COMMERCIAL CULTURE OF ASIAN SEA BASS Lates calcarifer

Grant No.: 2002-33610-12403 (X-03361012403-02)

EXECUTIVE SUMMARY

The overall purpose of the USDA/SBIR Phase II research and development effort for the project
entitled, Commercial Culture of Asian Sea Bass (Lates calcarifer), was to remove all further
technical impediments to the profitable commercial culture of Asian sea bass in Hawaii. The
Phase II research plan was designed to investigate the major questions remaining on Asian sea
bass culture in Hawaii to enable Hawaii Fish Company (HFC)’s Phase III commercialization to
proceed and succeed. Imported larvae were used to accomplish the Phase II objectives and will
be used for the initiation of Phase III commercialization.

The major accomplishments of HFC’s Phase II research and development efforts were:

• Successfully tested and determined larval rearing and nursery methodologies suitable for
commercial Asian sea bass production in Hawaii, with nursery survival as high as 100%
and the weaning of fry onto commercially available dry feeds.
• Successfully tested and determined tank and cage culture growout methodologies suitable
for commercial Asian sea bass production in Hawaii, rearing fingerlings to both plate-
and fillet-sizes of 400-600 g and 1–3 kg, respectively, and achieving growth rates of
greater than 1.2 g/day and tank and cage biomass levels of greater than 18 kg/m3.
• Successfully test-marketed Asian sea bass at a fine dining restaurant in Hawaii, supplying
them fresh-chilled fillet-sized fish on a weekly basis and achieving a high level of chef
and customer enthusiasm and satisfaction.

The ultimate goal of this Phase II effort was for Hawaii Fish Company (HFC) to obtain the
technical information necessary to establish a successful commercial Asian sea bass production
capability in Hawaii. The overall objectives of the project have therefore been met, and the
activities undertaken under the auspices of the project form the basis of this final report.
 COMMERCIAL CULTURE OF ASIAN SEA BASS Lates calcarifer

FINAL PROJECT REPORT

Grant No.: 2002-33610-12403 (X-03361012403-02)

Prepared By: Ronald P. Weidenbach

Hawaii Fish Company
P.O. Box 1039
Waialua, HI 96791-1039
hawaiifish@gmail.com

Clyde S. Tamaru, Ph.D.

University of Hawaii Sea Grant Extension Service
2525 Correa Road, HIG 205
Honolulu, HI 96822
ctamaru@hawai.edu

Submission Date: July 1, 2007

Submitted Electronically To: sbir@csrees.usda.gov

INTRODUCTION

Hawaii Fish Company (HFC) was awarded a U. S. Department of Agriculture Small Business
Innovative Research (USDA/SBIR) Phase II grant effective 09/01/2002 through 08/31/2004.
The project was subsequently extended by three no-cost extensions of time through 08/31/2007.
The overall goal of the project was to remove all further technical impediments to the profitable
commercial culture of Asian sea bass in Hawaii. Asian sea bass are also known and marketed as
barramundi, the Australian Aboriginal name for this highly esteemed food and sport fish.

The Phase I feasibility research effort focused on innovative rotifer culture methods and early
larval nutrition and temperature effects on larvae from day 1 to day 15 post-hatch (PH). The
results of the Phase I research clearly demonstrated the feasibility of rearing rotifers and early
Asian sea bass larvae in a small inland hatchery in Hawaii, and provided the research
information and initial fish stocks necessary for HFC to plan and conduct the Phase II research
and development effort.

The purpose of the Phase II research and development effort was broken down into several
objectives which were:
• to refine air transport and early larval-rearing methodologies
• to test and determine the methodologies necessary and appropriate for advanced larval
rearing (day 16 to day 60 PH)
• develop nursery methods
• on site testing of various grow-out conditions

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 • develop post-harvest handling of Asian sea bass appropriate for commercial production
in Hawaii
• to conduct an economic analysis of the costs of producing this new aquaculture product
for the United States seafood market
• to test and determine, if possible, broodstock maturation and spawning methodologies
appropriate for use in Hawaii.

Together, the Phase I and Phase II research objectives and results are intended to provide HFC
the information necessary to establish a successful commercial Asian sea bass production
capability in Hawaii to produce a new premium food fish for the domestic seafood market, that
will be implemented in Phase III by non-SBIR funding.

PHASE II RESEARCH AND DEVELOPMENT

Phase II Overview

HFC reviewed the research and extension literature on the biology and culture of Asian sea bass
in Southeast Asia and Australia, and assessed the results of the Phase I feasibility research efforts
as a basis for developing the Phase II research and development plan. The plan was designed to
investigate the major questions remaining on Asian sea bass culture in Hawaii to enable Phase III
commercialization to proceed and succeed. Imported larvae from Australia and Southeast Asia
were used to accomplish all Phase II objectives and will be used for the initiation of Phase III
commercialization.

Phase II Technical Objectives

The specific technical objectives proposed for the Phase II research and development efforts on
Asian sea bass were:
1. To determine the best and/or most economical shipping methodologies for international
air shipments;
2. To determine the best and/or most economical saltwater/seawater source for optimum
larval survival and growth at an inland hatchery site;
3. To determine the best and/or most practical Artemia nauplii HUFA enrichment medium
(media) for feeding larval Asian sea bass;
4. To determine the best and/or most economical larval feeding methodology by comparing
growth, survival, stress resistance, and essential and fatty acid profiles of larvae;
5. To determine the best and/or most economical dry feed and weaning strategy;
6. To determine the best and/or most economical management method to control
cannibalism;
7. To determine the best and/or most economical nursery and grow-out methodologies;
8. To determine the best methods of post-harvest handling of live and fresh Asian sea bass;
9. To determine the production economics of each stage of the production cycle (hatchery,
nursery, grow-out) and the profitability of marketing live and fresh, chilled Asian sea
bass;

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 10. To determine market acceptance and price sensitivity of live plate- or fillet/cutlet-size
fish and fresh chilled fish of the same sizes killed and chilled by the best method(s)
tested; and
11. To determine, if possible, the best and/or most economical maturation diets and spawning
methods by comparing fresh and dry feeds, and by comparing environmental
manipulation (day length, water temperature, salinity) and hormone-induced maturation
and induction of spawning.

