Development of Zn and Mn enrichment pmethod in live feed and nutritional importance in marine fish larvaeimportance in marine fish larvae

ByyNguyen Van Tien 1,2, Seiji Matsumoto2, Yutaka Haga3, Tomonari Kotani 4, Hiroshi 

4 3Fushimi 4, Shuichi Satoh 3

1 Research Institute for Aquaculture No 1, Viet Nam2 Scientific Feed Laboratory Co. Ltd., Japan

3T k U i i f M i S i d T h l J3Tokyo University of Marine Science and Technology, Japan4 Fukuyama University, Japan

IntroductionGrowth and survival of hatchery‐raised finfish are affected by the nutritional valuefinfish are affected by the nutritional value of live feeds such as rotifers and Artemia.Manipulation of nutrient profiles of liveManipulation of nutrient profiles of live feeds has been improved growth, survival and quality of hatchery‐raised fingerlingsand quality of hatchery raised fingerlings (example: enrichment of live feeds with HUFAs, vitamin A, taurine….), , )So far, little effort has been directed toward the enrichment of live foods with mineralsthe enrichment of live foods with minerals

Zn and Mn are important minerals for growth and normal skeletal developmentgrowth and normal skeletal development

Stimulates bone mineralizationStimulates bone mineralization and bone protein synthesis

Deficiency causes dwarfism andZn

Deficiency causes dwarfism and disturbance of skeletal formation

Constitutes of enzymes and involves in carbohydrate, lipid and protein metabolismM protein metabolism

Deficiency causes impaired growth and abnormal skeleton

Mn

growth and abnormal skeleton

Vertebral deformity in hatchery‐raised red sea bream Hattori et al 2003

Normal

red sea bream Hattori et al., 2003

Normal

Deformed

8% 18% 7%

NormalDeformedOthers91% 81% 93% Others93%

1999 2000 2001

ObjectivesObjectivesDevelop new method to enrich Zn andDevelop new method to enrich Zn and Mn in rotifers and Artemia nauplii to the level of these minerals in naturalthe level of these minerals in natural zooplanktons.

i i h ff f dTo investigate the effects of Zn and Mnsupplementation in Artemia on early growth, body compositions and skeletal deformity of red sea bream larvaey

Experiment 1: Examination direct absorption of Zn by rotifers

C tif di tl b Z fCan rotifer directly absorp Zn from enrichment media? 

Experimental conditions• Zinc source: ZnSO4 (0, 5, 10 ppb)

• Organism: rotifer (Brachionus plicatilis) (L size)Organism: rotifer (Brachionus plicatilis) (L size)• Stocking Density:

rotifer: 500ind/mlrotifer: 500ind/ml

chlorella: 3×106 cell/mL

• Sampling Rotifer Culture media (water) Chlorella• Sampling: Rotifer, Culture media (water), Chlorella

at 0 h,12 hrs, 24 hrs

T k 30 L d h d k• Tank: 30 L round‐shaped tank

• Water temperature: 27• Replication: Duplicate

Fig 1. Zn accumulation in the rotifer (DM)

Enrichment duration (hrs)

The rotifers could not absorb and retain zinc inThe rotifers could not absorb and retain zinc in their body from enrichment media effectively 

Zinc concentration in Chlorella 

Enrichment duration (hrs)

Chlorella could absorb water‐bound zinc moreChlorella could absorb water bound zinc more effective than the rotifer

It is possible to enrich Chlorella with zinc as a first step of enrichment Thezinc as a first step of enrichment. The  Chlorella thus produced contained high levels of zinc and can be fed to rotifers.rotifers.

Zn

ZnZn

ZnZn

Zn

Zn

Zn

Experiment 2: Zn absorption by Chlorella

Aims

To examine the zinc accumulationTo examine the zinc accumulation ability of the ChlorellaT d i h i bl d iTo determine the suitable duration for Chlorella enrichment with Zn

3h6h

24h12h

1h3h

0 4 8 160mg 4mg 8mg 16mg

Experimental conditionsExperimental conditions

• Zinc source: ZnSO (0 4 8 and 16 mg/10• Zinc source: ZnSO4 (0, 4, 8 and 16 mg/10 g of condensed Chlorella regularis

• Sampling: 0h, 1h, 3h, 6h, 12h, 24h

• Replication: duplicateReplication: duplicate

• Temperature: 27oC

Zn accumulation in the Chlorella (DM)

Enrichment duration (hrs)

Chlorella could absorb water bound zinc effectivelyChlorella could absorb water‐bound zinc effectivelyand quickly within the first 1 h of enrichment.

