Preliminary spawning and larval culture trials for groupers from Guam and Palau :: The University of the South Pacific Library

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PACIFIC AQUACULTURE ASSOCIATION

PRELIMINARY SPAWNING AND

LARVAL CULTURE TRIALS FOR GROUPERS

FROM GUAM AND PALAU

FINAL REPORT

Prepared by

William J. FitzGerald, Jr., Mike Bauerlein and Clyde S. Tamaro*

January 1994

Guam Aquaculture Development and Training Center

Department of Commerce

Government of Guam

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TABLE OF CONTENTS

FINAL REPORT

SUMMARY OF FINDINGS

MEIBOD

· RESULTS

DISCUSSION OF RESULTS

CONCLUSION

Page

2

3

6

7

APPENDIXES

I

Collection I - March 1993

II

Collection II - May 1993

III Collection III - July 1993

IV Collection IV - August 1993

V Shipping Live Adult Epins::phelus _mic_ro_do_n Trial

VI Shipment of Adult Epinephelus fuscoguttatus to Guam

VII Aquarium Species of Groupers for Culture

VIII Observed Spawning Behavior of Epinephelus _mi_ cro_do_n in Captivity

'

IX GADTC Technical Report No. 16: Preliminary Spawning and Larval

Culture Trials for Groupers from Guam and Palau

*

Clyde S. Tamaru, Hawaii C's Aquaculture Consultant Services, 1157

Lunaapono Place, Kailua, Hawaii 96734

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PACIFIC AQUACULTURE ASSOCIATION

PROJECT REPORT

FINAL REPORT

PROJEdr TITLE

Preliminary Spawning and Larval Culture Trials of Groupers from Palau and

Guam

PERIOD COVERED BY THIS REPORT

July 1, 1992 thru December 3 1, 1993

GRANT PERIOD

July 1, 1992 thru December 3 1, 1993 (extended to June 1994)

PRINCIPAL INVESTIGATOR

William J. FitzGerald

Chief, Economic Development and Planning Division

Guam Department of Commerce

590 South Marine Drive

Tamuning, Guam 96911

Mike Bauerlein

Guam Aquaculture Development and Training Center

Guam Department of Commerce

PROJECT OBJECTIVES

The project consists of the following objecti\\(eS in the preliminary work on

groupers:

- To spawn selected species of groupers in Palau and Guam.

- To ship viable grouper eggs from Palau to Guam for larval culture.

- To conduct preliminary larval culture procedures adapting proven

methods for a selected species from Palau and Guam.

- To apply developed larval culture procedures to potential aquarium

species.

- To establish preliminary guidelines for developing grouper spawning

and larval culture in the region for an indigenous species.

PAA Grouper Year 2 Final Report

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ACKNOWLEDGEMENT

This project was funded by a grant from the Pacific Aquaculture

Development Program, Office of Territorial and International Affairs, U.S.

Department of Interior, GEN-52, which is administered by University of ·

Hawaii Sea Grant College Program (SOEST) under International Grant No.

NA89AA-D-SG063 from NOAA Office of Sea Grant, Department of

Commerce. The project was implemented as part of the Pacific Aquaculture

Association's (PAA) program which is to promote and assist the

development of aquaculture as a vehicle for economic and social

advancement in the Pacific islands with PAA membership.

Special thanks are extended to Clyde Tamaro for his assistance during the

project and to all of those individuals that contributed to the success of the

project. The assistance of Annie Orcutt of the Pacific Aquaculture

Development Program, especially in regard to expediting the necessary

aarssriasntagnecmeeonftsthfoerMCilcyrdoenTesaimanaMrua'snpcaurlttiucripeaDtieomn,oins sgtrreaatitolynaCpepnretecria(tMedM. DThCe),

Department of Natural Resources in providing facilities and staff support in

Palau is greatly appreciated. Special acknowledgement is made of the

contributions by Gerald Heslin�a, David Idip, Don Hanser, and Becky

Madraisau of MMDC. The assistance of Mr. John Gibbons, State Executive

Administrator of the Koror State Government facilitated the permitting of

the collection of groupers from the Ulong Channel reserve. The assistance

and support to the project provided by Nancy Wong in facilitating the process

through some of the bureaucratic steps in the implementation of the project

is greatly appreciated. The extraordinary fishing expertise of Thomas Taro

of Palau was instrumental in the success of the project.

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SUMMARY OF FINDINGS

Four species of groupers (Plectropornus areolatust Epinephelus fusco~ttatus, _E.

microdon, and ..,.C'e-£"-ph_ a!_..o..,..ph_o_ lis miniata) were utilized for the investigation into

the spawning and larval culture. Three species were targeted as food species

with the fourth, Cephalopholis miniatat targeted as an aquarium species.

Epinephelus _mic_ro_do_n was used as a model species for conducting the spawning,

development, hatching, and larval rearing work to obtain detailed

f£rmation. Similar work was carried out for _E. fuscoguttatus, but with less

detailed data collection.

Maturation of_E. microdon was induced with a two injection protocol (priming

and resolving) of HCG. The injection protocol was refined with injections at

p7r0o0d/u14ct0i0onIUof/kfegrftoilriztehdee1gsgtsapnedr2fnisdhinpjeercstpioanwsnrewsapsec1t.i1v3exly.10T6.heTahveelreanggeth x

weight relationship, size frequency distribution, sex composition by size, size at

sexual transition from female to male were identified. Characteristics of sperm

activation and extension was documented. Embryonic development was

Pdporaoclafuiumlete)onaGtnedudaopmphtoiwmteourgemraidpeehgngictdaifleilenyd.si.tCyIhn(aUitrJia?actlotfee2rei0sd,t0iinc0sg0otenf¥asgplssa/cw1)onfmeodpr aterrgaegndsscp(froayrtottapytraieocsniedrfvroedm

oyster trochophores, trochophores with rotifers, size selected rotifers and the

control of no feed, which resulted in no significant difference in treatments;

however, the results indicated an improved survival with oyster trochophores.

Intensive (20 larvae/I) and extensive (2 larvae/1) larval rearing was conducted

with larvae reaching day 6 and 15 for intensive and extensive respectively before

termination due to high mortality.

Transportation procedures for

transportation ofE. microdon

(a6d)ualnt dliv_Ce.gmroimupaetars(w44a)s

developed.

was carried

Successful

out to

establish initial broodstocks at GADTC.

METHOD

There were no collections· of groupers attempted in Guam, since the results of

the grouper resource survey in Guam at the end of the previous project indicated

an inadequate population for the reliable collection of spawners. Therefore, all

of the collection efforts were focused in Palau.

Spawning

The fish were caught by hand-line while bottom fishing at known aggregation

channels. Fish were maintained on-board the boat in live bait wells. They were

transported back to the Micronesian Mariculture Demonstration Center

hbCW(MelhaaloMddterdiiDrneorfnCtliawoc)nw.eGkrTaseonhadndeneadfafdileosauhrttnaredwotdiepoeurirnnwseiwen(tHnhgeterCaeninGnptdryr)uao.pcnveesirfdddeeesrdrprm.eadwiGcntronione2uge,p0dpe0lrer0os. catAehndadtutM4rhe,0aMs0duD0ati1CnlitziatnihnnfeklgasftHiaesthduMmwgaaMesnreDC.

The spawning procedure and results are provided in Appendix IX.

Pa1>e 2

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Shipping Eggs

Eg�s were shipped at a density of 5,000 eggs/I to Guam for hatching at GADTC.

Shipment density trials were carried out at MMDC to determine an optimum

density considermg egg viability and quantity shipped. The trials simulated the

shipping condition and duration. A range of 1,000 to 100,000 eggs/I was

considered. Appendix IX elaborates on the procedures used in the trial.

Hatching/Larvae CUiture .

Incubation of the eggs goes through four stages (initial stocking of eggs,

separation of eggs, quantifying the eggs, and stocking in larval rearing tank).

Eggs are stocked in the incubation tank with aeration to keep the eggs

suspended, with a water flow exchange at a rate of 100%/day. Separation of

viable fertilized eggs and dead eggs is carried out in the incubation tank by

turning off the aeration and allow the eggs to separate. The viable eggs float to

the surface. Increasing the salinity to 40 ppt assists in the separation. The viable

eggs are skimmed off and transferred to 100 I tank. The eggs are guantified in

the 100 I tank through replicate 100 ml samples. After quantification, the eggs

are transferred to the larval rearing tank at the desired density. Volumetric

transfer from the quantification tank assures proper stocking density.

The larvae culture procedures are identified in Appendix IX.

RESULTS

Collection I

Two GADTC staff members (Mike Bauerlein and Mark Norman) traveled to

Palau on March 15, 1993 for the initial spawning work. Preparations were made

at GADTC to accommodate the pending first shipment of grouper e�gs from

Palau. Hatching tanks and larval rearing tanks along with the biological filters

were set up for receiving the shipment. Coordination with Palau counterparts

including MMDC, private fishermen, and the Koror State Government was

carried out in advance of travel to Palau so that arrangements were made for the

project. Materials and supplies necessary for the spawning work in Palau were

obtained and organized. The first two days in Palau were devoted to obtaining

the necessary permits and finalizing arrangements with the fishermen and tank

space at MMDC. An initial fishing trip was made on March 17, 1993. The

results of the fishing and subsequent spawning attempt are attached as Appendix

I.

The catch was poor, since GADTC staff were not permitted to fish in the

aggregation channel. This resulted in an inadequate number of spawners. The

one female and three males were ripe. The fish were in poor condition, since

the catch was bloated (swollen air bladder) from fishing m deeper waters away

from the aggregation channel. Because of the inability to obtam permission to

fish in the aggregation site, as done in the previous year's project, the GADTC

staff terminated the trip early. As a result, the intended spawning work could

not be carried out on this trip and also resulted in the loss of future months

spawning work until the issue could be resolved. Further substantial negative

impacts on the project included the

elimination of the mtended original

elimination

main target

osfpreecpielisc-atPeletrcetraotpmoemnutssand

areolatus. These problems arose despite thorough efforts through the project

PAA Grouper Year 2 Final Report

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pcorooprdoisnaalt!1?0renpwairtahtiPoanlastua.ge and prior to implementation to assure full

As an alternative to Palau, if the above issues could not be resolved, contacts

were made with Pohnpei's Department of Conservation and Resource

Surveillance to determine the possibility of conducting the work in Pohnpei.

The initial response was very favorable and they were very excited about

working with us on this project. However, this would have substantially

increased the work and introduced a number of new uncertainties as opposed to

continuing the work in Palau where a significant amount of previous work had

been accomplished (PAA Grouper Year I) in establishing the base of this

current project. Unfortunately, according to information from Pohnpei their

spawning season was over for the year for the main target species.

Resolution of Permit and Collection Issues

The lead PI traveled to Palau in early May to resolve permit problems in Palau

that arose during staffs' trip in March. There was a resistance to allow fishing in

the Ulong channel which is a reserve. Permits had been issued for research

purposes to carry out the previous grouper project.

Meetings were held with various Palau officials to resolve some permit problems

awnitdhtDo aovbitdaiInd1ppe,rDmiirsesciotonrtooffNisahtiunrarlesReersvoeusrfcoersg, rtoouopbetrasi.n

A meeting was held

permission to fish for

groupers in the reserve to carry out the PAA project. In addition, a scientific

research and collection permit was requested for the upcoming collection in

May. Both of these objectives were successfully carried out. A meeting was held

with John Gibbons, Director, Koror State Government, to obtain a modification

in the previous permit received to allow for collection of groupers within the

reserve channels. This issue was also successfully resolved.

With the resolution of these major barriers to implementing the project, the

project could proceed as originally planned (with the exception of lost

opportunities for spawning

over). The recent passage

of

of new

legislationsm p.,Psainlacue

the season was nearly

banning the export of

fis

h

for a four month period (March - June) further complicated the issue; however,

it was clarified that the ban does not apply to research work.

The owner of a private aquarium fish collection business was not in Palau, so

arrangements for the collection of aquarium groupers could not be carried out.

In addition to resolving the permit issues,

with the private

fisherman that assists on the project was made the next trip by GADTC staff

to Palau. Tank space at MMDC, and room and car arrangements were similarly

made. Arrangements for Guam Aquaculture Development and Training Center

staff to travel to Palau to continue the project were also made.

Collection II

Mark Norman and Jerry Gardener of GADTC were in Palau from May 16'to

MaAttohtpteatepyhymeew2np1Uedt,rsi.lex.1o9nnIF9Igoi3sptChtroohvaaintdtneeesmslttprrheitepstTpedorehevsdetpeassairpplwesaasnowwuflnmtt�ehnddegficonroofalmtldehecethtqiseuoeancfitaorearnsneftddoicslghotarhltoflueeouscrr.petsisTwopunahalwtesdinncatwuigynr;osgthpaftoaiirlswweihadenilnvsiwn.egghrt,erinps

Phial Report

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staff discovered that the two intended males to be used were missing. A report

from the MMDC staff informed us that they had died that day.

New problems were encountered with permits. This included the issuance of a

Certificate of Origin. Staff were referred to the Conservation Office for the

issuance of a certificate. However, Mr. Dmier O'Tobed refused to issue the

permit regardless of all of the permits issued for the project to collect the

groupers for research. This was in reaction to the new legislation that banned

the export of all marine species during March to June. With the assistance of

various contacts in Palau the issue was resolved with the intervention of the

President of Palau; however, by the time the Certificate of Origin was issued it

was too late to ship the grouper eggs.

