The dietaryThe dietaryPROBIOTIC to PROBIOTIC to

secure the health secure the health of of shrimpsshrimps

• In hatcheries, Bactocell is particularly indicated to:– Maintain a healthy microbial environment – Improve larvae quality (reduce deformities) and

development (facilitate transition from Zoe to Mysis)– Improve resistance of larvae to opportunistic pathogens &

stressSuppress use of antimicrobial compounds

� A solution for high larval production

BACTOCELL applications in BACTOCELL applications in HatcheriesHatcheries

BACTOCELL® 100

The The productproduct

Concentrated formula PA100 for hatcheries(100 billion CFU/g (10x1010 CFU/g))

-Standard dosage:- Larval tanks: 1g/m3 of water- Live preys: 1 to 5g/m3 of culture water

The strain: Pediococcus acidilactici MA18/5M

Yeast MouldBacilles GRAM+

sporulated

Bacilles GRAM+

no-sporulated

Bacillus LactobacillaceaeEnterococcus

FungiBacteria

Pediococcus

MICROORGANISM

Coques GRAM+

no-sporulated

Pediococcus and other probiotics microorganisms classification

Pediococcus acidilacticiPediococcus acidilacticiMA 18/5MMA 18/5M

The The strainstrain

The The strainstrain

BACTOCELL ADMINISTRATION

Live Prey

Microparticules (transition feeds)

Nursery feeds

Larval feeds

Enrichments

Grow out conditions of use:

100-200g/kg PA10

1-5g.m3of the PA

100

Water

1g.m3 of the PA

100

5-10g.m3 Direct mixing: 10-20g/kg of

PA 100

The The productproduct

Larval tank Nursery tanks

TA

NK

SF

EE

DIN

G

Preparation dose Bactocell 100 at 0.1g.m-3

Preventive dose Bactocell 100 added directly in tank at 1g.m-3 every day

Tank filling PL5Zoe Mysis

Microencapsulated diet

Bactocell PA10 mix with feed: 1g.kg-1

Complete feedBactocell PA10 mix with feed: 2g.kg-1

A0

Artemia hatching tanks: Bactocell 100 at 5g.m-3

Feeding programs for shrimps - hatcheryBACTOCELL applications in AquacultureBACTOCELL applications in Aquaculture

Bactocell is active in seawater

1,0E+02

1,0E+03

1,0E+04

1,0E+05

1,0E+06

S11 S17 S22 S27

Total Marine Microflora (CFU/g)

Vibrio Flora (CFU/g)

Bactocell PA100 (CFU/g)

In sea water tanks Bactocell is active :- It ranged 10e4-10e5 CFU/ml seawater- Vibrio level are below the detection threshold

* : belowdetectionthreshold2.10e3 CFU/g

* * * *

Population in Water tanks (CFU /ml)

The The productproduct

Bactocell is active in Enriched Live preys

• Bactocell concentration in artemia enriched during 14 hours (Gatesoupe, 2002)

• Bactocell concentration in rotifers:– Use in the culture� (Marine

hatchery)– Use during enrichment only

(2 hours)� Bactocell reach 10e4-10e5

CFU/ml (IFREMER, 2010)

The The productproduct

Bactocell reaches concentration between 10e4-10e5 CF U/ml

6.8 E+037.6 E+06Day 2

1.4 E+042.1 E+03Day 1

2.5 E+051.2 E+04Day 0

Rotifers (CFU/ml)Culture water (CFU/ml)

Day of culture

6.8 E+037.6 E+06Day 2

1.4 E+042.1 E+03Day 1

2.5 E+051.2 E+04Day 0

Rotifers (CFU/ml)Culture water (CFU/ml)

Day of culture

Bactocell administration to larvae through level preys is efficient

The The productproduct

Bactocell reaches concentration between 10e4-10e5 CFU/g of gut

1,0E+02

1,0E+03

1,0E+04

1,0E+05

1,0E+06

1,0E+07

S11 S17 Gut

Total Marine Microflora (CFU/g)

Vibrio Flora (CFU/g)

Bactocell PA100 (CFU/g)

Microbial population in the gut

Bactocell count in the gut

Direct administration to larvae through feed or water is also anefficient way to deliver Bactocell in hatcheries

The The productproduct

Bactocell reaches again concentration between 10e4-1 0e5 CFU/g of gut

Stomach Digestive gland Gut

Bac

teria

l con

cent

ratio

n (C

FU

.g-1

)

1e+2

1e+3

1e+4

1e+5

1e+6

1e+7

1e+8

1e+9

1e+10

1e+11P. acidilacticiFlore totaleVibrio

Stomach Digestive gland Gut

P. a

cidi

lact

ici

(CF

U.o

rgan

-1)

1e+2

1e+3

1e+4

1e+5

*

IFREMER, 2009

Administration through the feed

Direct administration to larvae through feed or water is also anefficient way to deliver Bactocell in hatcheries