The progress towards each of these objectives during the time period September 1, 2002 to June
30, 2007, future prospects, and a brief summary of unanticipated research delays and requests for
no-cost extensions of time forms the basis of this final report. Given the many challenges HFC
faced from the start of the Phase I grant to the beginning of this Phase II grant, the Principal
Investigator decided to focus initially on the Phase II research efforts outlined in Objectives 7, 8,
and 10, as these would lead most directly to the ultimate goal of product commercialization, and
to address the remaining objectives during the final year of the grant.

HFC’s Phase II research effort was

initiated by taking the day 15 PH
larvae produced during the Phase I
trials and rearing these through the
cannibalistic nursery phase prior to the
start of the Phase II research period.
The resultant fingerlings were then
raised on through two successive 10-
tank (1.1 m3) grow-out trials. In the
first grow-out trial, the advanced
nursery fish were reared to the
previously targeted ‘plate-size’ of 400-
600 grams and above, over a period of
approximately 24 months. In the
second follow-on trial, the fish were
reared for approximately 24 additional
months to a larger fillet-size of 1.0-2.0
kg. and above, as recommended by Figure 1. Aerial photograph of HFC quarry facility
equipped with net pens. Source: Google Earth.
Foresight Science & Technologies,
Inc. in their Commercialization
Assessment Report USDA For Hawaii Fish Company (HFC) On Commercial Culture of Asian
Sea Bass (Lates calcarifer), April 4, 2003. The fish produced by the second trial above are
currently being held in their grow-out tanks on a maintenance diet for ongoing post harvest
handling and market research, and as potential broodstock for future maturation and spawning
trials. A second set of fish were reared in floating cages in HFC’s 3.3 ha. fish pond, Figure 1, to
evaluate this form of grow-out, and a third set of 135 fish were “free-ranged” on natural foods in
the same pond to evaluate the potential of an additional means of obtaining broodstock-sized fish
for future in-house maturation and breeding efforts.

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PROGRESS TOWARDS OBJECTIVES

Objective 1: To determine the best and/or most economical shipping methodologies for
international air shipments.

Progress Towards Objectives: HFC’s initial four shipments of Asian sea bass larvae were of
day 1 PH larvae shipped from Queensland, Australia and Singapore in plastic bags placed in
cardboard boxes or fiberglass coolers at a density of 25,000 to 40,000 larvae per 10 liter bag.
The landed cost per larvae was relatively low, being approximately US$0.01 each, but survival
upon arrival and/or to 12-24 hours after arrival was very low to non-existent (range 0 - 12.5%)
making this larvae supply option economically unattractive.

HFC subsequently obtained a shipment of day 15 PH larvae from Australia at a density of 5,000
larvae per 10 liter bag. Survival was high (e.g., 100%) but the landed cost of the larvae was also
quite high, being approximately US$0.30 each for the 7-9 mm larvae. In addition, this age group
is also known to have just reached the beginning their highly cannibalistic advanced larval phase
during which substantial losses are typically expected. This situation was subsequently
complicated by the actions of one or more large Australian commercial barramundi producers
that successfully negotiated exclusive long-term contracts with Australia’s major barramundi
hatcheries to support their national and international expansion efforts while in the process
blocking HFC and others from having access to most sources of Australian barramundi larvae.

Future Prospects: HFC has recently located an experienced source of day 15 PH Asian sea bass
larvae in Southeast Asia that can ship larvae to Honolulu at a density of 2,500-5,000 larvae per
10-liter bag, at a landed cost of US0.05 – 0.10 each for 10-15 mm larvae. Multiple shipments
from this source are expected to validate this supply option and packing density as the best
source and most economical shipping age and density for importing Asian sea bass larvae to
Hawaii. HFC’s initial commercial operations will be established using these imported day 15
PH larvae. Development of an in-house hatchery production capability will be pursued at a later
date when existing fish stocks reach reproductive size and age.

Objective 2: To determine the best and/or most economical saltwater/seawater source for
optimum larval survival and growth at an inland hatchery site.

Progress Towards Objectives: HFC previously had five options for obtaining seawater for its
inland hatchery site: 1) transporting pumped seawater from the nearby ocean and disinfecting it
with 12% sodium hypochlorite, and dechlorinating with aeration and/or sodium thiosulphate, that
was used for the successful Phase I larval rearing trials; 2) transporting “disease- and parasite-
free” pumped salt well water from the State of Hawaii, Anuenue Fisheries Research Center,
approximately 35 miles away; 3) reconstituting salt water made from Morton kiln-dried solar salt
and “disease- and parasite-free” fresh well water that was used during Phase I rotifer rearing
activities (see Figure 2); 4) reconstituting salt water made from Morton kiln-dried solar salt and
“disease- and parasite-free” fresh well water with the addition of a synthetic salt additive for
sodium chloride (ProLine Super Salt Concentrate), that was also used for successful Phase I
larval rearing; and 5) making artificial seawater from artificial sea salt, eg., Instant Ocean,
Crystal Sea, and “disease- and parasite-free”fresh well water. Each option was time consuming

5
and relatively costly, given the volume of water required during a hatchery larval rearing cycle,
and there were also concerns about the suitability of the least costly option (reconstituting salt
water made from Morton kiln-dried solar salt and fresh well water) relative to its suitability for
sustaining delicate early stage day 1-15 PH larvae.

Figure 2. Making of artificial seawater during Phase I SBIR activities.

Fortunately, a sixth option has since developed whereby a new seawater well has been developed
four miles away, and to which HFC has unlimited access. Additionally, the State of Hawaii,
Department of Land and Natural Resources has indicated initial approval of HFC’s request to
lease a contiguous coastal State property and to have a saltwater well drilled on the lower portion
of the property to provide a source of seawater from a well. This seventh option is anticipated to
be available within the next few years.

Future Prospects: The nearby seawater well and/or HFC’s plans for its own future sea water
well will be the best and/or most economical saltwater/seawater source for optimum larval
survival and growth at HFC’s inland hatchery site.

Objective 3: To determine the best and/or most practical Artemia nauplii HUFA enrichment
medium (media) for feeding larval Asian sea bass.

