This study evaluated the effects of supplementing krill oil in diets for Litopenaeus vannamei reared under high salinity conditions. The results showed that 1) shrimp fed diets supplemented with krill oil grew better and had improved feed efficiency compared to those fed soybean oil alone under high salinity, and 2) including krill oil at 25% above recommended levels led to the best growth performance. Supplementing krill oil in shrimp feeds can help counteract the detrimental impacts of elevated salinity on growth.

1. SUPPLEMENTATION OF
KRILL OIL IN THE
FEEDING OF
Litopenaeus vannamei
CAN COUTERACT THE
DETRIMENTAL
EFFECTS OF HIGH
SALINITY
Alberto J.P. Nunes1, Sigve
Nordrum, Otávio Serino Castro,
Marcelo V.C. Sá
LABOMAR*. Brazil
1E-mail: albertojpn@uol.com.br
WAS 2009 Meeting
*Part of Universidade Federal do Ceará Veracruz, Mexico
Shrimp Nutrition Special Session
September 26th, 2009
03:10 pm

2. Rationale
 In shrimp farms water salinity
can vary from less than 1‰ to
more than 50‰
 Salinity fluctuates mainly as a
response to season, pond
depth, water exchange rates,
pumping site Influence of pumping site to riverine water affects
 Ideal salinities for the rearing water sality
of L. vannamei is around 20‰
 Species can tolerate wide
range of salinities, but above
40‰, osmoregulatory ability
can be depreciated
 High water salinity leads to
increased feed intake, poor
FCR and slow growth
Grow-out pond in an hypersaline area with salt
accumulated on the bottom

3. Review Outside fatty acid
cell
 Liu et al. (2007) supplementing
vitamin E (dietary tocopheryl
acetate) at 600 and 1,000 mg/kg
of diet for L. vannamei
experienced an increase in
shrimp resistance to acute
salinity changes
 Hurtado et al. (2006) were able
to demonstrate that growth of L.
vannamei at high salinities was
enhanced when fed on HUFA- Inside cell
enriched diets
 Hurtado et al. (2007) observed The cell membrane is formed by lipid bi-layer.
that a higher proportion of HUFA Phospholipids are the major lipid group within
in gill membrane in shrimp fed the membrane and contain a range of fatty
the high-HUFA diet counteracted acids including eicosapentanoic acid (EPA).
the influence of salinity on water Source: AkerBiomarine ASA, Norway
content in gills during a long-term
salinity exposure

4. Objectives
1. To evaluate if
supplementation of Krill
oil improves the growth
performance of juveniles
of L. vannamei when
reared under hypersaline
water conditions
2. To determine optimum Krill oil (Qrill™, AkerBiomarine ASA,
inclusion levels of Krill oil Norway) produced from the Antarctic Krill
in diets for the Pacific (Euphausia superba)
white shrimp when
exposed to high salinity
rearing conditions

5. LABOMAR/UFC Pacoti River Estuary
Eusébio. BRAZIL
Indoor tanks Outdoor tanks
19/jan/2009

6. Rearing System
INDOOR
50 tanks
Clear water
500-L volume
0.57 m2 area
XXXX shrimp/m2

7. Formulas and experimental design
Experimental Design
DIETS Salinity EPA+DHA Req.
 Experimental diets:
FISH Both Satisfied*
• One diet containing a
combination of fish and KRILL Both Satisfied*
soybean oil (diet FISH) SOY Both Deficient
• One diet with Krill and soybean KRILL- HIGH 50% deficient
oil (diet KRILL) KRILL+ HIGH 25% above*
• One diet with soybean oil alone
*based on 80% of that required by Penaeus monodon
(diet SOY) (Glencross et al., 2002)
• Two diets containing low and
high inclusion levels of Krill oil
in combination with soybean oil FISH KRILL SOY FISH
(diets KRILL- and KRILL+,
respectively)
 Salinity conditions
• IDEAL (21 – 26‰) and HIGH KRILL SOY KRILL- KRILL+
(40 – 47‰) salinity conditions
• KRILL- and KRILL+ tested
under high water salinity alone

8. Tank Distribution
FA01 FB06 FC11 FD16 FE21 FF26 FG31 FH36 FI41 FJ46
FA02 FB07 FC12 FD17 FE22 FF27 FG32 FH37 FI42 FJ47
FA03 FB08 FC13 FD18 FE23 FF28 FG33 FH38 FI43 FJ48
FA04 FB09 FC14 FD19 FE24 FF29 FG34 FH39 FI44 FJ49
FA05 FB10 FC15 FD20 FE25 FF30 FG35 FH40 FI45 FJ50
IDEAL water salinity (20 - 26‰) HIGH water salinity (40 - 47‰)
FISH SOY KRILL KRILL- KRILL+
Six replicate tanks were assigned for each diet, except FISH and KRILL which
used a total of seven replicate tanks under IDEAL salinity conditions.
Allotment of feeds in rearing tanks followed a random block design