"' Difficulty was also encountered in the logistical arrangements for the shipping of

eggs. The expansion of the tuna transshipment activity in Palau had blocked off

most of the cargo space on certain flights. This made scheduling shipments

more difficult.

A June trip to Palau to spawn Plectropomus sp. was cancelled due to the

continued permit problems that GADTC staff were faced with.

Collection III

Arrangements were initiated in the later part of June to contract the services of

Dr. Clyde Tamaru (C's Consultants Hawaii) to assist in the spawning and larval

culture of groupers. A work plan was developed and submitted to the Pacific

Aquaculture Development Program. The scheduled commencement of the

consultancy was July with the assistance of PADP staff in expediting the

necessary paper work. The work in Palau for the remainder of the project

focused on Epinephelus fuscoguttatus and E. microdon, since the Plectropomus

ssppe. csii;e>aswonfingrgosuepaesorsnteanrgdeetdedinfoJrunthee.

In addition, work

aquarium trade.

would

be

initiated

on

the

The lead PI headed the next trip to Palau to continue the project in July. This

was to make sure that the logistics of the project were fully carried out and

ensure the maximum effort to resolve possible hindrances of the project. A

subcontract with Dr. Clyde Tamaru was implemented in July 1993. Dr. Tamaru

accompanied the lead PI on the following two collections in Palau during July

athnedpAroujgeucstt.weTrheesucocclleescstfiuolnlydcaatrariiesdproeustednuterdinigntAhipspterinpd. ixDIeItIa. ilTshoenotbhjeercetisvuelstsof

are presented in the GADTC Technical Report No. 16 (Appendix IX).

Since the spawning season of P. areolatus was over the target species were E.

fuscogu ttatus and E. microdon. In addition, a shipping trial of live adult E.

microdon was carried out in Palau. The results (Appendix V) indicate that live

adults can be successfully shipped. Utilizing this procedure, the shipment to

Guam of the smaller live adult _E. fuscoguttatus collected was carried out.

Results of this shipment are presented in Appendix VI.

An evaluation of potential candidate aquarium species of ~roupers was carried

out. Seven species of groupers collected during the July tnp offered some

potential as aquarium species. Details of the collection and the evaluation are

presented in Appendix VII. The three top priority species were Cephalopholis

miniata, ..C., urodeta, and _C. spiloparaea.

PAA Grouper Year 2 Final Report

Pages

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Collection IV

The lead PI and Dr. Tamaro made a final trip to Palau in August. This is the

last month of the spawning season for the targeted E.

and E.

Work was focused on E.

as the model species. Observed

spawning behavior of E. microdon was documented during this trip

VIII). A final report of this trip and the previous one with the results the

iwnoPrkalaaruewaittthacthheedgaasthAenp9negnodfixa

IX. All of the project objectives were carried out

substantial amount of data beyond the original

intent of the

This data provides a very strong foundation to the

refinement spawning, egg incubation, egg development, hatching, and initial

larval stages. This will substantially facilitate future work with groupers.

To establish a broodstock of.C miniata in Guarri to facilitate future work on the

maturation, spawning and larval rearing of this species, 35 adult fish were

shipped to Guam. This was successfully carried out following the shipping

procedures developed in Appendix V. On arrival at the Guam Aquaculture

Development and Training Center the fish were held in 2,000 1 tanks and treated

over a two week period with antibiotics (Prefuran) to reduce potential infections

ftoronmfibtheergslhaispsptianngkaannddhaan2d6l.8inmg.2

The broodstock was

concrete tank. The

divided between a 20 m

fish exhibited minimal

aggressive and territorial behavior in the smaller 2,000 1 tanks under crowded

conditions. However, aggressive territorial behavior was very apparent in the·

larger tanks. Various degrees of damaged fins resulted from the aggressive

behavior. Some fish lost major portions of dorsal and caudal fins. The damaged

fish were removed from the larger tanks, received antibiotic bath treatments and

were held separately until they recovered. Seventeen fish died as a result of the

aggressive behavior. Both of the larger broodstock tanks were divided into

sections. A mated pair was placed in each section with the intent of reducing the

aggressive interaction and stimulatin� mating within the mated groups. Monthly

cannulation of the broodstock was irutiated to collect data on maturation. An

egg collector was placed in each tank to monitor spawns. This specific long-term

maturation monitoring of C. miniata was not part of the current project;

however, the work will continue past the completion of current project.

DISCUSSION OF RESULTS

E.

is a large species reaching a weight of over 15 kgs and not as

common as E. microdon. Therefore, E. microdon was utilized as the model

species for the more detailed information collected and trials conducted. This

work included the following.

- Assessment of reproductive biological characteristics

- Induction of spawning

- Spawning behavior

- Temporal changes of grouper oocytes during final maturation

- Fatty acid profiles of spawned eggs

- Activation of grouper sperm at various salinities

- Sperm extenders

- Embryonic development of grouper larvae

transportation

of light on newly hatched larvae

Larval rearin�

- Live feed options

First feeding experiments

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- Intensive larval rearing

- Extensive larval rearing

- Effect of formalin on survival of live feeds

As mentioned, the first two collections in Palau did not result in eggs bein&

shipped to Guam for the larval rearing phase due to the various complications

mentioned. However, the last two trips were very successful in accomplishing all

aspects of the work in Palau and in shipping eggs to Guam. The protocol for the

lPaarlvaaul.reTahriisnlm,?; tcrliuadlseadnodbatarrinainnggemcreyonptsrewseerrevemdaodyestienratdrovcahnocpehoofrtehseatsrapvaerlt

to

of

the

initial feed trials. Details of the larval rearing trials are included in Appendix

IX. Extensive larval rearing trials were carried out in addition to the more

qontrolled tank based intensive larval rearing. The results and discussion are

\\ncluded in Appendix IX.

CONCLUSION

The key problems encountered in implementing the project were the following:

- The

site in

constraint

Palau was

in obtaining

a hindrance

m petrhmeisinsiiotinaltoimfipslhemwiethnitnattihoen

aggregation

of the project

and jeopardized the continuation of the project.

- The new law in Palau that bans the export of marine species during

March thru June caused confusion with the Palau officials on how to treat

scientific research projects. This resulted in difficulty in obtaining the

necessary permits to carry out the project.

- The expansion of the tuna transshipment industry in Palau with the

increased number of foreign longline vessels based in Palau utilized most

or all available cargo space on the commercial flights. This made the

shipping of the eggs to Guam difficult.

All of the project objectives were successfully carried out despite the above

logistical and procedural problems. Substantial data was collected during the

duration of the project which provides a strong foundation to future work on the

grouper species considered in this project. However, the difficulty in completing

the larval rearing cycle remains. Future wqrk will focus on the critical stages in

the larval rearing which includes the initial'feedin�, and the critical points

associated with days 2-3 and 6. In addition, the imtial broodstocks that were

established at GADTC of E. microdon and C. miniata need to be expanded.

The continued utilization of E. microdon as the model species is recommended.

Ohce key issues in the reproduction and larval rearing are refined this will

facilitate adoption of procedures to the other three target species.

PAA Grouper Year 2 Final Report

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GROUPER SPAWNING

COLLECTION I

MARCH 1993

March 15 to 19, 1993

Participants

Mark Norman (GADTC Marine Tech II)

Michael Bauerlein (GADTC Biologist Supervisor)

Moon Phases

Quarter Moon - March 15?

New Moon- March 23

First Day's Catch

Date:

Location:

of the Ngerumkaol Reserve

March 18, 1993

Barrier Reef, approximately one mile southwest

Depth:

30 to 40 feet

Tides:

Fished during the outgoing tide

Fishing time:

1050 - 1350 (about 3 hours)

Fisherman:

Presley Etibek

Moon phase:

5 days prior to new moon

Travel time:

About one hour each way

Fishing method:

as bait. Only one line out.

Hand line bottom fishing. Used small "sardines"

Species caught:

Plectropomus aerolatus (4).

Condition of the fish:

free flowing milt.

The female had ripe eggs. All of the males had

P. aerolatus

Female 1170 g died before first injection

Male 1545 g

Male 1980 g

Male 1985 g

Spawning results:

There was no spawn due to the death of the female.

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Palau trip report: March 15 thru March 19, 1993

Travelers: Michael Bauerlein and Mark Norman

Chronology of Events

March 15: Arrived in Palau at about 1930 local time.

Rented a car and drove to MMDC.

Met Tim Adams of the South Pacific Commission who was staying at

MMDC for a few days on his way back to New Caledonia.

March 16: Checked in with Gerry Heslinga, Becky Madraisau,

and David Idip (the facility director). Neither Becky or David were

expecting us. David thought the grouper project was completed. ·He

has a copy of the final report. Becky was very cooperative and

designated four tanks to hold any groupers we might catch.

Filled out "Scientific Research Permit" and paid $100 on MMDC.

(We were told this would greatly expedite the approval process.)

Made contact with Franny Reklai regarding charter boat reservation.

Bought supplies needed for project.

March 17: Received permit from David.

The permit forbade us to fish in the sanctuary.

We met with Franny again and he suggested we fish on the outer reef

about a mile southwest of the sanctuary. He arranged for a fisherman

to pick us up at MMDC the next morrung to fish for grouper.

Talked with Nancy Wong at the Fisherman's Coop.

She was interested in hearing about our success with penaeids.

Paid $50 for a fishing permit from Koror state which also barred us

from fishing in the sanctuary.

Phoned Dot to tell her that our permits restricted us from fishing in

the sanctuary and that Bill should not fly down on Thursday nighdt

unless we called with news that we had caught fish. She informe me

that Bill was not returning from Hawaii until Friday.

March 18: Discovered that we had not taken the HCG with us, so

phoned Dave at the hatchery and asked him to ship it to us on the

next Continental flight.

Fisherman Presley Etibek arrived at the MMDC dock at 0820. We

boarded and proceeded to a shallow mangrove estuary on the east

side of Koror to catch small sardines for bait. We collected about fifty

baitfish. We started fishing at 1020 in the lagoon just north of the

sanctuary. Presley said he had been diving there a few nights earlier

and had collected grouper. We had no luck, and proceeded to the

barrier reef southwest of the sanctuary. We fished for about three

hours at this site on the outer reef near the sanctuary. Presley caught

one female and three male Plectropomus aerolatus. Several other

2.4 Page 14

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fish were able to escape, and our incidental catch was two jacks

(papio) and two snappers. We tried moving to shallower water close

to the exposed reef, but had no luck there. All of the fish bloated

enough to cause them to float upside down in the bait well. We tried

using a needle to bleed the air but the water was too rough and the

fish wouldn't hold still. We caught the female first, at 1130, and she

was able to right herself after about twenty minutes. Only one of the

males righted itself before our return to MMDC. Both Mark and I

felt a little seasick due to the choppy sea conditions. We returned

with the fish to MMDC. We bled off the air from the two upside

down males at about 1600. The female and the other male were

righted but their dorsal fins were out of the water exposed to the air.

We made no attempt to release more air from these two. Mark

cannulated the female and found her to be ripe. Sent a fax to Dot

requesting hatchery personnel to expect a shipment of eggs on

Sunday. They needed to arrange for someone from Aquatics and

Wildlife to inspect the shipment upon arrival.

March 19: Found the female grouper dead in the morning. The

males were OK.

Phoned Linda Sablan with the message to ignore the fax I sent and to

relay the message that we could not send an egg shipment on Sunday.

Also, there was no need for Bill to fly down.

Contacted Franny immediately but had no luck chartering a boat.

Checked with MMDC about using their boat but none of the staff

were available to take us out. Mark contacted the Fish and Fins and

Blue Marlin charter boat operations to charter a boat on the 19th

or 20th but they needed a week's advance notice. Mark also asked

Becky if he knew anybody with a boat to charter, but Becky didn't

know of anyone. Picked up the HCG at the Continental office. When

it aJ?peared that chartering a boat for Saturday was questionable, we

decided to return to Guam on Friday night flight. We cleared up our

bills at MMDC.

I met with Gerry Heslinga and obtained one of his customer

Satisfaction Forms. He said he could,sell us coolers for egg shipments

at $15 each.

·

I met with Larry Shannon (sp?) regarding collection/spawning habits·

of aquarium grouper. Also talked about obtaining wrasses from him

for the clam project. He said he still owes Gerry Heslinga about 300

wrasses. He also said he knew only certain species of wrasses would

be allowed into Guam. He didn't sound very excited about providing

us with wrasses. But he was very interested in providing aquarium

grouper species for us.

List of phone numbers for future reference:

Larry Shannon fax 488-2305

home 488-1962 can be reached between 6 and 7 p.m.

Guam time

Franny Reklai home 488-2863

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work

Presley Etibek home 488-2873

Mike Bauerlein

Biologist Supervisor

Mark Norman

Marine Technician II

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APPENDIX II

COLLECTION II-MAY 1993

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GROUPER SPAWNING REPORT

COLLECTION II

by MARK NORMAN

FIRST DAY CATCH:

DATE:

May 17, 1993

LOCATION:

Ulong Channel

DEPTH:

18 - 20 feet

TIDE:

Going out

FISHING TIME:

1030 - 1300

FISHERMAN:

Thomas Taro

MOON PHASE:

Three days before new moon

TRAVEL TIME:

40 minutes each way

FISHING METHOD:

Hand line bottom fishing using sardines as bait

CONDIDON OF THE FISH: First catch was one shark and five snappers.

Moved to the shallower part and started catching female groupers but no males.