Progress Towards Objectives: Larvae fed diets deficient in highly unsaturated fatty acids
(HUFAs), particularly 20:5n-3, or EPA, become pale and, when stressed, swim erratically and

6
‘faint’, after which they either recover (presumably temporarily) or die (Dhert et al., 1990;
Rimmer and Reed, 1994; Rimmer and Russell, 1998). The best and/or most practical Artemia
nauplii HUFA enrichment medium (media) employed to date at HFC for feeding larval Asian sea
bass has been Artemia nauplii enriched with 80% Nannochloropsis oculata paste and 20% tilapia
green water fed twice daily. This is in part an off-shoot of using N. oculata purchased from Reed
Mariculture to culture the brackish water rotifer Brachionus rotuniformis and also the freshwater
cladoceran Moina macrocopa, Figure 3, which is to be discussed in the next section. The one
algal paste product would thus serve to enrich all of the live food organisms (e.g., rotifers,
Artemia, and Moina, Figure 3) used in the culture of Asian seabass at HFC. The resulting fatty
acid profiles of Moina grown and/or enriched with various products are summarized in Table 1.

Figure 3. Various live feeds being investigated for use in rearing Asian sea bass larvae.

Table 1. Fatty acid profiles of Moina fed or enriched. Values are in terms of mg/100mg dry
weight.
Fatty Acid Artemia nauplii Moina Fed Yeast Moina Fed Moina Enriched
Greenwater with DOCOSA
Gold (300 ppm)
14:0 0.06 0.08 0.23 1.47
16:0 0.82 0.59 1.95 2.77
16:1n7 0.32 0.83 0.79 1.35
18:0 0.53 0.31 0.36 0.32
18:1n9 1.52 1.33 1.16 0.85
18:2n6 0.49 0.30 0.98 0.55
18:3n3 1.94 0.26 1.10 1.22
18:4n3 0.28 nd 0.10 0.32
20:1n9 0.05 0.01 nd 0.02
20:4n6 0.17 0.21 0.15 0.40
20:5n3 0.36 0.53 0.64 1.12
22:6n3 nd nd nd 4.47
Total 6.55 4.46 7.46 14.90

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From the results obtained it can be seen that the cladoceran already possesses a relatively good
content of EPA (C20:5n3), the essential fatty acid that is important for Asian sea bass larvae.
However, Moina can be enriched and their fatty acid profiles can be manipulated to meet the
needs of the seabass larvae and or fry. This observation coupled with initial culture trials of
Moina indicate that this live food organism will be an important part of the hatchery technologies
for producing Asian seabass larvae for stocking into the nursery phase of the culture process.

Figure 4 Estralita Weidenbach with harvested Moina from a single culture tank using a simple batch culture
system.

The activities revolving around culturing Moina were undertaken because it has been reported
that freshwater cladocerans Daphnia and Moina have been used to supplement or replace
Artemia nauplii as foods for intensively-reared Asian seabass larvae (Tattanon and
Tiensongrusmee, 1984; NICA, 1986; Parazo et al., 1990; Fermin, 1991). Moina may be fed
starting from day 15 PH, although they are more often used as an Artemia nauplii substitute from
day 25 PH. Moina are fed at least four times/day at densities of 1 individual/ml or greater.
Salinity must be reduced below 10 ppt for cladocerans to survive (Parazo et al., 1990; Fermin,
1991).

Moina densities that were achieved in a simple static culture system consisting of four 40 gallon
Rubbermaid™ barrels (Figure 4) during the reporting period were 10 individuals/ml which was
similar to those reported by Tamaru et. al., (2004). When the culture system is managed as a
batch culture where one half of the working volume is harvested each day approximately 2.25 x
106 individuals per day are available for live feed. This output would be able to support 2.25 m3
of larval culture which is more than adequate to handle a single shipment of 5,000 day 15 PH
larvae. Likewise, the system has the advantage of being easily scaled up by simply adding
additional culture tanks since the algal source of feed is commercially available in a bottle. From
the activities conducted to date, the project work group feels confident in its ability to produce
sufficient live feeds to meet the quantitative and qualitative requirements of day 15 PH Asian sea
bass larvae that are to be imported for future commercialization.

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Future Prospects: It has been well documented that Asian sea bass larvae require a diet high in
highly unsaturated fatty acids (HUFAs), in particular, diets high in C20:5n3, or EPA. In culture,
this is most often accomplished by providing HUFA/EPA-rich feeds to the live feed organisms,
eg. to rotifers or Artemia nauplii, or by HUFA/EPA-boosting of the live feed organism just prior
to feeding the larvae. The results obtained during the Phase I and Phase II research activities
have been consistent with these findings and feeding protocols. Because of the current
availability of imported day 15 PH larvae, HFC’s Phase II research activities have focused on
Day 15 PH and older larvae, testing the performance of variously enriched live feeds in
comparison to formulated diets that comprises Objective 4 below.

Objective 4: To determine the best and/or most economical larval feeding methodology by
comparing growth, survival, stress resistance, and essential fatty acid profiles of larvae.

Progress Towards Objectives: Utilizing the new Southeast Asian source of Asian seabass fry,
a shipment of day 15 PH larvae were obtained and an experiment comparing the effectiveness of
various enrichment protocols and a commercially available pellet was conducted between June
18 – June 28, 2007. The experiment was conducted in 85-liter plastic tanks (with a working
volume of 70 L) and stocked with 50 larvae per tank, resulting in a stocking density of 0.7
larvae/liter. A total of five treatments were
used with each treatment having five replicates.
In addition, a 6th treatment with only two
replicates was added due to the availability of
additional fish. The commercially available
products tested were enrichment media
Algamac 2000 (Aquafauna Biomarine,
Hawthorne California), Instant Algae,
Nannochloropsis oculata (Reed Mariculture,
California) (Figure 5) and Golden Pearls
(Artemia International LLC, Texas). Algamac
2000 and Instant Algae were used as
enrichment media for the Artemia nauplii and
Golden Pearls is a revolutionary new larval diet
that is currently experiencing great success in
marine fish hatcheries in Europe. Tilapia green
water (TGW) was produced as described during Figure 5. Enrichment of Artemia with Algamac
the Phase I activities. 2000 (left) and TGW/N. oculata paste (right)

The treatments used on the day 15 PH larvae were:

1. Algamac enriched Artemia

2. Algamac enriched Artemia + Golden Pearls
3. Golden Pearls only
4. Tilapia Greenwater (TGW) + N. oculata enriched Artemia
5. TGW/N. oculata/Algamac/ enriched Artemia + Golden Pearls
6. TGW/N. oculata enriched Artemia + Golden Pearls

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The experiment was conducted over the course of 10 days after which the body length and
survival was obtained from each tank. In addition, a stress test (which consisted of suspending a
subsample of 10 larvae in a net in air for 30 seconds) was also conducted for each tank. No
significant differences were observed in water temperature measurements (average 26.5oC ±
0.2), and DO levels which were above 4 mg/l for all treatments.