9. Formulas (g/kg)
INGREDIENT FISH KRILL SOY KRILL- KRILL+
Soybean meal, 46% CP 350.00 350.00 350.00 350.00 350.00
Protein Ingredients
Wheat flour 298.71 299.80 300.00 300.00 291.46
Poultry by-product meal 100.00 100.00 105.00 100.00 67.50
Fishmeal, Anchovy 60.17 71.58 70.55 75.62 68.90
Soybean protein concentrate 32.48 19.10 16.65 16.65 20.00
Corn gluten meal, 65% CP 0.00 0.00 0.00 0.00 40.00
DL-methionine crystalline, 99% 8.00 8.00 8.00 8.00 4.61
Fish oil 26.65 0.00 0.00 0.00 0.00
Lipid sources
Krill oil (QRILL™) 0.00 48.28 0.00 14.53 55.00
Soybean oil 10.00 4.45 34.49 21.21 3.82
Lecithin, soybean 15.00 0.00 15.00 15.00 0.00
Cholesterol (Solvay) 0.00 0.00 1.31 0.00 0.38
Rice, broken 40.00 40.00 40.00 40.00 40.00
Phosphate monodicalcium 13.00 13.00 13.00 13.00 13.00
Fixed portion
Potassium chloride (KCl) 10.00 10.00 10.00 10.00 10.00
Squid meal, whole 10.00 10.00 10.00 10.00 10.00
Salt common 10.00 10.00 10.00 10.00 10.00
Mineral-Vitamin Premix (DSM) 10.00 10.00 10.00 10.00 10.00
Synthetic binder (Bentoli) 4.00 4.00 4.00 4.00 4.00
Ascorbic acid polyphosphate (DSM) 2.00 1.80 2.00 2.00 1.34

10. Chemical Composition FISH SOY KRILL KRILL+ KRILL-
Nutritional Levels
Crude protein¹ 351.8 354.4 353.5 353.1 351.8
Crude fiber¹ 12.0 9.0 13.3 12.0 13.3
Ash¹ 93.7 89.5 91.0 82.3 91.0
Gross Energy (kcal/kg) 4,184 4,273 4,209 4,212 4,306
Inclusion of Lipid Sources
Fish oil1 26.6 0.0 0.0 0.0 0.0
Krill oil1,2 0.0 0.0 48.3 55 14.5
Soybean oil1 10.0 34.5 4.5 3.8 21.2
Cholesterol1,3 0.0 1.3 0.0 0.4 0.0
Soybean lecithin1 15.0 15.0 0.0 0.0 15.0
Ratio Marine:Plant Oil 2.7 0.0 11.0 14.4 0.4
Nutrient Levels
Total lipid content1 88.80 94.00 80.80 91.30 80.50
DHA (C22:6n-3)4 2.54 0.28 1.60 1.37 0.59
EPA (C20:5n-3)4 5.10 0.65 5.35 5.07 0.00
Ʃ HUFA4,5 7.64 0.93 6.94 6.44 0.59
LOA (C18:2n-6)4 28.32 44.71 16.21 17.97 33.66
LNA (C18:3n-3)4 3.40 4.91 1.52 2.03 3.94
Ʃ EFA4,6 39.36 50.54 24.68 26.44 38.19
Astaxanthin (ug/100 ul) 13.80 14.70 21.40 17.30 ---
1in g/kg of diet as wet basis.
2QRILL™ oil, Aker Biomarine ASA (Oslo, Norway).
3Cholesterol XG, Solvay Pharmaceuticals BV (Weesp, Netherlands).
4in % of total lipid content in the diet.
5sum of highly unsaturated fatty acids (DHA+EPA).
6sum of essential fatty acids (DHA+EPA+LOA+LNA).

11. Study Set-up
NURSERY
 PL12 reared in
nursery tanks of 3,000
PL Stocking Header tank L at 2.4 PL/L for 48
20,000 L
days when they
reached 0.65 ± 0.28 g
(n = 152)
Rearing in 3,000 L tanks
CONDITIONING PERIOD
Salt dilution  140 shrimp/m2 (80
shrimp/tank) and raised for 22
days for a conditioning period
to water salinity
 IDEAL: from 25 ± 0.9‰ (3.4%
CV) to 24 ± 0.4‰ (2.0% CV)
 HIGH: from 36 ± 0.8‰ (2.2%
Harvest CV) to 40 ± 0.4‰ (1.0% CV)
Stocking and acclimation