We moved to deeper water, about 30 - 35 feet in depth, and started catching male

groupers but the sharks kept taking them away. We were able to catch!'. aerolatus:

3 males and 13 females. We also caught 3 E. fuscoguttatus, weighing about 10 kg

each, which we released without cannulating. All thel'. _aerolatus females were

cannulated on the boat. The eggs looked very small to me and did not separate.

Males had free-flowing milt. Males and females were separated into two

compartments aboard the boat. The day was sunny and hot.

SPAWNING: When we returned to MMDC, we separated the males and females

into two different tanks. None of the fish had bloated. At 1600 five females were

weighed and received their first injection. Their average weight was 2.2 kg. The fish

were transferred into a new tank. This time we had two tanks for females. Fish

were checked periodically to make sure they were alfight.

On Tuesday, the fish received their second injection. On Wednesday morning at

0100, females were cannulated to check the condition of the eggs. Eggs looked good,

although small, and did not separate yet. All of them were swollen. At 0200 we

cannulated them again and started preparing our supplies. At 0400 one was

cannulated and found that she was getting ready to spawn; we could not delay the

spawning (strip) any longer if we were to meet the flight schedule. So we proceeded

to strip the females. First, we added 100 ml of sea water to a beaker and put it aside,

squeezed eggs into a dry beaker, and immediately squeezed the milt on to the eggs.

Then we poured the 100 ml of seawater into the container with the eggs and started

mixing. After the e�s and milt were mixed, we transferred them into a JO gallon

bucket for the washmg or separation. First, we waited until the e�s came to the

surface. Then we skimmed them into a styrofoam cooler contairung 50 liters of sea

water. From there, we would do our final separation into the bags. At 0800, I took

2.8 Page 18

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samples from each spawn and checked them with the microscope. The eggs did not

look like they were fertilized. I did not see signs of develoJ?ment. Three hours after

that I looked again; the e!lsl looked clear with nothing inside. After five hours I

looked again. There was still no sign of development. No larvae were observed to

hatch from any of the

Egg counts from two the spawns: 400,000 and 186,000.

SECOND DAY CATCH

DATE: May 18, 1993

LOCATION:

Ulong Channel

DEPT:

25 - 30 feet

TIDE:

Going out

FISHING TIME:

0900 - 1300

FISHERMAN:

Thomas Ta o

TRAVEL TIME:

40 minutes

MOON PHASE:

2 days before new moon

FlSIDNG METHOD:

Hand line bottom fishing

The 6 males were caught from the deeper part and 10 females were cau.(lht from the

shallower area, about 15 - 20 feet deep. None of the fish bloated. At this time we

only caught one shark and no snappers, even though we fished in the deep part of

the sanctuary. Fish were transpo ted to the facility and placed with the 3 males

caught the first day. At 1600, the other five females received their first injection.

Fish were checked periodically to make sure they were alright following the

injection.

On Wednesd!l at 1600 they received their second injection.

The weights these five fish were 2.6, 2.2, 2.3, 2.4, and 2.4 kg.

On Thursday morning at 0100, the fish were cannulated and the eggs looked small,

but separated. At 0300, the fish were swollen and ready to spawn. At 0400 we were

getting ready to strip when we found out that two of our males were missing and the

two that were left had no milt (possibly extracted by someone else?).

2.9 Page 19

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GROUPER SPAWNING REPORT IN PALAU

MAY 1993

OBJECTIVE. The purpose of the trip was to harvest grouper eggs and ship them to

GADTC for hatching.

Project began on May 16. 1993, with our arrival in Palau around 2000; we collected

our �ear{bags and equipment) and proceeded to MMDC. Upon our arrival at the

facility, (MMDC), Mark and I looked for the manager, Mr. lfeslinga. When we

found him we were greeted with hostile words sprinkled with profanity. We quickly

separated ourselves from this individual and went to the dorm area to sleep.

At the time of our arrival we also had called Nancy Wong about the Fishing Permit

for the sanctuary; she had not acquired it. The morning of May 17, we again called

Nancy about the Permit, she still had not received it and indicated that we should go

fishing without it. We were told by Nancy Wong and Franny that it would be OK.

During that morning we also encountered Gerry Heslinga, the MMDC manager.

He apologized for his in~ppropriate greeting but said we should have not come to

his area at that time of rught, which was about 2045.

Thomas, the boat operator, had been contacted early in the morning; he arrived at

0800. By 0915 we were on our way to the sanctuary. We stayed in the sanctuary for

4.5 hours; during that time we caught 13 females and 3 males. Returning to

MMDC, we followed the protocol, paying close attention to the times for the fish to

settle and for the first injection. The average weight of the groupers was 2.2 kg. We

still had not obtained our fishing permit by the end of the day.

Tuesday. May 18: We went fishing again in the sanctuary without a permit, leaving

the facili? at 0800. Fishing time was 5 hours. Six males were brought back; we

now had males. I had also inquired of Gerry about the Certificate of Origin

that would be needed in order to ship the eggs; he indicated that he could arrange

that. By early Wednesday morning that had changed and Gerry said we would need

to meet with the Chief Conservation Officer, who was not pleased with this project.

'

Again on Tuesday, we followed the project procedure, weighing, injecting and

separating the groupers as specified.

Wednesdafi. May 19: We begin at 0100 the procedure to collect and fertilize the

eggs. We ad also encountered another problem with shipping: there wasn't any

room on the morning flight. The eggs would have to stay at MMDC until 1600.

As I already mentioned, we still did not have the Certificate of Origin. We have

reason to believe that the President of Palau intervened on our behalf to have the

Certificate of Origin granted. We were called to the office of Dernier O'Tobed,

Chief Conservation Officer, and told that we were breaking the law of Palau. This

was not the legal time to export fish and whoever gave us the r.ermission to do so

·was also breaking the law. The Certificate was not issued unttl 1530. We had been

detained long enough for the schedule to fall apart. There was no way to return to

MMDC in time to make the shipment. On top of this, early Wednesday morning

around 0230, I received my second scolding from GerryJor disturbing his sleep.

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(The dogs were barking as we were walking between the dormitory and grouper

holding tanks, and this was keeping Gerry awake.)

Thursday, May 20: We still had 4 males from Wednesday; we had also injected four

females. We went to the grouper tank to commence the spawning, but to our

amazement we discovered that two of the 4 males had been removed from the tank

and the two that were left had no milt. This ended the project. We reported the

theft to Gerry and Becky when they arrived to work, but neither seemed to

be surprised. Friday, May 21, was a clean up day and we returned home.

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Proposed checklist/schedule for the May grouper spawning in Palau: Mark Norman

and Jerry Gardner

Sunday. May 16:

GADTC pickup truck with Mark and supplies leaves for airport at 5:00 P.M.

Upon arnval in Palau:

Rent a car from King rental.

Drive to MMDC.

Call Nancy Wang (the Koror state permit has not been issued) to see if she

has the permit.

Call:Franny Reklai at home and arrange time for fishing on Monday (if you

have' obtained the Koror permit). If you have not obtained the

inform Franny that you must obtain the permit before you can in the

sanctuary. Therefore, you will not be able to fish early in the morning.

Monday. May 17:

You need to check in with the Director, Becky, and Gerry. Keep these visits brief so

you have time to prepare the holding tanks and go fishing.

Check in with Director ldip. Ask him how you can arrange to rent the

MMDC boat for a half day later in the week to return any fish that are

caught on Monday and Tuesday.

Obtain the original copy of the research fishing permit from his secretary.

Check in with Becky. Ask him which tanks you can use. Let him know that

when you spawn the fish, you may need five aquariums (depending on your

catch), an electric light (drop light) for working at night, and a simple

microscope.

Check in with Gerry Heslinga. Let him know that you will need to purchase

styrofoam coolers for shipping the eggs,

50 small bags, and you may need to use

fooxuyrgoefnhfiosrtpaanckksinfogrthhoelde�mgs,

about

spawners. Ask him about using the freight forwarder to ship the eggs. (Cost,

etc.)

Obtain the Koror state fishing permit from John Gibbons (if you don't have

it already).

Prepare to fishing. Supplies needed:

data sheet and pencil

Battery aerators with airlines and airstones

Nets

Lunch, drinks

Copies of the Permits

Catch:

5 females and 6 or 7 males. If the fish bloat, wait until you return to MMDC

before bleeding the air bladder.

If possible, make a second trip the same day to catch 5 more females and 6 or

7 more males. If you catch more fish on Monday, you will need to put the

females in a separate tank from the first catch.

Otherwise, make arrangements to fish again the next day.

Remember that you need to inject the females before 4:00 P.M. for the

Wednesday morning spawning. Therefore, you must return from fishing by

3:30 P.M.

Upon return to MMDC:

Separate males and females into two holding tanks.

3.2 Page 22

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Bleed the air bladders of any bloated fish by piercing their abdomen with an

injection needle. Do not ~queeze the abdomen to force the air out. Allow

the fish to swim around with the needle in its body for a couple of hours (or

until the bladder has returned to normal).

Later (before 4 P.M.): Weigh the female and inject appropriate amount.

Formula: Add 10 ml sterile saline to HCG vial. Inject female with (0.7 X

the weight of the fish in kilograms) ml HCG.

Place female in a new tank. Continue this procedure until all females have

been injected.

Check flight schedule and/or contact the freight forwarder.

Start �athering the supplies you will need for the spawning and shipping: five

aquariums, electric light, four new coolers, etc. Reserve the MMDC boat for

returning the spent spawners to the sanctuary on Friday.

Tuesday. May 18:

You will be going fishing again today if you didn't catch enough fish on

Monday. Follow the same procedures for holding the fish except you will

need four new tanks to keep these fish separate. You may have to use

Gerry's tanks for this purpose.

Let the fish acclimate for an hour or so while bleeding the air from any that

are bloated.

·

Give Tuesday's catch or Monday's second catch their first injections.

Give Monday's first catch their second injections 24-hours after their first

injections.

Try to get the Certificate of Origin from the Department of Agriculture on

Tuesday or else you will have to get it on the way to the airport Wednesday

morning.

Get some sleep because you will be up very early to spawn the fish.

Tuesday night:

Keep a record of the hours you work at night. This is the time you will be

able to take off when you return to Guam.

Please note that all sea water used in fertilizing, washing and packing the

eggs should be filtered throu�h a 5 micron filter bag.

Please read through AppendJX 1 of the 1991 report and follow the

instructions given in that report if they differ greatly from the instructions

given here.

Some exceptions are that in the report they say to wait five hours before

skimming the eggs, but obviously you cannot wait that long. Ignore that part

and skim the eg�s when they are ready. Another point is that the 1991 report

recommends shipping at 15,000 eggs per liter. Please use the densities

outlined in this report instead.

Cannulate the females nine to ten hours after the second injection to check

the egg condition.

Mark should use his best judgement to determine when the eggs are ready.

You must

packed in

start

time

stripping the

to make it to

females by 4:00

the airport.

A.M.

in

o. rder

to

have

the

eggs

When the eggs are ready, strip them from the female into a plastic bucket.

Return the female to a new tank.

Use a syringe to collect at least one ml milt from a male. Return the male to

the new tank.

A?d 100 ml seawater to the bucket with the eggs. Immediately squirt in the

rmlt ai;id gently mix for two to three minutes. This may not work well if you

are usmg a large bucket.

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Alternate procedure: Transfer the eggs to a glass beaker and add 100 ml

water to the glass beaker.

Immediately squirt in the milt and gently mix for two to three minutes. Don't

let the eggs pack into a mass.

Wash the eggs as described in Appendix 1:

Place eggs in a two to four liter container to allow the eggs to float to the top.

Strip eggs from the next female and fertilize as before. Place fertilized eggs

in a two to four liter container for washing.

Repeat for the other three fish. Remember to keep the eggs from each fish

separate.

Wash the eggs from the first fish by gently pouri.ng or pipetting the floating

eggs into another collecting beaker and discardmg everything on the bottom.

Repeat for each group of eggs.

Wash each group of eggs a second time.

After the second washing, transfer the eggs into a ten gallon aquarium (if

available) for separation of the

eggs for shipping.

Determine the % fertilization the eggs using the procedure described in

Appendix 1 (if you have time).

Increase the salinity of the water to 38 or 40 ppt (if necessary) to get the eggs

to float.

Scoop off the floating eggs and place in a clean aquarium (or bucket) until

time to pack. Aerate gently.

.

Preserve a 2-ml or 3-ml sample from each spawn in a labeled vial to bring

back to Guam for a count. (If there are enough extra eggs).

Start ba�ging the eggs at around 6:00 A.M. if possible or as soon as Gerry

opens his work area. On Wednesday, you will be packing six coolers with

eggs for direct stocking into the GADTC larval tanks and a seventh cooler

for the egg shipping density vs. survival trial.

Prepare the six coolers for direct tank stocking by doubling the bags you

brought from GADTC and place three doubled bags into each cooler. Add

six liters of filtered sea water to each bag. Measure the water very carefully;

if anything, add a little bit less than six liters or you will exceed the 45 pound

weight limit and Continental will not accept the cooler for shipment. Bubble

oxygen into the water in each bag before adding the eggs.

For the six coolers with eggs for direct stocking into the larval culture tank,

skim the eggs from the surface of the tank and pour into a 500 ml or one liter

cylinder. Measure the volume of eggs floating m the cylinder. Record the

volume. There are about 1,000 eggs per ml. Add 18 ml of eggs (18,000 e�gs

per 6 liters in a bag = 3,000 eggs per liter) from the �linder to each bag m

the cooler. Do this by pipettin� the eggs from the cylmder into the bag.