Larval survival was high for all treatments with no statistical differences being detected amongst
the various treatments. The lowest percent survival was 95%, which was observed in one
replicate tank in treatment 5. Likewise, there were no differences observed in stress resistance
with all treatments exhibiting 100% survival one hour following the 30-second stress test. There
were, however, differences in growth that were statistically significant and are summarized in
Figure 6.

Figure 6. Summary of resulting growth in response to various enrichment and feed combinations for day 15
PH Asian seabass larvae. Bars that do not share an alphabet are statistically different (P<0.05).

Although the larvae that were fed Golden Pearls exclusively were found to be the smallest at the
end of the experiment, survival was 100% in all replicate tanks. The results clearly indicate that
although this commercial product originally designed for shrimp culture may not be ideal when
used alone with Asian seabass larvae, it certainly can be used in combination with the Artemia
enriched with the various methods used in the current investigation. Treatment 5, which

10
consisted of enriched Artemia nauplii using Algamac 2000, N. oculata paste, TGW and
supplemented with Golden Pearls, resulted in the highest average size of all of the treatments
examined. Overall, the current results are consistent with the those obtained during Phase I
activities with day 1-15 PH larvae where enrichment media high in the essential fatty acid EPA
(C20:5n3) was shown to be the best for hatchery operations with early Asian seabass larvae. In
the current experiment, Artemia enriched with N. oculata paste (Instant Algae) were consistently
larger than larvae that were not enriched with this media, with the exception of Treatment 1
Algamac 2000, which provided similar results. However, Algamac 2000 is also high in EPA in
comparison to other enrichment media (e.g., Algamac 3050, DOCOSA Gold, Super Selco).

Future Prospects: The results obtained to date will form the basis of future hatchery design and
operations at HFC with regard to Asian sea bass larvae. These results document the benefits of
properly enriching live food organisms that are to be used in Asian sea bass hatchery operations,
although the use of the commercial pellet, Golden Pearls, needs to be examined further as to its
utility as a diet to wean Asian sea bass larvae off of live foods. Nevertheless, the investigators
feel confident that a hatchery protocol for rearing Asian seabass larvae under the conditions in
Hawaii has been defined and can be used at commercial scale.

Objective 5: To determine the best and/or most economical dry feed and weaning strategy.

Progress Towards Objectives: Larvae were maintained in 85-liter early nursery tanks for
approximately 60 days, to approximately Day 75 PH, reaching a size range of 5-10 cm TL. The
best and/or most economical dry feed and weaning strategy determined to date has been to
initially supplement the enriched Artemia nauplii diet with Encapsulon #3 microencapsulated
pellets, sold by Argent Laboratories, Redmond Washington. This was accomplished at each
feeding and was soon followed by transitioning the larvae/juveniles to enriched Moina and
sequentially to Rangen #1 and #2 Trout and Salmon Starter. During this time period, the salinity
was reduced to freshwater in order to
accommodate the use of Moina and also provide
for adequate tank flushing with the introduction of
commercially prepared diets. This process was
then followed with the introduction to Rangen
1/16” Extruded 450 Floating Trout pellets and
was done according to the increasing size of the
fry/juveniles. The fry/juveniles were fed at least
twice daily to satiation. Because there was only
one trial made during the project with this size
class, additional testing will be conducted to
validate the observed results. One of the
surprising findings during the initial reporting
period was that the fry readily adapted to
commercial diets Figure 7 and this also indicates
a high probability of expanding the culture
process.
Figure 7. Asian seabass fry being fed
commercially available pellet.

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Future Prospects: Validate the results obtained to date and in addition, test alternative
commercially available diets to define the most cost effective weaning strategy.

Objective 6: To determine the best and/or most economical management method to control
cannibalism

Progress Towards Objectives: The larvae were maintained in the 85-liter early nursery tanks
for approximately 60 days (e.g., 75 days PH) reaching a size range of 5-10 cm TL. Salinity at
the beginning of the trial was at 15 ppt and was decreased to full freshwater over the course of
the initial 4-5 days. All nursery tanks were receiving a continuous flow of freshwater over the
course of the nursery phase. During the early nursery period, the larvae were graded every 3-5
days by passing the larvae through submerged stainless steel sieves of varying mesh sizes,
according to observations of size differential and the first evidence of cannibalism. Observed
shooters were removed daily by dip net and transferred to separate tanks of equivalent sized
fishes. The daily movement of “shooters” (fast-growing fish) and the twice weekly grading and
movement of fish to similar-sized cohorts precluded record keeping on individual tanks. Growth
was noticeably slower and the early nursery period longer than reported for Southeast Asia and
Australia (e.g., typically to Day 45 PH), due most likely to decreasing water temperatures with
increasing water flows (e.g., 22-24 OC). In addition, the conservative feeding rates utilized by
HFC in order to help maintain favorable water quality also contributed to a slower than
anticipated rate of growth. Average survival was approx. 65% with a production of
approximately 4,350 juveniles. Periodic grading continued on a weekly to bi-weekly basis
during the advanced nursery stage with submerged sieves and by hand, as indicated by disparate
growth rates within tanks, until no visible cannibalism was observed.

Future Prospects: The results obtained during the time period prior to the Phase II start date are
being repeated with additional relevant trials to insure that the initial results are valid. This is
important especially with the anticipated shift in source of larvae to 15 day PH larvae.

Objective 7: To determine the best and/or most economical nursery and grow-out
methodologies.

Progress Towards Objectives: On April 5, 2002, approximately 3,000 5-10 cm TL juveniles

were stocked into ten 1,140-liter polytanks (900 liter working volume each, Figure 8) at 300
juveniles per tank (i.e., 1 juvenile per 3 liters of water). The average body weight of and
individual juvenile stocked was 5.6 grams. This activity was initiated before the beginning of
the Phase II award period but took advantage of the products already available from the Phase I
award and targeted the advanced nursery and grow-out research objectives/trials. Five of the
1,140-liter polytanks were randomly selected and covered with translucent polyethylene covers
and fitted with 1,000-watt thermostatically-controlled submersible water heaters, to help
maintain elevated water temperatures, and five were covered with ¼” square mesh bird netting,
to determine growth under ambient water temperatures.