12. Data Collection
1. After 22 days of acclimation started on
experimental diets at 2.79 ± 0.60 g
2. Density reduced to 70 shrimp/m2 or 40
shrimp/tank
3. Fed twice daily in feeding trays at
0730 and 1600 h on a consumption
basis
4. Daily water analysis
• pH, temperature, salinity and
dissolved oxygen
5. 22-24 day interval – 10 shrimp/tank
were weighed
6. After 64 days shrimp were counted
and individually weighed
1. weekly growth rate (g/week)
2. Final body weight (g)
Feeding protocol used to adjust the amount of feed
3. final survival (%) delivered based estimated consumption from trays
4. yield (g/m 2) % Amount of Feed Ration Adjustment
Feed Left in Trays
5. food conversion ratio (FCR) < 10% No change in feed ration
> 10% Deliver 75% of original feed ration
No feed remains Increase 10% of original feed ration

13. 50 8.6
IDEAL water salinity tanks
Water Quality 8.4
Salinity (ppt) - Temperature (oC)
45 Salinity (‰)
Temperature (ƒC) 8.2
pH 8.0
 Trend towards increasing 40
7.8
salinity, from 22‰ to 25‰
pH
35 7.6
under IDEAL salinity tanks 7.4
30
7.2
and from 41‰ to 45‰ under
7.0
HIGH salinity tanks 25
6.8
 No differences between 20 6.6
treatments for pH, salinity Days of Rearing
and temperature 50
HIGH water salinity tanks
8.6
8.4
 Significant differences 45
Salinity (ppt) - Temperature (oC)
8.2
between HIGH and IDEAL 8.0
40
for water salinity Salinity (‰)
Temperature (ƒC)
7.8
pH
35 pH 7.6
7.4
30
7.2
Par. IDEAL HIGH 7.0
25
pH 7.27 ± 0.33 7.35 ± 0.21 6.8
20 6.6
Salin. 23 ± 1.2* 44 ± 2.0* 1 3 6 8 11 14 16 18 21 23 27 29 31 34 36 39 42 44 46 50 52 55 57 60 63
Days of Rearing
Temp. 27.3 ± 0.46 27.5 ± 0.46

14. Final Shrimp Survival (%)
P = 0.896
100.0% P = 0.720
96.3% 95.0% 95.0%
93.8% 94.2%
91.8% 92.5%
90.0%
90.0%
80.0%
70.0%
60.0%
FISH SOY KRILL FISH SOY KRILL KRILL - KRILL+
IDEALwater salinity HIGH water salinity
1. Chronic exposure to high salinity did not deteriorate shrimp survival
2. Increasing n-3 HUFA provided no additional benefit to shrimp survival

15. Shrimp Yield (g/m2)
Yield for KRILL 12-13%
higher compared to FISH,
SOY and KRILL-
650
P = 0.550 P = 0.370
598
600
579
569
555
550 536
533 531
529
500
450
400
FISH SOY KRILL FISH SOY KRILL KRILL- KRILL+
IDEALwater salinity HIGH water salinity
1. Final shrimp yield did not vary significantly among different diets regardless of
the salinity concentration

16. Shrimp Final Body Weight (g) at 1.45% KRILL- could not
counterbalance the effects of
KRILL able to promote a significantly higher shrimp high salinity
growth compared to FISH and SOY, regardless of salinity
No growth
12.50
improvements by
further increase in
12.03 KRILL+ at 5.50%
P < 0.0001 11.91
12.00
11.79
11.52 c
B B
11.50
11.12
b 10.96
10.86 10.88
11.00
a
A
A
A
10.50
P < 0.0001
10.00
FISH SOY KRILL FISH SOY KRILL KRILL- KRILL+
IDEAL water salinity HIGH water salinity
Factorial analyses:
both water salinity and diet type had a significant effect on shrimp body weight

17. Weekly Growth Rate (%)
10.0
8.33% WEIGHT GAIN DEPRESSION
AFTER SALINITY STRESS (%)
8.0
Y = 1.451X2 - 24.84X + 109.0
R² = 1.000
6.0
4.0 3.22%
2.94%
2.0
0.0
SOY FISH KRILL
As water salinity increased from IDEAL to HIGH there was a growth
depression, less significant with animals fed the KRILL diet

18. Conclusions
SALINITY
1. The higher the salinity, the more important was n-3 HUFA (DHA + EPA) to
boost shrimp growth
2. Under 21 – 26‰ shrimp did not appear to require diets with high n-3 HUFA
(DHA + EPA) levels as they performed well when fed a diet containing only a
vegetable oil source (i.e., soybean oil)
SOURCES
3. KRILL oil delivered an increased shrimp growth under both regular (21 –
26‰) and hypersaline (40 – 47‰) rearing conditions compared to the other
lipid sources tested
LEVELS
4. Under persistent hypersaline conditions, daily exposure to KRILL oil
appeared to be more important than an increased inclusion level
5. Under hypersaline water, n-3 HUFA, particularly DHA had the greatest
impact on shrimp growth. Best final body weight was achieved when
projected DHA achieved 1.6% of total lipid content
6. No further enhancement in growth was observed when DHA levels
exceeded this threshold for a salinity of 44 ± 2.0‰.