Label each bag in the cooler with the female ID number and number of eggs.

Fill the bags with oxygen and seal with rubber bands as we practiced.

Remember to set aside some eggs from each spawn to determine their hatch

rate.

Try to make two coolers each from three different females = 108,000 eggs =

18,000 eggs per bag X 6 bags per cooler. These six bags will be stocked into a

3,000 liter culture tank.

If this works, only the eggs from three females giving the most eggs will be

needed.

For the shipping density trial, eggs from a single female will be packed into

small bags (twelve small bags in all) in a single cooler. Use cardboard frame

(provided by Gerry) in the cooler to support the bags. Double the bags.

Label the bags: four bags will have 1,500 eggs each (1,000 eggs per liter);

3.4 Page 24

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four bags will have 7,500 eggs each (5,000 eggs per liter); four bags will have

15,000 eggs each (10,000 eggs per liter).

Add 1.5 liters water to each bag (again, measure the water very carefully to

make sure you won't exceed the weight limit.) Bubble oxygen into the water.

Add the appropriate volume of eggs to each bag as labelled (1.5 ml = 1,500

eggs; 7.5 ml = 7,500 e�s; 15 ml = 15,000 eggs). Fill the bags with air and

z seal. Remember that with the small bags there are two layers in the cooler,

so leave room for the second layer.

If there is a scale handy (I think Gerry has one in the packing area , weigh

the boxes to make sure they are under 45 pounds (20.45 kilograms each.

Seal them with tape and take them to the airport or to the freight orwarder

depending on what arrangements you have made.

Obtain the Certificate of Origin on the way to the airport if you did not get it

the day before. This must be included with the shipment or the eggs will be

confiscated upon arrival in Guam. If you have to declare how many eggs you

are shipping, put 400,000 or a lesser number (our imports only allow us to

bring in up to 400,000 eggs per shipment).

After delivering the eggs and permits to the airport or freight forwarders,

return to MMDC and using the leftover eggs, count the number of eggs in a

milliliter. Repeat several times and take an average. This will allow you to

calculate the number of mi's to put in each bag for the next morning's

stohiepsmtiemnatt(ethfoerntuhme bfiersrtmshaiypbmeednitf)f.erent from the 1000 eggs per ml you used

Wednesday afternoon

Twenty-four hours after the first injection of the Tuesday catch (or the

second batch of fish from Monday), give the females their second injection.

Follow the previously described procedure.

Wednesday night (Thursday morning)

If possible, phone Mike at home, 734-4232, at around 11:30 P.M. Palau time

to find if the eggs hatched OK. This will determine if you need to send extra

boxes on Thursday.

Spawn the fish as done the previous night.

Pack at least two coolers from a single spawner for direct stocking (three

bags per cooler with 18,000 eggs = 18 mis of eggs per bag). If possible, pack

another cooler for the egg density trial.

Repeat the procedures used the previous morning. Again, obtain a

Certificate of Origin for this shipment if you don't already have one.

After delivering the eggs, get. some sleep.

Friday, May 21:

Return the spent spawners to the sanctuary with the MMDC boat. Pay any

outstanding bills at MMDC and prepare to return to Guam.

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APPENDIX III

COLLECTION III - JULY 1993

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SPAWNING GROUPERS IN PALAU

III

July 12 thru July 18, 1993

OBJECTIVES

- To

selected species of

in Palau and Guam.

- To viable grouper eggs from

to Guam for larval culture.

- To conductpreliminary larval culture procedures adapting proven methods for a selected

species from Palau and Guam.

- To apply developed larval culture procedures to potential aquarium species.

- To establish preliminary guidelines for developing grouper spawning and larval culture in the

region for an indigenous species.

FUNDING

Pacific Aquaculture Association, U.S. Department of Interior

·

PAA- June 1992 thru December 1993

TICIPANTS

William J. FitzGerald (EDP, Guam

of Commerce)

Clyde Tamaru (C's Consultants,

Christine Tamaru (C's Consultants, Hawaii)

Mike Bauerlein (EDP, Guam Department of Commerce, GADTC)

·

Full Moon- July 4, 1993

Last Quarter Moon- July 12th

New Moon- July 19

CATCH

seaward jutting reef

VEL TIME:

July 13, 1993

Ngerumekaol (Ulong) Channel: outer third of channel to the southern point

Low tide 2.1 ft at 0812, High tide 4.2 ft at 1353,

One day after last Quarter Moon

1030-1430 (4 hours);

Thomas Taro

45 minutes each way

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FISHING METHOD:

Handlines (3); sardines, squid and reef fish for bait

SPECIES CAUGHT:

Plectropomus areolatus (5 total), Epinephelusfuscoguttatus (ll total - 9

female, 2 male), E. microdon (33 total, 5 females, 28 males),

CONDIDON OF FISH: P. areolatus males were not ripe. E. microdon males were ripe. E.

fuscoguttatus female were ripe.

A total of nine female and male E. fuscoguttatus were caught and examined on July 13, 1993. Males o

fuscoguttatus species were difficult to obtain from the wild and only two individuals were caught durin

the project.

Five P. areolatus were caught and examined for their state of maturity. Table below. Previous data .

regarding the spawning season for this species and the results of the gonadal biopsies indicate that th .

peak in the spawning season had already passed. After the fish were examined they were returned

spawning channel.

Table. p, areolatus state of matud

Weight

· Sex

State ofMaturity

2.42

Unknown

2.78

M

Cann sperm

1.66

Unknown

2.52

M

Cann sperm

1.42

F

PV

SPAWNING RESULTS: E. microdon females possessing vitellogenic oocytes were injected on July

1993 with a

injection of 2500 IU HCG/kg body weight between 16:30 and 17:30 hours and

into a 4000

tank equipped with running seawater and continuous aeration. Individual fem

were marked

administered

awistihngvlaeriionjuescctiuotns

to

of

the caudal

HCG at a

fin for

dosage

identification.

of 1000 IU/kg

Five males were

body wei�ht and

chosen

placed

at random,

with the ·

females. Ovarian biopsies were repeated on all females 24 hours after the first (priming) injection

oocyte

preserved. Also at that time, the females were administered a second (resolving)

injection

at a dosage of 700 IU/kg and allowed to spawn naturally.

Fish

Weight

(KG)

Oocyte

·. Diameter (um)

First Injection

Second Injection

IU/kg ·.

#

Eggs Spgwned

" lO ·

Feit,

TCBW

.79

414

2500

700

8

99.9

95

BC

.87

427

2500

700

8

99.9

95

BW

1.03

454

2500

700

8

99.9

95

BC

1.11

427

2500

700

8

99.9

95

BCBW 1.21

420

2500

700

8

99.9

95

TrJFeuheslrepyteit1lcoi5ztt,ie_av1dle9nle9yug3.mg.OsbTfverheroermotwfhoasepntecaerowfutneermemsdeapleoeegfrEgathst.ueamrnneidcearstonthddteowspnoaelwitrnoceierttneyhtrdianeegetetehhcoeotfuesfprdesar,twtotihlnibezineeogduthnteaedgnregkrfsegwwomeeinarrelgee2sfdi7erwst°teeCrrcemlaenoiandbvesa3deg4refvpreaopdtmt0, 2th:3e0 on

PAGE2

3.8 Page 28

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aws ninatghnetdannthukem. hTbaehtrecohffercfaeutmnedafriltoeysmo(fftihivniesd)gi.vriToduhupealsppfeiasrwhcenwniantasggewesatoismfafaelstroetidloibbzeyseddrievvgeidgdsintwogabtshefeovteuorntydahltionguhbme(ib.veee.r,ryo>fh9isgp5ha%w()in..ee.d,

were treated as described for E. microdon with the exception of the dosage of HCG

An ovarian biopsy performed on female BC at approximately 04:30 on July 15, 1993,

her to have ovulated completely. She was then strip spawned and eggs were manually fertilized at

using

the dry method. The resulting fecundity,

in Table below. Females identified as

TfeCrt,iTlizWat,ioBnW, a, nadndpeTrCceBnCt hwaetcreh

of the spawned eggs

allowed to complete

,

maturation in the same tank. However, they did not spawn and the ovulated eggs were manually

on the afternoon of July 16, 1993, as the ovulated oocytes by this time period were overripe and

. nviable.

E. Juscoguttatus males were administered HCG as described for E. microdon and were found to

spond in similar fashion. In addition, a single male E. fuscoguttatus held captive at MMDC for public

. ibit was examined for milt. The gonad biopsy, however, was negative.

OF EGGS: Fertilized eggs of E. microdon and E. fuscoguttatus were packed at a density of

000 eggs/I and air-transported to GADTC to conduct larval rearing trials.

ECOND DAY'S ATCH

July 14, 1993

West Channel (7 • 16.9'N 134 • 15.2'E)

Low tide 1.9 ft at 0924, High tide 4.1 ft at 1517,

PHASE:

Two days after Last Quarter Moon

ISHING TIME:

900-1300 (4 hours)

Thomas Taro

TIME:

45 minutes each way

ISHING METHOD:

Handlines (2); sardines & tuna for ,bait

SPECIES CAUGHT:

E. microdon (2 females)

OF FISH: On July 14, 1993, an additional two E. microdon females were caught and

with the same group of males that participated in the first group spawning.

uSddePontsAdeaecWgrteweNdeoInffNto7Grh00yT:dICrTUaBht/Cieokignro,hnfosoJhlrluemolwydoien1dd6en,2to1r49teh9asp3otm,auawrepsnnpl.tarwtoexarismbiynaitateiralyetes1od2lvohinonugthrisantjaefstcaetmiroetnhdeoafrye1bs4oe0lt0vwiIneUgen/ikn1j'efe6,.c:3tSi0poaanwn. dnAi1nl7tgh:3ow0uagasht

a

TC

GROUPER SPAWNING PALAU July 12 thru July 18, 1993

PAGE 3

3.9 Page 29

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Fish

TCBC

TC

Weight .

1.23

1.29

dcicyte . First Injection Second Irijectfon#EggsSpawned

niamete((um)

IU/Kg .

JU/kg

< x lO 6

415

700

1400

1.6

369

700

1400

NO SPAWN

Fert.

%

32.6

· Hatch·

THIRD DAY'S CATCH

DATE:

July 17, 1993

LOCATION:

• 15.2'E)

Patch reefs inside barrier reef Rock Islands 7 Beaches area (7 • 16.9'N 13

TIDES:

Low tide 0.6 ft at 1218, High tide 4.9 ft at 1842,

MOON PHASE:

5 days past Last Quarter Moon

FISHING TIME:

0930-1230 (3 hours);

FISHERMEN:

Thomas Taro

TRAVEL TIME:

1 hr 15 minutes (45 minutes to release fish in Ulong Channel)

FISHING METHOD:

Handlines (2); sardines & tuna for bait

EuSserPpoxEi)dn,CeePptIalEheec(SlltruCuospnAfoadUsmectGuieasrHtmuleTsio:np(eladrudsnuedsxeC)(t,eelpVrumhanraiidlnooelepatdeharoslmelbixiismn)m.eaidrngsiiaentxaa)t,a(P5(llelicvuternodupenotdmeerutmesrianmreeidnoelsadetxus)se,x(V,2a3uridnoedlaaedtleo),rumCtiei(np3ehdalsoepxh),olis

CONDffiON OF FISH: Two Aethaloperca rogata reported by the fisherman to have eggs. This

species is reported to spawn year round. The three dead C. miniata were dissected and examined as

sex and state of maturity. One was in transition from female to male. The other two were one female

and one male.

Table. C. miniata sex and state of maturi

Date

Total Length Weight

.Sex

Maturity

7/17/93

20.5

183

26.2

452

28.5

516

F

F/M

M

PV + Vit

Transitional

Cann sperm

SPECIES INFORMATION:

A. Plectropomus areolatus P. truncatus)

1.

2.

3.

PMStraoerfsttesrcroemdmspoencisepsefcoiresfooofdP.leScMttarotaeprdochmtotuhhsrausvpeJsu.aninheirgeheefrfrfoant tcsoanntedncthaanndnteelns dinerPearlaflue.sh.

12 thru July 18. 1993

PAGE 4 ·

3.10 Page 30

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4. In spawning season catches of P. areolatus consisted of 60% to 85% of the total number of

fish caught.

B. Plectropomus leopardus

1. Starts aggregating/spawning March thru June

2. Preferred species for food. Stated to have a higher fat content and tenderer flesh.

3. Potentially of higher market value than P. areolatus in the Japanese market, since the fish

has a bright red coloration when chilled (P. areolatus has a grey-green color with black spots).

C. Epinephelus microdon (was E. microdon)

1. Aggregates/spawns May thru August, but mainly June/July

2. Flesh can be tough.

D. E. fuscoguttatus

1. Aggregates/spawns May thru August, but mainly June/July

2. Preferred of the Epinephelus sps, mainly because of its bigger size.

SPAWNING

:lUPER

PAIAU

July

12

thru

July

18,

1993

PAGES

4.1 Page 31

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APPENDIX IV

COLLECTION IV - AUGUST 1993

4.2 Page 32

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SPAWNING GROUPERS IN PALAU

IV

August 12 thru August 18, 1993

GENERAL INFORMATION

PROJECT OBJECTIVES

- To spawn selected species of groupers in Palau and Guam.

- To ship viable grouper eggs from Palau to Guam for larval culture.