All juveniles were fed Rangen Extruded 450 Floating Trout pellets to satiation twice daily,
starting with 1/16” pellets and increasing according to fish growth to 1/8”, 3/16”, and ¼” pellets.
Initially, periodic grading continued on a weekly to bi-weekly basis, as indicated by disparate

12
Figure 8. Photographs of tanks used in advanced nursery and growout trials at HFC.

growth rates within tanks. This was done until no visible cannibalism was observed. Water flow
was increased as necessary to maintain favorable water quality as all tanks were being provided
with commercially prepared diets. However, it became clear that increasing water exchange
rates over time also minimized the differential temperature benefit of using the submersible
heaters. This minimal benefit initially, if any, combined with the increasingly frequent power
supply problems and high electrical costs at our temporary airfield research hatchery, and the
onset of warm summer air temperatures, led to the decision to terminate the use of the heaters.
However, the poly covers were maintained in place throughout the duration of the advanced
nursery and grow-out trials to see if any resulting temperature differential produced any
discernible growth benefit. In short, all tanks were found to have similar water temperatures
(e.g., approximately 21oC in winter, 24 OC in summer) and apparently no benefit was observed to
having the plastic covers.

At the start of the formal Phase II grant period on September 1, 2002, the 10-tank advanced
nursery/grow-out trial had been underway for approximately 5 months and the cannibalistic
nursery period had passed. Survival in the polytanks to this point in time was estimated at
approximately 75%, resulting in an average of 225 fish remaining per tank. However, over time,
the increasing Asian sea bass biomass in the tanks combined with periodic power failures
resulted in periodic partial or total fish kills in the research tanks. By March 7, 2003,
approximately 11 months after initial stocking, two tanks of research fish had been entirely lost
and partial losses had occurred in the other tanks.

On July 8, 2003, another several-hour power outage resulted in the combined loss of 175
additional fish from three of the remaining tanks. These fish were weighed and measured to
determine average fish size to this point in time. Average body weight for all fish was 0.41 ±
0.10 kg body weight and the biomass in the tanks was estimated to range between 81 – 106
kg/m3 which is very high for tank culture and provided some indication of the flushing rates
needed to maintain water quality, eg., open ocean cage culture trials with the Pacific threadfin
support between 13 – 20 kg/m3 and is thought to represent unlimited rate of flushing (Helsley
2001). This date was used as a starting point for conducting a continuing growout trial using the
same tanks. As of this date, the fish had been in culture for 458 days with a resulting average

13
body weight of 0.41 kg. This equates to an estimated rate of growth of 0.9 grams/day which as
mentioned before is very low for this species and a reflection of the on site culture conditions
that were employed.

Between August 2 and August 12, 2004, all of the fish in the ten grow-out tanks were counted
and individually weighed and measured to determine growth and survival. Percent survival
ranged between 6.7% – 28.3% for each tank for the duration of this growout period. The results
were statistically analyzed and a summary of the growth observed is provided in Figure 9. The
average body weight of 0.41 kg from above was used as the initial starting size for the fish used
in this grow-out trial. There was some variability detected in the average resulting body weight
for three of the tanks (e.g., Tank 3L, Tank 4L and Tank 5R) as these were significantly larger
than those observed for the other tanks. Overall, the resulting growth rates (calculated at 1.2
g/day) and survival was less than reported in the literature for Australia and Southeast Asia.
However, the results are consistent with the sub-optimal 21-25oC tank water temperatures
resulting from the high water exchange rates with the research hatchery’s 21oC well water. As
mentioned previously, a high flushing rate was necessary to maintain favorable tank water
quality as the resulting biomass in the tanks ranged between 23 – 64 kg/m3. As also mentioned,
these values are relatively high in comparison with other culture technologies and species. The
results obtained were therefore considered very encouraging in that the fish had reached or
exceeded the target “plate size” of 400-600 grams as commonly sold in Australia and Southeast
Asia.

Figure 9. Summary of average body weight observed from each of the growout tanks used in the
trial.

14
The statistically significant differences in average body weights observed for the three tanks is a
reflection of having a lower number of individuals present due to a lower percent survival
observed for these three tanks. A summary of the relationship of observed survival and average
body weight from the August, 12, 2004 results is presented in Figure 10. The three tanks with
the lowest percent survival also resulted with fish with the highest average body weight.

Figure 10. Relationship between observed survival and average body weight observed from the
August 12, 2004 sampling of fish. The value Y = average body weight and X = % survival.

The biomass within each of the ten tanks was then equalized by adding and subtracting fish as
necessary to achieve an average biomass of 49.8 ± 12.4 kg/m3, and a second grow-out trial was
begun to try and raise the surviving fish under the same culture conditions on to a larger fillet
product size of 1-3kg. The subject fish were again fed ¼” Rangen Extruded 450 Floating Trout
pellets to satiation twice daily. After 24 months, all the fish in each of the ten tanks were again
counted, weighed, and measured and the results of average body weights obtained per tank is
also summarized in Figure 9. As with the previous grow-out period, the same tanks were found
to produce significantly larger average size fishes and again due most likely to the lower number
of individuals that were present in the tanks. Average body weights of over 1 kg were achieved,
with the exception of Tank 1L. Overall rate of growth was again slower than that reported in the
literature, and the culture conditions in the tanks used, i.e., cool water temperatures, clearly
contributed to the poor performance. As summarized in Figure 11, the condition factor (Body
Weight/Body Length), which is a measure of robustness used in fishery science, had already
peaked after the first grow-out period. Continued use of the tanks after this point was apparently

15
Figure 11. Summary of changes in condition factor in tank growout trials.

counterproductive, relative to condition factor, but was necessitated by being the only culture
tanks permitted by the landlord at the time. Nevertheless, while the overall rate of growth was
again slower than that reported in the literature, it was encouraging to know that this species can
adapt to being raised under high densities and under the conditions found on site. It should also
be pointed out that these tank-reared fishes are now forming the basis for the marketing success
that is being experienced under the auspices of this project. A photograph of one of the
harvested individuals is provided in Figure 12.