The The productproduct

Bactocell reaches again concentration between 10e4-1 0e5 CFU/g of gut

Villamil et al., 2010

Administration through water

Protocol for Artemia Enrichment• Protocol defined based on the work conducted with the IFREMER

(Gatesoupe, 2002)

– Bactocell solution preparation • Weigh the right amount of Bactocell in a bucket• Add the sea water, natural salinity (no chlorinated water) • Stir for 2-3 minutes with the enrichment product • Check the consistency of the mixture. • Distribute directly the solution at the water inlet tofavor dilution in the tank

– Enrichment step:• Cysts are decapsulated and kept 24 to hatch, • we obtain AO (nauplii) which are then rinsed 20min. • Enrichment:

– 300 A0 / ml– Salinity natural – 14 to 24 hours enrichment – Recommended dose: the objective is to work 5.10exp 5 CFU / ml of enrichment medium for

300 A0 / ml – Dosage = 5g Bactocell per m3 of enrichment media (In the case of bad quality culture In the case of bad quality culture

BactocellBactocell can be increase up to 10g/mcan be increase up to 10g/m 33))• Then follow the standard enrichment final procedure and distribution protocol

The The productproduct

The The strainstrain

BACTOCELL EFFECTS

Pediococcus acidilactici MA18/5M prevents the colonization and development of pathogenic bacteria and

positively influence microbial communities

The The strainstrain

Prevention of pathogenic bacteria development (Vibrio, pseudomonas,…)

- Acidification of pathogens interior- Metabolism perturbations

Bactocell

Lactic acid

Pathogens

Pathogens growth

XpH

• Antagonism against Vibrio:– in vitro

– In vivo

Ex: Live feed afterenrichment with Bactocell:Decraese in Vibrio countaby 10 to 100 times

ExperimentalExperimental studiesstudies

Number of active live preys/ non mobile ones

Bactocell cycles� direct benefits

• Antagonism against Vibrio:– In vivoEx: Live feed culture with Bactocell: Decrease in Vibrio count

by 10 to 100 times

ExperimentalExperimental studiesstudies

Vib

rio (

logC

FU

.g-1

)

1e+7

1e+8

BactocellControl

Before feeding After feeding

Vib

rio (

logC

FU

.g-1

)

1e+7

1e+8

BactocellControl

Before feeding After feeding

P<0,05

ExperimentalExperimental studiesstudies

• Antagonism against Vibrio:– In vivo

Ex: Decrease in Vibrio concentration in the gut of the shrimps after feeding with Bactocell

Bactocell group

L.stylirostris, IFREMERP. Monodon

Aquac. Sc. Inst. CHINA

LOCATION : DAC-IFREMER New Caledonia/ 2005.

OBJECTIVE : Bactocell evaluation as antibiotic replacement in larval production

EXPERIMENTATION

Administration mode : directly into the waterInclusion rate : 3 doses: 107 CFU/100l (L1), 2.107/100l CFU/g (L2) and 3.107 CFU/ 100l (L3)Duration: 10 daysRearing system : 500L tanks and cages in pondDensity: Feeding frequency:

ANIMAL

Specie : Larvae of Litopenaeus stylirostrisStade of development: from mysis to nauplii

(Pham D., 2005)

ExperimentalExperimental studiesstudies

0%

20%

40%

60%

80%

100%

120%

E L1 L2 L3 T-Treatment

Sur

viva

l (%

)

J0 J4 J6 J9

• Effects on survival:– A significant dose effect of Bactocell– Bactocell at 3.107 CFU/100l lead to the same results

than antibiotic treatment (E: erythromycine, T- : control)

ExperimentalExperimental studiesstudies

• Effect on larvae developement:– Bactocell doses between 2.107 CFU/100l and 3.107

CFU/100l allows to enhance the proportion of mysisstage compared to the control.

Proportion of the various larval stage for the diff erent treatment after 9 days

0%

20%

40%

60%

80%

100%

120%

140%

160%

T- L1 L2 L3 E

Treatment

Pro

porti

on %

ZoéIII Mysis I Mysis II Mysis III

ExperimentalExperimental studiesstudies

LOCATION : Aquaculture Science Institute Shengzhen DapengTrial Center, China, 2003

OBJECTIVE: Bactocell evaluation on intensive breading type experiments

EXPERIMENTATION

Administration mode : via the feedInclusion rate : 106 CFU/g of feedDuration: 7 weeksRearing system : 2,5-m³ tanks Density: 2500 PL 15/m³

ANIMALS

Specie : Penaeus monodonStade of development : PL 15

ExperimentalExperimental studiesstudies

50

60

70

80

90

100

110

CONTROL BACTOCELL

% S

urvi

val

CONTROL

BACTOCELL

94,6

73,3

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

1,1

Indi

vidu

al W

eigh

t (g)

CONTROL

BACTOCELL

0,4

0,8

• Effects on survival– Increases survival by

29% (P<0,05) after 7 weeks.

• Effects on growthparameters– Significant increase in

weight by 50%(P<0,05)

– Non significant increases in length by 16% (P>0,05)

ExperimentalExperimental studiesstudies