- To conduct preliminary larval culture procedures adapting proven methods for a selected

species from Palau and Guam.

- To apply developed larval culture procedures to potential aquarium species.

- To establish preliminary guidelines for developing grouper spawning and larval culture in the

region for an indigenous species.

PROJECT FUNDING

Pacific Aquaculture Association, U.S. Department of Interior

PROJECT DURATION

PAA - June 1992 thru December 1993

PARTICIPANTS

William J. FitzGerald (EDP, Guam Department of Commerce)

Clyde Tamaru (C's Consultants, Hawaii)

Christine Calstrom-Trick (C's Consultants, Hawaii)

Mark Norman (EDP, Guam Department of Commerce, GADTC)

MOON PHASES

Full Moon - August 2, 1993

Last Quarter Moon - August 1 1th

New Moon - August 18

FIRST DAY'S CATCH

DATE:

August 12, 1993

LOCATION:

West Channel: Ngeremlengui (NW of Ngetpang Bay)

TIDES:

Low tide 2.1 ft at 0828, High tide 4.2 ft at 1430,

MOON PHASE:

1 day after last Quarter Moon

FISHING TIME:

0945-1400 (4 hours);

FISHERMEN:

Thomas Taro

TRAVEL TIME:

45 minutes each way

FISHING METHOD:

Handlines (3); tuna and reef fish for bait

rSePsEt nCoIEt rSipCeA). UEG. fHusTc:oguttatEuspi(n1efpehmelaulse)microdon (14; 4 females, 10 males - 2 ripe 2 marginal and the

4.3 Page 33

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CONDITION OF FISH: Eggs were

from all of the females.

None of the fish were injected on the day catch. Waiting to get a male E.

to spawn.

Species

Epinephelus microdon

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Epinephelus fuscoguttatus

1

Sex

Weight (kg)

F

0.64

F

1.03

F

1.19

F

1.27

M

1.38

M/F

1.35

M

1.58

M

1.50

M

1.36

M

1.39

M

1.47

M

1.53

M

1.33

M

1.59

F

4.35

Total Length (cm)

Died next day

42.55

47.63

46.36

45.09

46.36

44.45

49.53

SECOND DAY'S CATCH

DATE:

August 13, 1993

LOCATION:

Ulong Channel (7 ' 16.9'N 134 • 15.Z'E) and Rock Isl. area

TIDES:

Low tide 1 .8 ft at 0953, High tide 4.3 at 1610,

MOON PHASE:

2 days after Last Quarter Moon

FISHING TIME:

0915 start (2 hours at Ulong and 2 hours on patch reefs in Rock Isl)

FISHERMEN:

Thomas Taro

TRAVEL TIME:

45 minutes each way from T-dock

FISHING METHOD:

Handlines (2); reef fish, sardines & tuna for bait

SPECIES CAUGHT:

At Ulong channel - E. microdon (7 were kept 2 females and 5 males; 9 males

caught measured and checked for condition of maturation then released). At patch reefs in Rock Isl. - C.

miniata (6), P. leopardus (2), Variola louti (2), Cephalopholis urodeta (2), Aethaloperca rogaa (1),

Cephalopholis sonneratti and a number of snappers (6) and trigger fish - Sufflamen chrysoptera (1).

GROUPER SPAWNING PAl.AU August 1 2 thru August 18, 1993

PAGE 2

4.4 Page 34

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-Species

Epinepilelus microdon

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

Sex

M

M/F

M

F

Cephalopholis miniata

1

F

2

M

3

Unk

4

Unk

5

Unk

6

M

Variola louti

1

2

Cephalopholis urodeta

1

2

Aethaloperca rogaa

1

Unk

Plectropomus areolatus

1

2

Unk

Cephalopholis sonneratti

1

Unk

Weight (kg)

.

Total Length (cm)

44.45

47.63

45.09

49.53

35.56

45.72

46.36

48.26

2.05

50. 1 7

1.46

46.06

1.45

45.72

1.24

44.45

1.51

48.26

1.34

43.82

1.33

43.82

.37

27.94

.54

33.97

.49

32.07

.47

30.48

.32

30.48

.33

28.58

.99

40.%

.50

33.02

.50

35.24

.52

31.75

Condition (cannulated)

Sparse

Negative

Sparse

Can. sperm

Running

Can. sperm

Vit

Can

neg

?

Neg

GROUPER SPAWNING PALAU August 12 thru August 18, 1993

PAGE 3

4.5 Page 35

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CONDITION OF FISH:

I_nJ'_e_ ctio_n _ of f_ ish_ fro_ m _ 1st _ and_2n_ d d__ajy'...!f!§isrrhiLQng_g_ _ _ _ _--:------:---------:-----------

Species

Sex

Weight (kg)

- 171s0t0I(~/~~/~3)

2nd injection

- 1600 (8/15/93)

Epinephelus microdon

TC

F

TW

F

BW

F

M

2.05

.15

.17

.26

.1

.30

.34

.52

M

1.46

M

1.45

M

1.24

.1

M

1.51

.1

Primin� injection for females dose was 700 IUI kg HCG

Resolvm,g injection for females dose was 1400 IU/k,g HCG

Males in3ected at approximately 500 IU/kg HCG (smgle injection)

STBafPW htAeerW faetnhcNdeuITnrNedWGsitoywlRvweiEnargeSsU9i1n.8Lj1.e6T5c%xSti:o1a0nn6dAaetlg9lag8tps.h3p/r%freiosexhffiemefromtariltaiBezlleeW ydf2iras3ehn3s0dsppe(TaBcwWtWivn.ee)T,ldy0.h.2eT3Tf0hheee(cTuiTnnWCddu)ithcyaaendfddoasr0pTf5ae0Cwr0tnwil(siTazosaCct1c)io..u1nr3Trrexhadet1e50sp�taofewg71gn02ss./1hf%foriosu.mhr.s

SHIPPING: The shipping density was as presented in the table below. Egg density in the incubation

tank was 5,545 eggs/I. Seven liters of 40 PPT seawater was the water volume in each large shipping bag.

Two large bags per styrofoam cooler. One liter volume in small bags with 10 bags per cooler. Packing

time was 1 hr 30 min. The total time in the bag from time of packing to being placed in the designated

tank at GADTC was 9 hrs.

Targeted

GADTC tank

# of Eggs

Shipped

/! Density

in b

(eggs

1)

Tank Volume

(liters)

Larvae Density

in tank* *

(larvae/I)

LRT 1

70,000

10,000

1,000*

35

LRT 2

70,000

10,000

1,000*

35

LRT 3

70,000

10,000

1,000*

35

LRT 4

70,000

10,000

1,000*

35

Extensive tank

280,000

20,000

15,000

9.3

S-7 Extensive tank

420,000

20,000

150,000

1.9

Experimental tanks

15,000

1,000

15

33.3

(15 tanks @ 1,000/tank))

Total

995,000

*Initial volume to allow for proper density of oyster trochophores in first feeding. Total working volume

is 3,000 I.

* *Based on estimated 50% hatch.

HATCHING: The hatch rate measured at MMDC was 91.4% for the eggs shipped to Guam. The hatch

rate measured at GADTC was > 90%.

SHIPPING OF FISH: Two.live E. microdon (males) were shipped as check in baggage with the return ol

one of the GADTC staff. Fish were cooled to 24 C prior to packing. They were placed one fish per

GROUPER SPAWNING PALAUAugust 12 thruAu~st 18. 1993

4.6 Page 36

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aoconnodalwrerreivirnael

13.5 1 of seawater at 24 C. Doubled bagged. A bag of blue ice was added

were in excellent condition. The water temperature on arrival was 22 C.

feeding the following day when food was offered.

to each box. The fish

The fish were active

JllIRD DAY'S CATCH

DATE:

August 16, 1993

LinOsiCdeAlTagIOooNn::

Mesikm,

Inside barrier

Miich, Edldukl

reef

from just

north

of

Ngetpang

Bay

to

south

on

patch

reefs

TIDES:

Low tide 0.5 ft at 1239, High tide 5.6 ft at 1857

MOON PHASE:

5 days past Last Quarter Moon

FISHING TIME:

1000-1500 (5 hours);

FISHERMEN:

Thomas Taro

TRAVEL TIME:

(45 minutes)

FISHING METHOD:

Handlines (4); sardines, squid & tuna for bait

SPECIES CAUGHT:

coming in, two additional

The target species was C. miniata.

fish died prior to departure). Other

s4p7eocifeCs.cmauignhiattianwcleurdeecdaEu.gmhte(rr3ad(i4ed),

Plectropomus /eopardus (8), C. spi/oparaea (6), E. macrospilos, and numerous other reef fish (totalling

approximately 150 lbs).

CONDITION OF FISH: The fish were not checked, since they were week from the trip in and they

were intended for broodstock in Guam. However, the 3 fish that died on the trip in from fishing were

examined and all three were females. One indicated recent (within a couple days) ovulation and another

indicated near spawning. The third fish was an immature female. The two additional fish that died while

being held in the tank were not checked. Information on these samples are presented in the table below.

Fish

Total Length

� Wei ht

Sex

Condition

(cm)

(g

1

21.5

122

F

Ripe

2

23

175

F

Recent spawn

3

23

151

F

Not mature

4

5

SHIPPING FISH: Sixteen C. miniata were shipped by air cargo on 8/17/93. Five larger fish were sent

1 fish/box, four boxes

Were sent 3 fish/box.

with medium

The shippin$

size

ba$s

to small fish

consisted of

were sent 2 fish/box, and

two Continental Airlines

one

bags

bfiollxewd iwthiththr1e2e1

fish

of

water cooled to 23 ° C. The sh1ppmg time was approximately 6 hrs. All fish survived. All of the inner

bags had leaked water; however, sufficient water remained in the inner bag to keep the gills wet. All of

the fish will undergo a two week treatment of antibiotic baths.

An additional 28 C. miniata were shipped to GADTC on 8/18/93. Packing temperature was lowered to

f�i0sh°

C. The fish were acclimated from

survived the 9 hr transport time.

28°

C to 23°

C over a one hour period prior to packing.

All of the

... ... ...... . GR01 ll>J::D <:'D : A \\lll\\Jll\\_lf'"! PA I 1 1

�. 1'1 •'--· A ··�·-· 10 1<Vl"

4.7 Page 37

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Five E. microdon were

fish/box. Two smaller

shipped as check in

fish were shipped 2

baggage

fish/box.

on 8/17/93. Three larger

All fish survived in good

cfiosnhdwiteioren.shTiphpeesdhip1ping

bags consisted of two Continental Airlines bags filled with 12 1 of water cooled to 23° C. The shipme . ,

time was approximately 4 hours. The water was cooled to 24• C for shipment. Blue ice was placed

inside the cooler at one end to maintain the reduced temperature.

Two E. microdon juveniles (estimated to be 2-3 months old) and four juvenile E. merra were shipped

part of the above checked in baggage. All six fish were placed in one bag with 4 1 of water. All fish

survived the 4 hour shipment. The water temperature was not lowered prior to packing.

GROUPER SPAWNING PALAU August 12 thru August 18, 1993

PAGE

4.8 Page 38

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APPENDIX V

SHIPPING LIVE ADULT .EPINEPHELUS MICRODON TRIAL

4.9 Page 39

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SHIPPING LIVE ADULT _ EPI_ NE_ PH_ EL_ US MICRODON TRIAL

PURPOSE:

To determine if adult groupers could be transported to Guam to establish a

broodstock.

METHOD:

Two adult E. microdon were used as the test animals. They were placed in a

30 gallon aquarium filled approximately half-way. The initial temperature was

taken then the water temperature was lowered to the target transport temperature

using ice floated in sealed plastic bags. When the target transport temperature was

reached, the fish were transferred to large plastic bags (3 mil thickness, Continental

Airlines plastic bags used to cover ice coolers). The shipping bags were double

bagged. The inner bag was filled with 12 liters of water (lowered to the target

temperature). One fish, which was acclimated at the lowered the shipping

temperature, was placed in each shipment container. Oxygen was added and the

bags were sealed. Two initial shipment temperatures were used (25 and 22° C). Ice

was added to the cooler to help maintain the shipping temperature. The condition

of the fish were examined every two hours after the initial four hours of the trial to

determine if they were still alive.

The lowering of the water temperature was done over a 45 minute period

from the ambient 28.5 to 25° c. An additional 10 minutes was taken to bring the

temperature to 22 degrees.

RESULTS:

Both fish survived the trial shipping. The fish were transferred to a holding

tank at the termination of the trial. Both fish recovered immediately and were able

to maintain their equilibrium and swim; even though, their reaction time was

depressed initially.

summarv 0f Transvort Tn.a1

Time

Condition of Fish

25• c

22· c

1600 Initiation

live

2000

live

2200

live

2400

live

0200*

live

0400 Termination

live

Initial Water Temperature

28.5

Final water Temperature

19

23.5

Weight of fish

0.91 kg

0.66 kg

*The inner plastic bag of the 22 degree C tnal was punctured and bag was partially

deflated. Outer bag maintained its integrity.

4.10 Page 40

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The temperature in the 25• C target transport decreased during the trial to

19• C . This was due to excess ice placed in the cooler chest to maintain the

lowered temperature.

CONCLUSION:

The fish tolerated the lower water temperature which slowed down their

movement and metabolism and reduced the stress of transpo:tation and handling.