A cage culture grow-out trial was initiated on 9/17/03, stocking four 3M3 floating cages with 75
Asian sea bass juveniles each in HFC’s 3.3 ha pond. A fifth cage was stocked on 12/4/03 with
another 75 fish. During January 2004, a several day period of low dissolved oxygen levels in the
pond resulting from extended windless overcast weather resulted in substantial losses of fish in
these cages. As it was apparent that all of the remaining fish would likely die with another night
of cage confinement, the decision was made to release the 135 surviving fish from the cages into
the pond to facilitate their chances for survival. The length-weight data from the dead cage
cultured fish was recorded to determine Asian sea bass growth capabilities under cage culture
conditions in Hawaii and is summarized in Table 2.

The performance of the fish in the cages are the most encouraging of the grow-out trials since
survival, up to the point of the inclimate weather conditions, and observed growth were very
good and consistent with the results reported in Southeast Asia and Australia. At the time of the
incident, a total of 105 days had elapsed under cage culture conditions. The rate of growth of the
fishes in the cages was calculated at 7.2 grams/day as compared to that observed in the tanks,
which was calculated at 1.2 g/day. Resulting biomass in the cages are equivalent and exceed

16
Figure 12. Photograph of Asian sea bass individual from the tank culture system and being
measured on August 24, 2006.

results mentioned for open ocean cage culture and underscore the characteristics that make the
Asian sea bass extremely attractive for commercial production.

Table 2. Summary of performance of Asian sea bass grown in cages in HFC’s quarry pond.
Cage Number Average BW Average BW Survival Biomass
(kg) (kg) January 1, (%) (kg/m3)
July 8, 2003 2004
1 0.40 ± 0.12 0.94 ± 0.24 100.0 23.4
2 0.40 ± 0.12 0.98 ± 0.18 100.0 24.4
3 0.40 ± 0.12 0.93 ± 0.19 81.3 18.9
4 0.40 ± 0.12 1.07 ± 0.25 100.0 26.9
5 0.40 ± 0.12 1.04 ± 0.28 94.7 24.7

The released “free ranging” fish were intended to provide a future indication of growth potential
of Asian sea bass in HFC’s pond (Figure 1) for their possible future use as hatchery broodstock.
However, during the week of December 6, 2006, another extended period of windless overcast
weather resulted in a major fish kill in HFC’s pond and cages, resulting in the loss of the free-
ranging potential broodfish (Figure 13). A subsample of twenty-six of the fish were recovered,
weighed, measured and examined internally for evidence of gonad development. The average

17
Figure 13. Photographs of Asian sea bass losses sustained on December 6, 2006 at HFC's quarry
pond.
body weight and length of the individuals recovered were 9.7 ± 1.1 kg and 90.2 ± 2.7 cm,
respectively. Samples of gonadal tissue have been preserved and are being histologically
processed to assess if sexual maturation is being achieved. Asian sea bass are protandrous
hermaphrodites and the presence of spermatocytes or ultrastructures that resemble testicular
development are to be used as an indication that sexual maturity is being achieved in the quarry
pond. Although Asian sea bass broodstock need to migrate to or be conditioned in saltwater to
complete their sexual maturation, it is hoped that histological examination of the collected
“gonadal” tissues will indicate initial gonad development.

Irregardless of the losses, the growth by the “free-ranging” fish was excellent with many of the
collected or observed fish exceeding 10 kg in weight, providing optimism that future broodfish
can be obtained on-site to support future maturation and reproduction efforts. A comparison of
the rate of growth observed for the three culture processes that the Asian sea bass were exposed
to at HFC is summarized in Figure 14. Clearly, the use of HFC’s quarry pond will provide a
significant advantage in the grow-out of the Asian sea bass but only if the pond can be cost-
effectively aerated to circumvent the annual changes in weather conditions. Nonetheless, the
results obtained to date are extremely encouraging that commercial production of Asian sea bass
can be achieved at HFC.

Whenever possible, body measurements (length and weight) were made over the course of the
project. The data was used to construct a length weight relationship (Figure 15) that has utility in
that in can be used to convert body weight from body length and vice versa and is to be used in
future activities. The statistical model is: Y = (0.002 * X2) – (0.071 * X) + 0.968 where Y =
body weight in kilogram and X is body length in cm.

18
Figure 14. Summary of growth rates achieved in tank, cage and free ranging conditions over the
course of the project.

Figure 15. Length-weight relationship of Asian seabass at cultured at HFC.

19
Future Prospects: While the initial goal of the two successive tank grow-out experiments was
to compare fish growth rates between ambient and elevated water temperatures, the high water
exchange rates required to maintain favorable water quality conditions in these tanks with their
high resident biomasses, resulted in lowered the water temperatures in all tanks. Ultimately, the
lower water temperatures reduced fish growth rates and eliminated any differential water
temperatures and growth rates between the treatments. Nevertheless, the trials were very
successful in establishing achievable biomass levels for commercial Asian sea bass production
under Hawaii’s sub-tropical conditions. The results achieved clearly demonstrate the hardiness
of these fish under crowded production conditions, in cooler than anticipated waters. The cage-
culture and “free ranging” trials provided similar positive results, and the mortalities obtained
throughout all of these trials can and will be overcome as electrical service is provided and
emergency generators are established for the site to support commercial Phase III production.

Objective 8: To determine the best methods of post-harvest handling of live and fresh Asian sea
bass.

Progress Towards Objectives: HFC has identified three potential product forms and they are:

• live 400-600 gram plate-sized fish for the local Asian ethnic live seafood markets and
restaurants
• 400-600 gram plate-sized fish for the broader fresh chilled seafood market
• 1-3 kilogram fish for the even larger fillet market, initially targeting Hawaii’s high-end
white tablecloth restaurants.

However, by placing live fish on display in Hawaii’s live seafood markets and restaurants, HFC
would be alerting its potential competitors as to its new product line prior to being ready to meet
initial market demand, thereby encouraging early competition at a time when HFC is unprepared
to adequately respond. The same would be true with initial fresh chilled whole fish sales
whereby demand would be demonstrated to markets and wholesalers prior to HFC being able to
provide adequate product to meet demand, thereby encouraging markets and wholesalers to seek
out other international sources of this product. Such a process could undercut HFC’s planned
Phase III expansion and production. As such, HFC decided to test market its Asian sea bass or
barramundi at a high-end fine dining restaurant catering to out-of-State visitors where the
culinary characteristics of the HFC-produced barramundi could be evaluated, recipes could be
developed, and customer responses could be evaluated, with minimal risk of HFC’s competitors
being alerted.

