnac Cedex − France
species originally modulation which in turn affect Barnwell, the health the PE8
Peterborough, Research
5PL e-mail: aqua@lallemand.com Tel.: +33 (0)5 62 74 5555 Fax:+33 (0)5 62 74 5500
Na2SeO3), 0.09 Tel: +44 (0)1832 270831 Email:
from USA but and commercial productive status of fish. Institute
welcome@fes-ltd.com for www.lallemandanimalnutrition.com
intro- This latest experimental trial in
If you use extrusion equipment we can save you money! Channel F i s h e r i es www.lallemand.com

adv viv americalatina 132x190a.indd 1 25-06-2010 14:09:21

10 30 | InternatIonal
| InternatIonal AquAFeed
AquAFeed | July-august
| July-august 2010
2010 July-august
July-august 2010
2010 | InternatIonal
| InternatIonal AquAFeed
AquAFeed | 11| 31
 F: Growth performance

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
GROWiNG iS
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

Southwest U n i ve r s 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.
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.
beautiful
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 bacteria
Approximately 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
Table 2: WG, SGR, FCR, PER and survival of Channel catfish fed different diets with feed additives performance
Diets WG1 (g) SGR2 (%/d) FCR3 PER4 Survival Survival rate of the experi-
ment was high (>95%) and unre-
lated to dietary treatment (see
Control 102.63±2.22a 1.95±0.04a 1.94±0.092a 1.49±0.08a 95.83±2.89a Table 2). Data on the growth
Car+Thy 116.77±2.49b 2.15±0.05b 1.84±0.03a 1.58±0.02a 95.00±2.50a performance of channel catfish,
OS 127.53±6.40c 2.24±0.12b 1.64±0.04b 1.77±0.07b 98.33±1.44a including weight gain (WG), spe-
cific growth ratio (SGR), feed
Data were presented as mean±SD (n=2). Value in the same column having different superscripts is
conversion ratio (FCR) and pro-
FRANCE / 00 33 2 97 40 42 09

significant (P<0.05).
1 WG (Weight gain, g) = final body weight − initial bodyweight
tein efficiency ratio (PER) are
shown in Table 2.
2 SGR (Specific growth ratio, /d) = (loge average final weight - loge average initial weight) / no days) x
WG of fish fed the OS diet Our hydrolysates have a superior concentration of Natural Active NutrientsTM
100 was significantly higher than
3 FCR (Feed conversion ratio) = feed consumed (g, dry weight) / weight gain (g) those of fish fed the Con diet Thanks to their biological action on fish and shrimp growth mechanisms,
4 PER (Protein efficiency ratio) = weight gain (g) / protein intake (g) (P<0.05). they boost your feed performance and help you substituting fish meal.
SGR of fish fed the OS diet

contact@aquativ-diana.com - www.aquativ-diana.com
32 | InternatIonal AquAFeed | July-august 2010
 F: Growth performance
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
Figures 5-7: Composition of Aerobic and Anaerobic the Orego-Stim®
bacteria species in the Anterior, Mid and posterior diet.
intestine of Channel Catfish Anaerobic
bacteria
The three intes-
were significantly higher than those of fish tinal anaerobic bacteria identified with
fed the control diet. FCR and PER were also the identification plate (VITEK-ANA) of
affected by OS. anaerobic bacteria were Bifidobacterium
Lowest FCR and highest PER values (Bif.), Lactobacillus (Lac.) and Bacteroides
were observed for catfish fed the OS diet (Bact.), respectively. The predominant
compared to catfish fed the control diet intestinal anaerobic bacteria were Bact. Bif.
(P<0.05). and Lac. (Figure 7).
In the AI, MI and PI numbers of Bif. and
Composition of bacteria Lac in the Orego-Stim® group significantly
flora - Aerobic Bacteria increased compared to that of the control
Bacterial numbers are shown in tables (P<0.05). Numbers of Bact. significantly
5-7. decreased in the Orego-Stim® group com-
Intestinal aerobic bacteria of 4 families pared to that of control (P<0.05).
(genera) of channel catfish were identi-
fied. The intestinal predominant aerobic Discussions and conclusion
bacteria were Enterobacteriaceae Ent.）, As there is evidence that intestinal com-
Aeromonas (Aer.), Bacillus (Bac.) and Vibrio petitive bacterial exclusion and bacterial
(Vib.) (Figure 5-6). antagonism are the unique modes of action
In AI, the numbers of Enterobacteriaceae that help the immune response of fish, it is
in fish fed Orego-Stim® decreased sig- pertinent to understand how Orego-Stim®
nificantly compared to control (P<0.05). can beneficially balance the gut flora in
34 | InternatIonal AquAFeed | July-august 2010
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.
Dry with 20-50% less energy
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 Geelen Counterflow / T +31-475-592315
Technology Park MK43 0BT, United Geelen Counterflow USA Inc. / T +1-772-559-4338
Kingdom Geelen Counterflow América Latina / T +54-9-2362-418899
E info@geelencounterflow.com / I www.geelencounterflow.com
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