The fish that was initially at 25• C, but dropped to 19· C durmg the trial period,

was noticeably slower moving during initial recovery and during the periodic checks

the gill operculum movement was slower than the other fish. Further trials are

needed to determine what would be the optimum water temperature to maximize

the transport time while maintaining the fish in good condition. Additional trials

a(wreein�ehetd).edTrtoiaolspotinmoiztheetrhsepsehciiepspiifisnytsetreemstwfohrilceurlteidvuatciionng

the total cost of shipping

should be conducted to

facihtate the establishment of a broodstock in Guam. This would reduce future

costs involved in working with these species (travel to Palau and associated

collection and operation costs in Palau). It would also allow for additional work to

be conducted that is not carried out in Palau due to the requirements of meeting

flight shipment schedules and time constraints.

5.1 Page 41

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APPENDIX VI

SHIPMENT OF ADULT EPINEPHELUS FUSCOGUTIATUS TO

GUAM

5.2 Page 42

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SHIPMENT OF ADULT EPINEPHELUS FUSCOGUTTATUS TO GUAM

PURPOTSoEe:stablish broodstock of Epinephelus fuscoguttatus at Guam- Aquaculture Development

and Training Center.

METHOD:

Three female .E. fuscoguttatus were selected based on size. Smaller fish were selected from

the catch made on July 14, 1993 from the West Channel in Palau. The fish ranged in size from 2 -

3 kg.

The fish were prepared for shipment by lowering the water temperature from the ambient

temperature of 28' C to 23' C over a one hour period. Ice sealed in plastic bags and floated in the

tank was used to lower the water temperature. The packing method followed the trial shipment of

_E. microdon.

RESULTS:

Two of the fish survived the shipment. The time from sealing for shipment and the opening

of the boxes was approximately 5 hours. The temperature in the shipment water dropped in all

boxes below the intended 18-20' C. Temperature dropped between 9 -13' C in the shipping water.

The fish were transferred to a transfer tank upon arrival. The fish were initially not

responding and had to be massaged and kept moving to resuscitate them. Approximately 1-hour

of resuscitation was required. The fish were very weak. Attempts to feed the fish over the next

two days were unsuccessful. The two surviving female fish died 3-days later.

Shipping Summary

Fish 1

Fish 2

Fish 3

Weight (kgs)

sc) Sex

Initial Ambient Temperature (C)

Initial Packing Temperature

Termination Temperature (C

Reacclimatization Temperature (C)

Shipping time (hours)

Survival

2.69

F

28

23

9

27

5

y

3.21

F

28

23

12

27

5

y

3.35

F

28

23

13

27

5

N

CONCLUSION:

The temperature in the shipping boxes dropped too low. Excess ice was placed in the

container. It is recommended that future shipments utilize blue ice placed on the top or side of

the container. This would avoid direct contact with the shipping water. Crumpled newspaper or

foam packing material could be utilized as insulation.

5.3 Page 43

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APPENDIX VII

AQUARIUM SPECIES OF GROUPERS FOR CULTURE

5.4 Page 44

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AQUARIUM SPECIES OF GROUPERS FOR CULTURE

PURPOTShEe :identification of appropriate species of groupers that would potentially be suitable for

cultivation for the tropical aquarium fish trade.

METHOD:

With the assistance of a private fisherman, approxi~at~ly 3 hours were spent fishing (not

counting time moving between sites) on patch reefs ms1de the barrier reef on the west side

of Palau around the Rock Islands and 7 Beaches area. The fishing method consisted of

handlines (3) with mackerel and squid for bait. The fish were transferred live to the

Micronesian Mariculture Demonstration Center (MMDC) and held in tanks. Some fish

had extended gas bladders and were partially relieved of the pressure with a hyperdermic

needle.

The criteria for selecting an appropriate species for the aquarium trade consisted of the

following:

- Colorful

- Active swimming behavior or non sedentary or cryptic

- Behavior suitable as pet in aquarium

- Adaptability to living in captivity in a tank

- Broodstock readily available (male and female)

- Larval culture feasible

RESULTS:

Seven species of groupers were collected (July 1993) that offered some potential as

aquarium species. The initial collection consisted of the following:

Table 1.

collected

Species

No.

Sex

July August

Potential aquarium species ofgroupers

Cephalopholis _ min_ia_ta

8

53

Cephalopholis _ uro_ det_a

1

2

Cephalopholis spiloparaea

1

6

Cephalopholis _ son_ ner_ati

1

Epmephelus _ fas_ cia_tus

1

Epinephelus merra

4

Vario a louti

3

2

_Va_rio_la albimarginata

1

3-M, 5-F, 1-T..undetermined

undetermined

Non-aquarium species ofgroupers

Plectropomus leopardus

1

8

Plectropornus _ aer_ ola_tus

2

Aethaloperca rogaa

2

1

Epinephelus _ mic_rod_on

4

16

Other species include various snappers, goat fish, and wrasses.

undetermined

female - ripe

11-M, 2-F, 1-T, 4 undetermined

5.5 Page 45

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A description of each potential aquarium species after Myers (1989) with additional

physical description is provided as follows.

Cephalopholis _ min_iat_a

The coloration of both

- Standard length to

juveniles and adults

3 1 cm.

consist

oCfolamrgmeornounnamd eev-eCnloyraspl aGcreoduper.'

black outlined blue spots on a red background. It typically inhabits channels and

outer reef slopes in areas of rich coral growth and clear water at depths of 3 to 150

m or more. It occurs solitarily or in small groups. It is common on the leeward

barrier reefs of Palau and the Carolines, but rare in the Marianas.

It is reported to occur in pairs and to spawn year round with spawning at dusk or

dawn (personal commumcations, Larry Sharon).

Ce_phalopholis _ uro_ det_a - Standard length to 19 cm. Common name - Flagtail

Grouper. This small grouper occurs on lagoon and seaward reefs at depths of 1 to

36 m. It is particularly common on shallow clearwater seaward reefs at depths of 3

to 15 m. It is generally solitary and feeds on small fishes and crustaceans.

Ce_phalopholis _son_ne_ra_ti - Standard length to 47 cm. Common name - Tomato

grouper. This colorful grouper inhabits deep lagoon reefs and steep outer reef

slopes at depths of 12 to 150 m. It is uncommon in less than 30 m, but show up

regularly in catches of bottom fish. It feeds on small fishes and crustaceans

including shrimps, crabs, and stomatopods. Females mature at 28 cm, and males

mature at 34 cm and spawning coincides with the April-May and October rainy

seasons in E. Africa. The one specimen caught was from less than 30 m inside the

barrier reef. It was a deep maroon crimson color. Approximately 35 cm long.

Cephalopholis spiloparaea - Standard length to 19 cm. Common name Orange-red

pigmy grouper. The color of this little grouper may range from a dull orange to a

deep maroon or crimson. It is the deep water counter part of C. urodeta, inhabiting

steep outer reef slopes at depths of 16 to at least 108 m. It is rare in less than 30 m,

but quite common deeper.

Variola louti - Standard length to 56 cm. Common name - Lyretail Grouper.

Coloration is different between juvenile, sub-adults and adults. The juvenile and

sub-adults generally are very attractive with a purple/maroon background and white

spots. The long trailing tail is an attractive characteristic. It inhabits deep lagoon

pinnacles, channels , and seaward reefs at depths of 4 to at least 150 m. Adults seem

to prefer areas of rich coral growth below 15 m; but juveniles may occur in as little

as 4 m. Its diet consists primarily of fishes, including relatively large spiny or

venomous species such as squirrel fishes and scorpion fishes, and to a lesser extent

on crabs or spiny lobsters. Large individuals may be ciguatoxic in certain areas.

The fish behaved were very skittish in the holding tank. When a net was placed in

tchoelotraantkiotnofcaadtecshtoothaevrefriyshwtahseheVdaoruiotlacowlooruwldhferneqmueanltilgyhjtutmanpko.ut of the tank. The

_Va_rio_la albimarginata - Standard length to 45 cm. Common name - Whitemargin

Lyretail Grouper. It is relatively rare. It inhabits outer reef slopes and coastal reefs

at depths of 12 to 90 m.

bGEeprloionuweppe1h5re. mluT.sh_fIiasnsi_pcsiroao_tntueescot-efSdtthabenadmyasoradsntldceonlamggtmhoootnons2g, 9ritocmumpa.eyrCos coocmfuomruaotsenrsnhraaemellfoewsl-oBapslea4scmkat-tadipneppdethhdass

5.6 Page 46

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been fished from as deep as 160 m. It is generally solitary and feeds primarily on

crustaceans and fishes, and to a lesser extent on octopods and ophiuroids, by day as

well as at night. Off East Africa females of _E. fasciatus mature at 16 cm, males at

17.5 cm, and spawning takes place seasonally prior to the major rainy season.

Epinephelus _ me_ rra - Standard length to 22.5 cm. Common name - Honeycomb

Grouper. The honeycomb grouper is a common resident of shallow lagoon and

semi-protected seaward reefs that occasionally occurs as deep as 50 m. Juveniles

often occur in thickets of staghorn Acropora corals. E. rnerra feeds primarily on

fishes as it grows.

The three _C. rniniata that died were opened to examine the gonads to determine the sex

and if they were mature. The results are presented in Table 2. Examination of the gonads

revealed that they had spawned in the last 1-2 months. Sperm could be activated.

Table 2. C. miniata

Specime.n

.·.· ·

·. . .

.· ·.

1

2

3

·

SL(cm)

.

26.3

28.5

20.5

Weight (g)

452

516

183

Sex

M/F

M

F

Comment

Transitional female

Sperm activated with seawater

PV and few vit eggs

CONCLUSION:

_C. rniniata is the first choice for cultivation for the aquarium trade. C. urodeta is the

second choice. V. louti would have the added advantage of use as a food species, since it

occurs in the commercial fisheries market. Of the species collected the two most

outstanding species based on appearance

uniform between the different sizes of.C

were.C

miniata .

r.nYi,nliIaDtialal dnedmVo.nlsmtrialtl.edThcoelocorlpohraatsieosntwhaats

it apparently goes through as it matures. It also faded drastically to a pale color in a light

colored tank.

r The priority order of the potential aquarium species examined is presented in Table 3.

This is a reliminary evaluation of the species considered based on only the first four

factors o the critena for selection as an appropriate aquarium species. The latter two

criteria could not be adequately evaluated at this time. However, C. miniata seems to have

an adequate readily catchable broodstock and was the dominate species in the catch of

aquarium type species. Some of the other species (e.g., _V. albirnarginata) are considered

rare and would be difficult to obtain adequate numbers of adult fish as broodstock.

s Table 3.

Priority

list for

.

Species

1

Cephalopholis rniniata

2

Cephalopholis urodeta

3

Cephalopholis spiloparacea

4

Variola louti

5

Variola albirnarginata

6

Cephalopholis sonnerati

7

Epmephelus fasciatus

8

Epinephelus merra

5.7 Page 47

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Based on desirable behavior and "friendliness" the.C .miniata was the superior species.

Wild caught species showed signs of curiosity when an observer looked in the tank. The

fish would come to the surface and look at the person. .C miniata held in captivity at

MMDC for approximately three months would come to the surface when bemg fed and

take the food from the attendant's hand. Other species showed degrees of behavior from

sitting on the bottom and not moving to that described for.C miniata. .C urodeta

demonstrated behavioral characteristics that also make it a preferred aquarium species.

However, V.

demonstrated a nervousness in the tank and frequently jumped out of

the tank when a net was placed in the tank.

A second effort to collect aquarium species the following month (August 1993) was made

for the selected priority species of C. miniata. The catch consisted of 53 C. miniata. Of

these, 44 were sent to Guam to establish an initial broodstock for this species.

5.8 Page 48

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APPENDIX VIII

OBSERVED SPAWNING BEHAVIOR OF EPINEPHELUS

MICRODON IN CAPTMTY

5.9 Page 49

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OBSERVED SPAWNING BEHAVIOR OF EPINEPHELUS MICRODON

IN CAPTIVITY

The spawnmg behavior of E. microdon was observed during two separate

spawnings Of groups of_E. .microdon held in a tank at Micronesian Mariculture

Demonstration Center. The spawnings were induced through injections of HCG to

mature females (2 injections) and males (1 injection). The chronology of the

spawning behavior was recorded in the second group of fish spawned and is

presented in Table 1.

Table 1. Chronology of Spawning and Spawning in Epinephelus microdon

.

. A. c. ti. .vity,

'.fifu:e . ·

" ::· :- .·:"

Day

Primin� Injection

Resolvmg Injection

Initial Movement of Males

Initial Movement of Female

Onset of tail waging and head shaking

Increased

Initial upward swimming

Spawning

1800

0100-0200

0400

0430

0530

0545

0640

7/13

7/14

7/15

The first group consisted of 5 females. All females spawned naturally in the

tank approximately 12 hours following the resolving injection. These females were

injected with a high priming injection of HCG (2,500 IU/kg) and a lower resolving

injection (700 IU/kg).

The second group consisted of 2 females and 7 males (same 5 males used in

j the previous trial plus 2 additional males). Only one of the females spawned

naturally. The second fish responded to the HCG in ections (i.e., ovulation), but it

did not result in a natural spawn. Both females received a priming injection of 700

IU/kg followed 24 hours later with a resolving injection of 1400 IU/kg. The second

female did not exhibit the spawning behavior nor did it elicit the spawning behavior

by the males. The female that did not spawn nor demonstrate the spawning

behavior possessed the smallest e� diameter that could possibly indicate a critical

oocyte diameter of > 400 um for this species. Three female _E. fuscoguttatus that

were injected with 700 IU/kg for the priming injections and 1400 IU/kg resolving

injection as well as two that were injected at 250 IU/kg and 500 JU/kg for the

priming and resolving injections respectfully did not display spawning behavior nor

did natural spawning occur. One control female was not injected with HCG and did

not ovulate or display spawning behavior. The ovulated fish had to be stripped

spawned. Fish that were not stripped spawned near ovulation had to be forcibly

stripped (approximately 48 hrs later) to eliminate the degenerated eggs.