HFC has begun test marketing the Asian sea bass produced at its farm site in collaboration with
Turtle Bay Resort’s (Figure 16) Twenty One Degrees North restaurant. Approximately six
months of sales consisting of 5 fish per week ranging between 1.0 – 1.3 kg in size has been
taking place under the auspices of the project and the results have exceeded expectations. An
initial version of Turtle Bay Resort’s promotional flyer that is being provided to restaurant
customers was included in the 2007 North Shore Restaurant Guide, is found in Appendix I.

Future Prospects: Future prospects for HFC’s Asian sea bass/barramundi look outstanding.
Turtle Bay Resorts on the North shore of Hawaii is ready to launch a promotional campaign

20
Figure 16. Photograph of Turtle Bay Resorts located on the North Shore of Oahu and site of
Twenty One Degrees North, the fine dining restaurant marketing BARRAMUNDI, “raised in
Mokuleia right here on the North Shore” (see promotional flyer Appendix I).

when HFC has sufficient supplies of Asian sea bass/barramundi on hand to meet their anticipated
customer demand. Turtle Bay Resorts have also offered to assist HFC in promoting the
company’s Asian sea bass to chefs of other high-end restaurants, including to the inter-island
cruise ships, so long as Turtle Bay Resorts is provided an exclusive supply arrangement for the
North Shore of Oahu.

Objective 9: To determine the production economics of each stage of the production cycle
(hatchery, nursery, growout) and the profitability of marketing live and fresh chilled.

Progress Towards Objectives: The production economics Asian sea bass culture in Hawaii
have not been determined as the production conditions and growth rates obtained during the
project have been sub-optimal, as explained throughout this report, and therefore have not been
representative of realistic commercial production costs and growth and production rates.
Nevertheless, given the glimpses of growth and production potential of this fish under Hawaii
conditions, the positive customer responses to date, the very favorable pricing currently being
obtained to date, and the apparent high demand for this a high-end fish product, the production
economics appear to be very favorable.

Future Prospects: The introductory price offered to HFC for its initial Asian sea bass sales are
equal or greater to the highest prices currently paid for other seafood products sold in Hawaii.
Although HFC’s initial production growth rates and survival were lower than typically obtained
in Australia and Southeast Asia for this fish, these problems relate primarily to HFC’s current
inability to recirculate warm waters to achieve optimum growth, and to its current non-existent
or unreliable electrical supply problems at the farm and temporary fish hatchery, respectively,
both of which will be addressed in HFC’s upcoming new production facilities. With these
adjustments in hand, future production costs will be competitive, and, with the anticipated high
dollar value of barramundi in the Hawaii marketplace and HFC’s high anticipated total

21
production and sales, HFC expects that our future Asian sea bass production efforts will be both
favorable and profitable.

Objective 10: To determine market acceptance and price sensitivity of live plate- or fillet/cutlet-
size fish and fresh, chilled fish of the same sizes killed and chilled by the best method(s) tested.

Progress Towards Objectives: HFC initiated weekly sales of fresh chilled 1.0-1.3 kg whole
Asian sea bass/barramundi in January 2007 to Turtle Bay Resort’s (Figure 16) Twenty-One
Degrees North fine dining restaurant on the North Shore of Oahu, at an introductory wholesale
farm-gate price of $17.60/kg ($8.00/lb). Under the present arrangements, the restaurant chef
picks-up the fish at HFC every Thursday afternoon for their weekend catch-of-the-day specials
that are conveyed to their customers verbally by their wait-staff so long as weekly supplies last.
Restaurant staff fillet HFC’s Asian sea bass/barramundi with the skin-on, obtaining an average of
four serving portions per fish, and are preparing the fish for a weekly gourmet seafood dish.
Customer response has been outstanding, with the weekends’ supply of fish typically selling-out
on Friday nights, at times in as little as two hours after the restaurant opens. Turtle Bay Resort’s
Executive Chef was formerly the Executive Chef at one of Sydney, Australia’s leading five star
hotels/restaurants, with Asian sea bass/barramundi being his former signature dish. His senior
chef has repeatedly said to HFC that the Company’s Asian sea bass/barramundi are now by far
Twenty-one Degrees’ number one seafood item, beating out Hawaii’s wild-caught deep-water
snapper, opakapaka, yellow-fin tuna (ahi), and swordfish, as well as farm raised salmon and
Kona kampachiTM.

Future Prospects: HFC anticipates that the current strong positive response will only grow as
increased supplies of HFC’s Asian sea bass are produced and made available for sales.

Objective 11: To determine, if possible, the best and/or most economical maturation diets and
spawning methods by comparing fresh and dry feeds, and by comparing environmental
manipulation (day length, water temperature, salinity) and hormone-induced maturation and
induction of spawning.

Progress Towards Objectives: As reported above, in early 2004 a several day period of low
dissolved oxygen levels in HFC’s pond resulted in substantial losses of fish in the research grow-
out cages. As it was apparent that all of the remaining fish would likely die with another night of
cage confinement under such low dissolved oxygen conditions, the decision was made to release
the 135 surviving fish from the cages into the pond to facilitate their chances for survival. The
released “free ranging” fish were intended to provide a future indication of growth potential of
Asian sea bass in HFC’s pond for their possible future use as hatchery broodstock.
Unfortunately, during the week of December 6, 2006, another extended period of windless
overcast weather resulted in a major fish kill, resulting in the loss of all of the free-ranging
potential broodfish, ending any hope of successful maturation and spawning of these fish during
the current Phase II grant. However, a subsample of twenty-six of the fish were recovered,
weighed, measured, and examined internally for evidence of gonadal development. Tissue
samples were initially frozen and have recently been undergoing preservation and histological
preparation in collaboration with UH Sea Grant College Program and the Hawaii Department of
Agriculture Aquaculture Development Program.