Experiment 3: Enrichment of rotifer h h d hl llwith Zn‐enriched Chlorella 

• Objective:Objective:– To investigate incorporation ability of Zn in rotifer by feeding with Zn enriched Chlorellay g

• Experimental conditionsCondensed freshwater Chlorella vulgaris– Condensed freshwater Chlorella vulgaris

– Supplemented with 0.0; 0.8 and 1.6 mg Zn g−1

– Incubation duration was 12 hours at 25oCIncubation duration was 12 hours at 25 C– Organism: rotifer (Brachionus plicatilis) (L size); 300 ind/mL/

– Chlorella density 1.8x106 cell/mL

Zn content in enriched rotiferZn content in enriched rotifer

Summary experiment 3Summary experiment 3• Zn in rotifer was successfully improved byenrichment with Chlorella that pre‐accumulated with Zn.

• Zn content in the rotifer was elevated with Znsupplementation in the Chlorella.

• The Zn content in the 1.6 mg Zn group after 24 h enrichment was 640.3 μg g−1, almosth enrichment was 640.3 μg g , almost equivalent to Zn content in natural Acartiaclausi which contains 700 μg g− 1 zinc (DM)clausi which contains 700 μg g zinc (DM)

Exp 4: Enrichment Artemiawith  Zn and Mnp

Objectives• To determine the incubation duration of

Marine ω A® with Zn and Mn

• To investigate incorporation ability of Zn and Mn in ArtemiaZn and Mn in Artemia

MaterialsMarine ω A ®: A commercial enrichment material made byenrichment material made by microalgae that had been removed th ll ll (Ni hi M i T hthe cell-wall (Nisshin Marine Tech Co., Ltd., Japan)Artemia: Newly-hatched naupliiArtemia franciscana (E G gradeArtemia franciscana (E.G. grade, INVE, Belgium) Mi l Z d MMinerals: Zn and Mn

Methods• Incubation of marine ω A®.

Zn levels: 0, 1.6, 3.2 mg/10g marine ω A®, , g gMn levels: 30 and 60 µg/g marine ω A®

Incubation duration: 0 1 3 and 6 hoursIncubation duration: 0, 1, 3 and 6 hours• Artemia enrichment

Z l l 0 0 4 0 8 d 1 6 / iZn levels: 0, 0.4, 0.8 and 1.6 mg/g marine ω A®

Mn levels: 0 12 0 24 mg/g marine ω A®Mn levels: 0.12, 0.24 mg/g marine ω A®

Incubation duration: 2 hoursEnrichment duration: 24 hours

Incorporation of Zn and Mn in Marine ω®

6000

7000 0 1.6 3.2

120

140 Mn 30 Mn 60

4000

5000

6000

(µ/g

) 80

100

120

(µg/

g)

1000

2000

3000Zn (

20

40

60

Mn

(

0

1000

Initial 1h 3h 6hEnrichment duration (h)

0Initial 1 3 6

Enrichment duration (h)

Incubation 1-2 hrs was enough for

Enrichment duration (h) Enrichment duration (h)

Marine ω A® to incorporate Zn and Mn

Zn and Mn content in Artemiaat 24 hrs enrichment (DW)at 24 hrs enrichment (DW)

900 Mn 0.12 Mn 0.24 17

18 Mn 0.12Mn 0.24

500600700800

g g-1

)

13141516

g g-1

)

Mn 0.24

200300400500

Zn (µ

g

89

101112

Mn

(µg

0 0.4 0.8 1.6Zn level (mg g-1)

0100

0 0.4 0.8 1.6Zn level (mg g-1)

678

Zn level (mg g 1)

Enrichment Artemia with Marine ω A®

l t d ith Z d M ld

Zn level (mg g )

supplemented with Zn and Mn could increase Zn and Mn contents in the nauplii

Nutritional important of Zn andNutritional important of Zn and Mn in marine fish larvae

Experiment 5: Effect of Zn and MnExperiment 5: Effect of Zn and Mnenrichment in Artemia on growthand vertebral deformity in red seaand vertebral deformity in red seabream larvae

ObjectiveTo investigate the effects of Zn and Mnsupplementation in Artemia on growth, pp g ,body compositions and skeletal deformity of red sea bream larvaeof red sea bream larvae

Feeding scheme & experimental conditionsRotifer  L strain 5 ind mL‐1 (twice daily)