Prior to the onset of spawning behavior the fish laid inactive along the

bottom side of the tank. They were often 2 - 4 fish deep or bunched together. The

male fish showed the first activity by moving away from the side and bottom of the

tank and started to slowly swim about the tank. This was followed later by the

female movin� away from the bottom side; even though the female was upright,

swimming activity was still limited to small movements around the bottom area of

5.10 Page 50

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the tank. The next significant behavior change was the onset of tail waging as the

males swam slowly past the female. Similarly, head shaking was initiated as the

males swam closely past the female. Increased territorial behavior the males

against other males was also observed at this time. The frequency tail waging and

head shaking increased over the next two hours prior to actual spawning. The

culminating behavior was an upward swimming to the surface by the female which

was followed by 2 to 4 males. The males swam close to the female usually in

physical contact. A swirling swimming behavior was also observed at the surface.

This upward swimming behavior by the female occurred three times with actual

spawrung with the complete release of the eggs on the third upward swim. Between

each of the upward swimming behaviors the female would settled back to the

bottom of the tank. Males would continue the tail waging and head shaking

behavior during this interlude as well as the territorial behavior.

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APPENDIX IX

GADTC TECHNICAL REPORT NO. 16: PRELIMINARY SPAWNING

AND LARVAL CULTURE TRIALS FOR GROUPERS FROM GUAM

AND PALAU

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Preliminary Spawning and Larval Culture Trials

for Groupers from Guam and Palau

TECHNICAL REPORTNO. 016

December 1993

TECHNICAL REPORT SERIES

OF THE

GUAM AQUACULTURE DEVELOPMENT AND TRAI NING

CENTER

DEPARTMENT OF COMMERCE

Government of Guam

6th Floor ITC , Suite 601

590 South Marine Drive

Tamuning, GU 96911

6.3 Page 53

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Prel iminary Spawning and Larval culture Tri a l s

For Groupers from Guam and P a l au

Final Report

July 2 - 2 4 and August 6 - 2 4 , 1 9 9 3

Prepared for :

D iv i s i on o f Economic Development and P l anning

Guam Department of Commerce

6th F loor GITC Bldg . , Suite 6 0 1

5 9 0 South Marine Drive

Tamun ing , Guam 9 6 9 1 1

Prepared by :

C lyde s . Tamaru , Ph . D .

Edited by : Chr ist ine Carlston-Trick

Hawa i i C ' s Aquaculture Consultant Services

1 1 5 7 Lunaapono Place

Kai lua , Hawai i 9 6 7 3 4

November 5 , 1 9 9 3

6.4 Page 54

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ACKNOWLEDGMENTS

Special thanks are extended to William J. FitzGerald, Jr., of the Department of Commerce,

Guam, for inviting me to take part in the consultancy. Special thanks are also extended to Anne

Orcutt and Joan Yamada for expediting the administrative process to make this consultancy a

reality. Assistance from the staff at the Guam Aquaculture Development and Training Center,

especially Mike Bauerlein, Mark Norman, and Wing-Kai Wong, was critical in meeting the

objectives ofthe consultancy. Likewise, David Idip, Gerald Heslinga, and Becky Madraisau at

the Micronesian Mariculture Demonstration Center played a major part in the achievements in

Palau. A warm Mahala is extended to the fisherman extraordinaire, Thomas Taro from Palau,

who made sure we were able to obtain the species of groupers under study.

During the course of the consultancy, assistance was also received from: Drs. Fred

Kamemoto, Samula R. Haley and Gordon Grau, Department ofBiology and Department of

Zoology, respectively ofthe University ofHawaii at Manoa, Dr. James A. Brock, Aquaculture

Development Program, State ofHawaii, Tom Iwai, Department ofLand and Natural Resources,

State ofHawaii, Ryan Murashige, Uwajima Fisheries Inc., JeffTellock, Provesta Corporation,.

Special thanks are extended to Dr. Harry Ako and his staff at the Department ofEnvironmental

Biochemistry, University ofHawaii at Manoa for conducting the fatty acid analyses. Lastly, a

warm Mahala is extended to Christine Carlstrom-Trick for her field assistance and moral support

during the consultancy as well as the editing and final preparation of the report.

This report is funded by a grant from the Pacific Aquaculture Development

Program, Office of Territorial and International Affairs, U.S. Department of I nterior,

GEN-52, which is administered by the University of Hawaii Sea Grant College

Program (SOEST) under International Grant No. NA89AA-D-SG063 from NOAA

Office of Sea Grant, Department of Commerce. The views expressed herein are

those of the author and do not necessarily reflect the views of NOAA or any of its

subagencies. The project was im plemented as part of the Pacific Aquaculture

Association's program which is to promote and assist the development of

aquaculture as a vehicle for economic and social advancement in the Pacific

islands with PAA membership.

Coverphotograph by William J. FitzGerald, Jr.

6.5 Page 55

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TABLE OF CONTENTS

EXECUTIVE SUMRMA Y

8

INTRODUCTION

10

REPRODUCTION

•

.

•

12

Biological Characteristics of E. microdon

12

INDUCTION OF SPAWNING

15

E . microdon

•

15

E. fuscoguttatus

18

SPAWNING BEHAVIOR •

19

TEMPORAL CHANGES OF GROUPER OOCYTES DURING FINAL MATURATION

20

FATTY ACID PROFILES OF SPAWNED EGGS

20

ACTIVATION OF GROUPER SPERM AT VARIOUS SALINITIES

23

SPERM EXTENDERS . • • . . . . . . . . .

24

EMBRYONIC DEVELOPMENT OF GROUPER LARVAE

25

EGG TRANSPORT •

26

LARVAL REARING

26

LIVE FEEDS PRODUCTION AT GADTC

28

Oyster Trochophores

•

28

Phytoplankton Production •

29

Rotifer Production • • • .

29

Harvest of copepods

•

32

Artemia Nauplii Production

34

Penaeus stylirostris nauplii

35

Sea Urchin , Tripneustes gratilla

36

EFFECT OF LIGHT ON NEWLY HATCHED LARVAE

36

FIRST FEEDING EXPERIMENT

36

INTENSIVE LARVAL REARING

38

EXTENSIVE LARVAL REARING

40

EFFECT OF FORMALIN ON. SURVIVAL OF LIVE FEEDS

42

OTHER GROUPER SPECIES • • •

44

Plectropomus areolatus

44

Cephalopholis miniata

44

3

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TRANSPORT OF ADULT GROUPERS TO GADTC

47

E . microdon

.

•

47

E . fuscogu ttatus • • •

48

Cephalopholis miniata

48

SUMRAM Y AND RECOMMENDATIONS

•

48

Reproduction •

49

Larval Rearing

49

LITERATURE CITED

51

4

6.7 Page 57

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LJ:ST OF TABLES

Table 1 . Summary of spawning trials for E . microdon

conducted in July 1993 at MMDC.

•

.

•

...

15

Table 2 . summary of induction of spawning trials for

E . microdon in August 1 9 9 3 at MMDC. • • • • • •

16

Table 3 . Summary of hormone treatment for maturation

of E . microdon males . . . . . . . . . . . . . . .

17

Table 4 . summary of spawning trial for E. fuscogu ttatus .

18

Table 5 . Fatty acid profiles of spawned eggs from

E . microdon and E . fuscoguttatus

•

.

22

Table 6 . Fatty acid profiles of spawned eggs from

various marine teleost s .

•

.

•

22

Table 7 . - Activation of E . microdon and E . -fuscoguttatus

sperm at various salinities

•

23

Table 8 . Summary of various sperm extenders on

maintaining sperm of E . microdon at 5°C. • . . . . . .

25

Table 9 . Temporal changes in oocytes undergoing final

maturation , and developmental series of stripped eggs

from "BC" E . fuscoguttatus at 2 7°C and 34 ppt .

25

Table 1 0 . Summary of rotifer production using two

variants of the batch culture method at GADTC . . . . .

30

Table 1 1 . Comparison of fatty acid profiles from rotifers

produced at GADTC using two batch culture methods

31

Table 12 . Comparison of production and fertility

of rotifers cultured using three different methods .

32

Table 13 . Summary of harvesting copepods at night from

Raceway #2 at GADTC .

•

34

Table 14 . Fatty acid profiles of newly hatched and

enriched Artemia nauplii • • • • • • • • • • • . . . .

35

Table 15 . Summary of data obtained by gonadal biopsies

o f P . areolatus captured on July 1 3 , 1993 . • • • •

44

Table 1 6 . Summary of gonadal biopsies obtained from C .

miniata . . . . . . . . . . . . . . . . . . . . . . .

45

5

6.8 Page 58

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LJ:ST OP' P'J:GURES

Figure 1 . Annual landings of - 11temekai11 between 1985 and 1990

in Palau . . . . . . . . . . . . . . . . . . . . . . .

11

Figure 2 . Size frequency distribution of E. mi crodon caught

and purchased during July and August 1993

•

.

•

.

13

Figure 3 . Length x weight relationship of E . mi crodon caught

and purchased during the second phase of the

consultancy . . . . . . . . . . . . . . . . . . .

13

Figure 4 . Sex composition of E . microdon caught during July

and August 1 9 9 3 in Palau • • • • • • • . • • • • •

14

Figure 5 . Changes in the oocyte diameters of "BC" female E .

fuscoguttatus undergoing induction of spawning . • .

21

Figure 6 . Summary of hatched E . microdon larvae stocked at

various egg densities under s imulated transport

conditions • •

27

Figure 7 . Relationship of spawned egg diameter versus total

length of hatched larvae from various species under

culture . • . • • . . . - . . .- . . . .- . . . . . . . .

28

Figure 8 . Schematic representation of the two batch culture

methods for culturing rotifers as GADTC

•

.

•

.

•

30

Figure 9 . Size frequency distribution of s-type rotifers

being cultured at GADTC

...•..

....

33

Figure 1 0 . Effect of l ight on survival of E . microdon

larvae . . . . . . . . . . . . . . .

...

37

Figure 1 1 . Summary o f small-scale larval rearing trial o f E .

mi crodon . . . . . . . . . . . . . . . . . . . . .

39

Figure 12 . Effect of various concentrations of forma lin on

l ive feeds and medusae • . • • • • • • . • . • . • .

43

Figure 13 .

Length x weight relationship of C . miniata

captured during July and August 1993 .

•

46

Figure 14 . S ize frequency distribution of oocytes from two

female C . miniata caught on August 16 , 1993

•

47

6

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LXST OF APPENDXCES

f

Appendix 1 . Pictorial essay of the methods of capture ,

transport , induction of spawning , and larval rearing

tanks

•

.

•

.

•

.

•

.

•

.

•

.

•

.

54

Appendix 2 . Procedure for strip spawning and "dry method" o f

fertil i z ing eggs . . . . . . . . . . . . . . . . .

63 .

Appendix 3 . Spawning behavior of Epinephelus microdon

67

Appendix. 4 . ' Temporal chal}ges of oocY!-es during final

maturation , spawning , and embryonic development for

Epinephelus .fuscoguttatus

•

70

Appendix 5 .

Morpho logical changes o f Epinephelus

.tuscoguttatus larvae • • • •

..... . . .

74

Appendix 6 . oyster trochophores and a comparison o f rotifers

to trochophores and copepodites

•

.

•

78

Appendix 7 . Spawning protocol for the sea urchin , Tripneustes

gratilla . . . . . . . . . . . . . . . . . . . . . . .

�85

Appendix 8 . Various grouper species encountered during the

consultancy . . . . . . . . . . . . . . . . . . .

89

Appendix 9 . Gonadal tissue from Cephalophilis miniata

Appendix 1 0 . Procedures for the artificial propagation

of groupers in Micronesia

•

96

7

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EXECUTIVE SUMYAMR

This report summarizes the activities of the short-term

consultancy focused on the development of hatchery technology for

the artificial propagation of groupers in Guam and Palau . The two

1J

J

phases of the consultancy were July 2-24 and August 6-24 , 1 9 9 3 , and :!

were accompl ished at the Micronesian Mariculture Demonstration i

Center (MMDC) in Palau and at the Guam Aquaculture Development and

Training Center ( GADTC) in Guam .

Accomplishments during this

period were :

1 . Defined the state of maturity of female E . mi crodon

necessary to initiate hormone treatment for induction of final

maturation and spawning. Characterized the in vivo changes in

oocyte diameters that accompany hormonal induction of final

maturation and spawning. -

2 . Production of fertilized eggs ( 7 x 106) and larvae from

Epinephelus microdon by hormone ( i . e . , HCG) induced spawns .

3 . Defined the state of maturity of female E . fuscoguttatus

necessary to initiate hormone treatment for induction of final

maturation and spawning. Characterized the in vivo changes in

oocyte diameters that accompany hormonal induction of final

maturation and spawning.

4 . Production of fertilized eggs (1 . 5 x 106) and larvae from

E . fuscogu ttatus by a hormone induced spawn .

5 . Characterized length x weight relationship , size frequency

distribution , sex composition by size, size at sexual

transition from female to male for E . microdon and

Cephalopholis miniata .