22
Future Prospects: Although Asian sea bass broodstock need to migrate to or be conditioned in
saltwater to complete their sexual maturation, it is hoped that the histological examination of the
collected “gonadal” tissues from the dead potential broodfish will indicate that initial gonad
development had begun. Irregardless of these results, the growth by these “free-ranging” fish
was excellent, with many of the collected or observed fish exceeding 10 kg. in weight, providing
optimism that future broodfish can be obtained on-site to support future maturation and
reproduction efforts.

A BRIEF SUMMARY OF UNANTICIPATED RESEARCH DELAYS AND REQUESTS

FOR NO-COST EXTENSIONS OF TIME

HFC’s Phase I Asian sea bass project experienced a series of research delays resulting from
unanticipated reviews and challenges of HFC’s aquaculture tenancy and research activities at
Dillingham Airfield by HFC’s landlord, the Hawaii Department of Transportation Airports
Division (DOTA). HFC was finally given approval to set-up its initial Phase II research tanks in
August 2002, just prior the start of the Phase II Asian seabass research effort, to begin the Phase
II grow-out trials.

Disappointingly, in May 2003, HFC was thanked for its many years of tenancy at the airfield and
notified that the airfield ownership was being transferred from the U.S. Army to the DOTA, and
that this required that HFC’s office, hatchery, and research facilities be vacated by October 14,
2003 to allow for their scheduled demolition. HFC’s airfield office, laboratory, workshop, and
feed storage buildings, and the research tanks located between these buildings were demolished
as planned in November 2003. Fortunately, the new DOTA Oahu manager agreed to allow HFC
to continue to conduct our Phase II research trials then in progress, in tanks unaffected by the
demolition efforts, while we endeavored to obtain permits and construct new facilities and
research tanks at the adjacent quarry property. These efforts have been underway throughout the
term of the Phase II grant, but the ongoing construction boom in Hawaii has tremendously
backlogged the County and State’s permitting processes as well as the scheduling of architectural
and construction work. HFC has moved forward on these permitting and construction efforts to
the best of our ability, but we still have quite a ways to go before all permits are in hand and our
new facilities are completed

Throughout the initial two years of the grant and during the first two no-cost extensions, HFC
continued to conduct grow-out trials comparing the effects of ambient and elevated water
temperatures on Asian sea bass growth rates. These trials established the biomass levels that
could be obtained using HFC’s initial tank size and water exchange rates, and also demonstrated
that Asian sea bass feed and grow on a year round basis in Hawaii’s ambient water temperatures.
HFC also conducted a cage culture trial and a free-ranging trial for potential future broodstock,
installed a new well and wastewater disposal system at our future hatchery and farm site, and
have continued with our engineering design efforts for the future aquafarm and minor
construction efforts while pushing slowly forward with our continuing land lease and permitting
challenges.

23
The two major technical constraints faced by HFC during the Phase II research efforts that
frequently slowed progress were the continuing operational challenges of working in temporary
facilities at the airfield, and periodic power outages at the airfield resulting in periodic fish
losses. Mr. Weidenbach’s time was also periodically impacted by his need to attend to
unexpected family problems back in Florida, following Hurricane Charlie, in which his mother
received severe head injuries and in which her independent living complex in Port Charlotte, FL
was severely damaged. Mr. Weidenbach’s mother was evacuated to Tampa General Hospital,
but while recovering from her head injuries, was diagnosed and operated on for breast cancer.
Then, the following January, while undergoing follow-up radiation therapy, she suffered a
massive heart attack and, after several weeks, passed on. However, the physical repairs to her
apartment complex took approximately one year to reach the point where the buildings were
deemed safe for Mr. Weidenbach to return and complete the sorting, removal, disposition and/or
storage of her apartment belongings. Probate matters continued on into early 2007.

Although these various matters slowed HFC’s research progress, the researchers were
determined to prevail in the subject research and commercialization efforts. To this end, the fish
produced by the second grow-out trial were held in their grow-out tanks on a maintenance diet
for post harvest handling and market research, described above, under the Third No-Cost
Extension, with a secondary focus of the final grant year being to refine larval rearing
methodologies, as this is where the highest losses of Asian sea bass occur during the production
cycle due to the high levels of cannibalism during these periods. Together, these efforts are
enabling HFC to meet all Phase II technical objectives and provide the technical information
necessary for HFC to actively and successfully purse Phase III commercialization.

LITERATURE CITED

Dhert, P., P. Lavens, M. Duray and P. Sorgeloos, 1990. Improved larval survival at
metamorphosis of Asian seabass (Lates calcarifer) using omega 3-HUFA-enriched live
food. Aquaculture, 90 (63-74).

NICA, 1986. Technical Manual for Seed Production of Seabass. National Institute of Coastal
Aquaculture, Songkhla, Thailand.

Fermin, A.C., 1991. Freshwater cladoceran Moina macrocopa (Strauss) as an alternative live
feed for rearing sea bass Lates calcarifer (Bloch) fry. Journal of Applied Ichthyology,
7: 8-14

Parazo, M.M., L.Ma.B Garcia, F.G. Ayson, A.C. Fermin, J.M.E. Almendras, D.M. Reyes, Jr. and
E.M. Avila, 1990. Sea Bass Hatchery Operations. SEAFDEC Extension Manual No. 18,
Iloilo City, Philippines, 38 pp.

Rimmer, M.A. and A. Reed, 1994. Effects of nutritional enhancement of live food organisms on
growth and survival of barramundi, Lates calcarifer (Bloch), larvae. Aquacult. Fish.
Manage., Vol. 25, No. 2, p. 143-156.

Rimmer, M.A. and D.J. Russell, 1998. Aspects of the Biology and Culture of Lates calcarifer.
In: Tropical Mariculture, Chapter 14, p. 449-475.

24
Tamaru, C.S., H. Ako, L. Asano, and K. McGovern-Hopkins. 2004. Growth of Moina macrocopa
(Straus) for use in freshwater ornamental fish culture. Aquatips, Regional Notes, Center for
Tropical and Subtropical Aquaculture. Vol. 15, No. 2, June 2004.

Tattanon, T. and B. Tiensongrusmee, 1984. Manual for Spawning of Seabass, Lates calcarifer,
in Captivity. Food and Agricultural Organization of the United Nations, Rome.

25
APPENDIX I. Turtle Bay Resort’s Twenty One Degrees North Promotional Flyer

26