Artemia (thrice daily)

Artificial diet (once / 2 h)

Culture duration (day)0 5 10 15 20 25 30

Tank volume: 1000 L, triplicatesDensity: 20 000 larvae per tankDensity: 20,000 larvae per tankWater ToC: 20-23 oCPhotoperiod: 12 h dark : 12 h lightPhotoperiod: 12 h dark : 12 h lightCulture duration: 30 days

Artemia enrichment with Zn (0 1 mg/g) and Mn (2 4 mg/g)

Marine ω A®

incubation

with Zn (0.1 mg/g) and Mn (2.4 mg/g)

incubation 2 hours Control Z M ZM

Artemia enrichment

150 nauplii/mL26 oC4 g marine ω®/L

Harvesting 24, 29, 33 hours

Feeding to larvae

Zn and Mn contents in enriched Artemia (dry matter basis)Artemia (dry-matter basis)

Zn Mn

350400450

354045

200250300350

(µg

g-1 )

20253035

(µg

g-1 )

50100150200

Zn

5101520

Mn

050

Control Z M ZM

I iti l 24 h 29 h 33 h

05

Control Z M ZM

I iti l 24 h 29 h 33 hInitial 24 hours 29 hours 33 hours Initial 24 hours 29 hours 33 hours

Growth of red sea bream at 30 dph

19,5 20,0

abb

ab

18,018,5 19,0

mm

) a

17,0 17,5 18,0

leng

th (m

15 516,0 16,5

Tota

l

Z M ZMControl ZControl MZControl

15,0 15,5

Control Z M ZMZ M ZMControl ZControl MZControl

Survival of red sea bream during 15-30 dph

90

g p

607080

%)

a a a a

405060

urvi

val (

%

102030Su

010

Control Z M ZM

Zn and Mn content in whole body of red sea bream at 30 dph (dry-matter basis)

Mn90 Zn

red sea bream at 30 dph (dry-matter basis)

ab ab b

789

µg g

‐1)b

ab

85

90

g g‐

1 )

Zn

456

conten

t (µ

a a

ab

75

80

ontent (µ

g

123

Mnc

65

70

Zn co

01

Control  Z M ZM

60

Control  Z M ZM

Typical vertebral deformities in red sea bream at 30 dphp

1 2 3

Dentary Pugheadness Vertebral fusion

Lordosis

4 5 6

Kyphosis

Rib deformity 4 5 6

Caudal vertebrate Fin support

Rib deformity

Average percentage of fish with at least 1 skeletal deformity at 30 dph (%)skeletal deformity at 30 dph (%)

60 b

40

50

60

ity (%

)

aa a

b

30

40

defo

rmi

10

20

Skel

etal

0Control Z M ZM

Treatment

Percentage of major skeletal deformities of red sea bream at 30 dph (n=600)red sea bream at 30 dph (n=600)

35

40Control Z

25

30

35

%)

Control Z

M ZM

15

20

25

cent

age

(

5

10

15

Perc

0Jaw Vertebral

lNeural

iHemal

iDorsal fin

column spine spine ray

Occurrence of vertebral deformity in red sea bream at 30 dph (n=600)

18

%)

p ( )

121416

form

ity (%

68

10

tebr

al d

ef

0246

Vert

0

1 2 3 4 5 6 7 8 910 11 12 13 14 15 16 17 18 9 0 1 2

Abdominal vertebrae ZMZ 

MControl 

1 1 1 20 21 22 23

Vertebral numberCaudal vertebrae

Summary experiment 5• Mn supplementation in Artemia 12-42.8 µg g-1

(dry-matter) improves growth performance of (d y atte ) p o es g o t pe o a ce othe red sea bream larvae

• Zn and Mn supplementation in Artemial k l l d l f dpromotes normal skeletal development of red

sea bream larvae • Zn and Mn contents in whole body of red sea• Zn and Mn contents in whole body of red sea bream were affected by Zn and Mn content in Artemia

• Zn and Mn should be supplemented together to prevent decline of Mn in enriched Artemia

Conclusion• The Zn concentration in rotifers was

significantly increased indirectly by feedingsignificantly increased indirectly by feedingzinc-enriched Chlorella for 6 to 12 h.

• Zn and Mn contents in Artemia weresignificantly improved indirectly by feedingZn and Mn enriched marine ω A®

• Supplementation of Zn and Mn in Artemiaimproves growth performance and promotes

l k l t l d l t f dnormal skeletal development of red seabream larvae

Further readingsFurther readings