Chong M. Lee 1, Peizhi Lian 1, David Bengtson 2, Nick King 3 and George Nardi 4

1. Dept. of Nutrition and Food Sciences2. Dept. of Fisheries, Animal and Veterinary Science University of Rhode Island3. Skretting, Vancouver, BC4. GreatBay Aquaculture, Portsmouth, New Hampshire

a

Background 36,000 MT of squid (Loligo pealei and Illex illecebrosus) landed in Northeast of U.S. annually 40-50% turns into processing byproduct or waste currently not being utilized Presently, the Northeast of U.S. (RI, NY and NJ) generates ~10 million lbs (4,540 MT) squid processing byproduct/yr.

Squid byproduct characteristics Consists of head, fin and viscera + unclaimed mantles and tentacles Approximately, 11% protein, 2% lipid (11.6% EPA; 24.5% DHA), 1.2% ash and 86% moisture Unique features - Use of endogenous enzymes for hydrolysis

- Believed to possess a protein fraction associated with “growth factor” (Meyers, 1989), and high level of chemo-attractant betaine and gowth promoting taurine

- Squid hydrolysate may offer a potential as a specialty feed ingredient e.g. starter diet

Commercial scale production of squid hydrolysate

330 gal capacity86% M

73-78% M Yield: 47-54%

Vibrating screen

In-line viscometer

Evaporator

Hydrolysis

Raw squid byproduct

hydrolysate

Proximate composition of squid hydrolysate (concentrated – 76% moisture)

Moisture Protein Lipid Ash

76 % 18.63 % 3.30 % 2.19 %

•Squid hydrolysate contains 11.16% EPA and 24.45% DHA, while salmon oil contains 8.65% EPA and 10.67% DHA (on an oil weight basis).

IngredientsAmount (% dry wt basis)

Squid hydrolysate 73.33

Salmon oil 9.54

Lecithin 3.01

Vitamin- premix 0.44

Mineral premix 2.01

Starch 5.02

Yeast 4.02

Algae (spirulina: chlorella) 2.64

Proximate composition

Protein 64.66

Lipid 18.72

Carbohydrate 7.40

Ash 9.21

Energy (MJ/Kg) 19.12

Composition of squid hydrolysate-based basal microdiet

Pilot plant production of microparticulate feed

Grinding

homogenizng drum drying

sieving

sieving

(DHA)

(EPA)

Fatty acid profiles of microdiets

*The 100 g basal squid hydrolysate diet will provide 2.00 g EPA and 3.60 g DHA based on EPA/DHA distribution (% fatty acid/oil).

Field feeding trial on Atlantic cod larvae: weaning procedure

0.25 million of cod larvae/ production tank (5 m3 ). One tank for squid hydrolysate(SH)-larval diet Six tanks (control group) for a premium commercial diet Cod larvae upon hatch were on

- initially rotifer

- the combination of rotifer and Artemia

- co-feeding of Artemia and microdiet

Weaning 1 week later with gradual removal of Artemia, the fish were kept on the SH microdiet for another 2 weeks.

(The trial was conducted at the GreatBay Aquaculture in Portsmouth, NH)

Survival

70-75% of the fish on the SH microdiet survived

through the weaning period, which is considered excellent (in the same range for the control group - the premium commercial diet)

Overall, there was no real difference in survival among the production tanks during weaning.

Swimming behavior

Different behavior between the fish fed SH diet and the rest.

The SH fish had a lighter color. A darker color is often associated with stress.

The SH fish were very responsive as a sign of good health being more uniform in size.This has very significant ramifications as it relates to

cannibalism and grading. The fish were swimming together in uniform

manner and appeared to be in motion more so than those in the control diet tanks.

Tank hygiene

The SH diet was rated better than the premium control diet.

The SH diet appeared to stay very stable in the water without leaching. Leaching tends to cause foam on the surface (which was a problem with the control diet).

0

20

40

60

80

100

CS

I 60

EL3 - Squid diet

EL4 - GM

* GM: the premium commercial diet** Salinity at 65 ppt for 60 min (CSI: cumulative stress index)

The number of dead larvae counted every 3 min. At the end of 60 min, the % cumulative mortality was used as a Cumulative Stress Index (CSI-60). Indication of "condition“ after treatments, stress resistance, and screening.

42-45DPH

15.9 +/-

0.37

16.3 +/-

0.54

18.3 +/-

0.41

16.8 +/-

0.72

16.6 +/-

0.56

16.7 +/-

0.38

17.5 +/-

0.65

Salinity stress test for cod larvae (48 dph)

Total length (mm)

Microdiets from SH with different hydrolysis time on growth performance of Atlantic cod larvae (50 dph*) in 42 days feeding

Test diets

IBL cm IBW g IBCF BW-g BL cm BCF Survival SGR %/d

SH2 1.76 ±0.14

0.040 ±0.010

0.72 ±0.07

0.526 ±

0.04 4.07 ±

0.08 0.77 ±

0.02 67.7 ±

27.4 5.99 ±

0.10 SH2-1 1.76 ±

0.14 0.040 ±0.010

0.72 ±0.07

0.458 ±0.02

3.90 ±0.06

0.788 ±0.02

65.0 ±8.7 5.85 ±

0.03 SH1 1.76 ±

0.14 0.040 ±0.010

0.72 ±0.07

0.508 ±0.05

4.02 ±0.09

0.78 ±0.04

62.9 ±12.0

6.19 ±

0.33

SH 2: 2 h hydrolysis at 55C, followed by 30 min at 75CSH 1: 1 h hydrolysisSH 2-1: fed 2 h hydrolysate diet for 21 d followed by 1 h diet for 21 d

IBL: initial body length; BW: body wt; BCF: body condition factor;SGR: specific growth rate* 30 dph and 20 d acclimation

SDS-PAGE profiles of squid by-product during hydrolysis

Molecular marker (kDa)

5.0

10.0

15.0

20.0

25.0

0 60 120 180 240 300

Time (min)

DH

(%

)

VISCOSITY

DEGREE OF HYDROLYSIS

Diets Survival rate (%) Weight (mg) Length (mm) SGR

Artemia 81 2 a 29.8 a 12.2 1.0 a 2.86 0.55 a

Commercial 65 4 b 21.5 4.0 b 11.1 0.8 a 1.39 0.16 b

Squid only 92 3 c 26.2 4.9 ab 11.7 0.8 a 2.23 0.25 ab

Surivial, weight, length and specific growth rate

of summer flounder larvae (Paralichthys dentatus) after 22-day

Artemia Commercial Squid Hydrolysate0

20

40

60

80

100

Survival Rate

Specific Growth Rate

Survival and growth of summer flounder larvae (2-wk old) after 22-day feeding

Diets

Su

rviv

al R

ate

(%

); S

GR

(1

-10

, %)

* SGR = [(ln Wf/Wi)/ days of feeding] 100

Additional studies to be considered

Feeding trials on additional species Hydrolysate from finfish-squid combination Squid hydrolysate for broodstock nutrition Feed performance test in plant protein-

based aquaculture feeds

Acknowledgements

The present study was supported by the U.S. Dept. of Agriculture,

the Univ. of Rhode Island, Agricultural Experiment Station and the

National Oceanic and Atmospheric Administration (NOAA). The

information reported in this presentation has been filed for an

invention disclosure (7108PCT; May 17, 2004).