6 . Defined the salinity for the activation of sperm from E .

microdon and E . fuscoguttatus .

7 . Tested various solutions for use a s sperm extenders using

sperm from E . mi crodon .

a . Characterized the size frequency distribution of recently

spawned and mature C. miniata females to determine the

critical oocyte size for the induction of spawning .

9 . Characterized embryonic development for E . microdon and E .

fuscoguttatus .

8

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10 . Defined the optimum egg density for transportin�

fertilized grouper eggs . successfully transported 7 0 0 x 10

fertilized eggs from MMDC in Palau to GADTC in Guam .

1 1 . completed experiments for the transport of adult E .

mi crodon and E . fuscogut:tatus.. Successfully transported six

adult E . microdon and 46 adult Cephalopholis miniata

broodstock from MMDC in Palau to GADTC in Guam .

12 . Characterized the fatty acid profiles of spawned eggs from

E . microdon and E . fuscoguttatus .

13 . Reviewed and improved the production of rotifers at GADTC.

Impro�ed the nutritional ( i . e . , fatty acid prof ile) quality of

rotifers at GADTC . Established contact to obtain a smaller

size rotifer ( SS-type) for mass culture and use for larval

rearing of grouper .

14 . Establ ished the methodology for obtaining copepods from

shrimp broodstock raceways at GADTC for use in grouper larval

rearing trials . Characterized the relative abundance of

copepods in relation to standard raceway maintenance

procedures for shrimp broodstock .

15 . Completed small-scale ( i . e . , 15-liter) , intensive ( i . e . ,

3 0 00-liter) and extensive ( 2 0 , 000 - 100 , 00 0- l iter) larval

rearing experiments and rearing trials for E . microdon and E .

fuscoguttatus .

16 . Production of 15-day-old posthatched E . fuscoguttatus

larvae .

17 . Determined the effects of light on newly hatched E .

microdon larvae through first feeding .

18 . Determined the survival rate of rotifers , copepods ,

medusae and Artemia nauplii under acute formalin treatments .

19 . Collected and submitted oyster trochophores , newly hatched

and enriched Artemia nauplii , Penaeus stylirostris naup li i ,

copepods , and enriched rotifers for fatty acid analysis .

2 0 . Located source of the sea urchin , Tripneustes gratilla ,

and provided the spawning protocol for future use as an

initial larval feed.

2 1 . Production of a procedural guide for the artif icial

propagation of grouper.

9

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INTRODUCTION

Groupers are commercially important fishes supporting small

scale fisheries in many areas of the world . Capture of groupers is

by trawling , hook and line , trap , spear and gill nets . FAO f ishery

statistics indicate a reported annual worldwide catch in 1 9 8 8 and

1 9 89 of 8 5 , 045 and 8 0 , 7 6 0 metric tons , respectively. The catch of

groupers in Southeast Asian waters alone accounts for approximately

6 0% of the total catch (Anon . -, 1989 } . Although the grouper catch

in southeast Asia by weight is not represented in the top twenty

species harvested , it ranks 14th in dollar value ( Kohno et a l . ,

1990) .

In Palau , commercial export of live groupers from 1984 to 1988

produced an average annual yield ranging between 10 and 15 tons

(Kitalong and Oiterong, 1991) . Fishing for groupers was largely

concentrated in channels and capture was easiest during the large

annual spawning aggregations ( Johannes , 1 9 8 1 } . While seasonal

restrictions have been implemented in at least one spawning ground

( i . e . , since 198 0 , Ngerumkaol Channel ) , other areas where groupers

aggregate are continuously f ished today. F ishermen in Palau are

concerned over the dwindling numbers of groupers being caught as

exhibited in the catch records of "temekai " , over the past f ive

years ( Figure 1 ) . The term " temekai" actually is used for a

number of groupers ( i . e . , Epinephelus fuscogu ttatus , E . microdon ,

E . maculatus, E . tukula, E . dyctiphelus, E . poecilonotus, E . ongus,

E . merra and Anyperdon leucogrammus) and the catch records do not

distinguish between the various species ( F itzGerald et a l . , 1991) .

This downward trend , however , i s particularly noticeable for E .

fuscoguttatus , even when f ishing in the reserve area and during

their spawning season . The proposed total fishing ban on grouper

wi l l have a serious impact as Palau gains further autonomy and

continues to address means of becoming economically independent .

In Guam , the grouper populations have been severely over f ished for

many years and markets import groupers from other areas in the

South Pacific ( e . g . , Chuuk , Palau) to satisfy local demand

( FitzGerald , pers . comm. ) .

The popularity of grouper as a food fish has steadily

increased worldwide , resulting in commercial ventures , especially

in Southeast Asi a , to culture many species . Early culture attempts

show that groupers are fast-growing , euryhaline , hardy, disease

resistant and therefore profitable . Findings show that grouper

culture can be performed in earthen ponds as well as in net cages

(Tamaru et a l . , 1993 a ) . Net cage culture represents an opportunity

for expans ion of the industry , despite the ever increasing

competition for ava i lable land space . In Taiwan , groupers fetch a

farm gate price ranging from us $15 . 0 0 to $ 2 0 . 00 /kg (Tamaru et

10

7.3 Page 63

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50

0

.-1

><

OJ

�

"'

1985

1986

1988

YEAR

1989

19 9 0

Figure 1 . Annual landings of 11temekai 11 between 1 9 8 5 to 1990

in Palau.

Data summarized from Kitalong and

Oiterong ( 19 9 1 ) .

al . , 1993 a ) . The grouper culture industry , however , remains

dependent on seed from the wild . A single f ingerl ing commands a

price of US $2 . 00 to $ 3 . 00 and is currently the highest cost of

grouper culture (Tamaru et a l . , 199 3 a ) .

Hatchery technology for the artificial propagation of striped

mul let , Mugil cephal us , has been in place at GADTC since 1992 as

part of the overall objectives to provide seed for the

diversif ication of aquaculture activities in Guam . In keeping with

this mission, GADTC has been engaged in adapting the hatchery

technology for striped mullet to the production of grouper seed .

Seed production of grouper would provide the basis to begin

commercial growout and stock enhancement activities . Likewise , the

developed technology can be used for the production of grouper

species that are conducive to the aquarium trade . The knowledge

base and experience of the consultant was used to review, modify,

and improve existing hatchery techniques at GADTC with the major

11

7.4 Page 64

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focus on developing a suitable hatchery technology for groupers . A

pictoria1 presentation summariz ing the methods employed during this

consultancy for capture , ovarian staging , administration of hormone

for the induction of spawning, fertilized egg and hatched larvae

development , larval rearing and return of broodstock to the capture

site is presented in Appendix 1 .

REPRODUCTION

Understanding the reproductive characteristics of any target

species is a prerequisite for the production of fertilized eggs on

demand . such information provides the basis for control ling

maturation and the spawning of good quality eggs .

Biological Characteristics of E. microdon

A total of 63 adult Epinephelus microdon individual s were

collected by hook and line during both phases of the consultancy .

Captured individuals were transported live to the Micronesian

Mariculture Demonstration Center (MMDC) and weighed . In addition ,

four ( i . e . , <0 . 6 kg body weight) individuals were purchased from

the local fresh fish outlets on Koror , Republic of Palau . The

individuals that were purchased were of a smaller size range than

those that had been caught . Total length was obtained only from

the individuals col lected during the second phase of the

consultancy . The size frequency distribution of the collected

individuals is summarized in Figure 2 . All individuals were

biopsied using a polyethylene cannula and the state o f maturity

noted . oocyte samples were fixed in a solution of 5% formalin in

seawater and the average oocyte diameters determined using a

compound microscope equipped with an ocular micrometer . This

method has been validated for use in staging ovarian maturation in

female striped mullet (Mugil cephalus) and milkfish ( Chanos chanos )

( Shehadeh et a l . , 1973 ; Lee et a l . , 19 8 6 ) . All of the fish caught

from the wild were found to be mature, meaning that the females

possessed vitellogenic oocytes and from the males , sperm could be

extruded by applying pressure to the abdomen or obtained by

cannulation . It is obvious from the fish that were examined during

July and August that these months mark part of the spawning season

for this species . The beginning and ending months of the spawning

season remain to be determined .

A length x weight relationship was generated from fish that

were caught or purchased during the second phase of the consultancy

and is summarized in Figure 3 . The following model was found to

provide the best fit of the data :

BW

=

0

.

0R627

(TL) -

= 0 . 95

1 . 612

p = <0 . 001

12

7.5 Page 65

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20

16

12

•

4 II

0

0 . 3 - 0. 6

. ;~?:tf~~ili~

• rJ

0 . 15 · 0 , ,

0 . 9 - l. . 2

l. . 2 - 1 . s

l. . S • l. , 8

BODY WEIGHT (KG)

l. . 8 - 2 . l.

Fiqure 2 . Size frequency distribution of E . microdon caught

and purchased between July and August 19 9 3 .

2 . l.

1.9

l. , 7

�

� 1 .5

�

� l. . 3

�H 1 . l.

8 ·0.9

111

0,7

0.5

• NALE

+ NATURE 1"l!:MAL!I:

� DIMATURE 1"l!:MAL:I!

MODEL

•

WT•0 , 067 (TL) • 1 , 6 12

2

R • 0.95

P•<0 . 001

0,3

28

32

36

40

44

48

52

TOTAL LENGTH (CM)

Fiqure 3 . Length x weight relationship of E . microdon caught

and purchased during the second phase of the

consultancy .

7.6 Page 66

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The sex composition of the different sizes of groupers ( i . e . ,

0 . 3 kg classes ) obtained during both phases of the consultancy is

shown in Figure 4 . That females were found only in the smaller ·•

size groups ( < i . 2 kg in body weight) is consistent with protogynous

hermaphrodites .

Immature ( i . e . , possessing previtellogenic .

oocytes ) females were found only in the smallest ( < 0 . 9 kg) s ize

class while mature ( i . e . , possessing vitellogenic oocytes ) females •I

ranged between O . 9 and i . 2 kg . The latter s iz e range was also I

predominated by mature males-. From the data presented in this

(4)

100

10

60

40

(5)

(l.l.)

(.26)

(14)

(3)

.2 0

0

0.3

D.6

0 . 15

0 .9

1.2

1.5

1.8

D.9

l. • .2

1.5

l. . 8

2.1

BODY WEJ:GHT (:&:G)

- llATmtl! 1"l!IMALl'

- Dl'fl. l'll!RI P!Ll!MA

Fiqure 4 . Sex composition of E . microdon caught during July

and August i993 in Palau. Numbers in parentheses

equal the number of individuals in each size class .

report , it would appear that i . 2 kg body weight represents the

upper limit for females of this species . Individuals that weighed

less than 0 . 9 kg , whi le being definitely female , may not have

reached puberty or may have spawned recently. Whether this lower

size class represents a critical size for maturation of females

requires further investigation .

i4

7.7 Page 67

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~

1:

,.~- INDUCTION OF SPAWNING

~

:

microdon

Females possessing vitellogenic oocytes were inj ected on July

13 , 1993 with a priming injection of 2500 IU HCG/kg body weight

between 16 : 3 0 and 17 : 3 0 hours and placed into a 4 0 00-liter

fiberglass tank equipped with running seawater and continuous

aeration . Individual females were marked with various cuts to the.

caudal fin ( e . g . , TC = top cut , BC = bottom cut , TW = top wedge ,

and so forth ) . Five males were chosen at random , administered a

single inj ection of HCG at a dosage of 1000 IU/kg body weight and

placed with the females . Ovarian biopsies were repeated on all

females 2 4 hours after the first (priming) inj ection and oocyte

samples preserved .

Also at that time , the females were

administered a second (resolving) injection of HCG at a dosage of

700 IU/kg and allowed to spawn naturally.

Fertilized eggs from one �emale were de_tected to be undergoing

first cleavage at 02 : 3 0 on July 1 5 , 19 93 . The water temperature

and salinity in the spawning tank were 2 7°C and 34 ppt ,

respectively . Over the course of the next two to three hours , the

other females were observed spawning . The total number of spawned

eggs and the percentage of fertilized eggs were determined from the

one spawning tank . The fecundity of individual fish was estimated

by dividing the total number of spawned eggs by the number of

females ( f ive) . The percentage of fertilized eggs was found to be

very high ( i . e . , 9 9 . 9 % ) and the hatch rate from this group spawning

was also observed to be very high ( i . e . , >95%) .

ble 1 .

r MMDC.

tFIiDsh

'"-

t: TCBW

f

r

i,

BC

,·

BW

BC

BCBW

TCBC

TC

Summary of spawning trials for E . microdon conducted in July 1993

BW

(kg)

0. 79

0 . 87

1. 03

1 . 11

1.21

1 . 23

1 . 29

Egg

Diameter

( µ.m )

4 14

427

454

427

420

415

369

First

Inject.

( IU / kq)

2500

700

I 700

Second

Inject .

( IU / kq)

700

1400

I 1400

# Eggs

Spawned

Bxl05

8X105

- Bx105

Bx105

I 1. 6x106

I I no spawn

Fert .

(%)

99 . 9

32 . 6

-

Hatch

(%)

>95%

I 0I

I-

On July 14 , 1993 , an additional two E . microdon females ( and

marked as TCBC and TC, Table 1 ) were caught by hook and l ine and

placed with the same group of males that participated in the f irst

15

7.8 Page 68

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group spawning. Their hormone treatment was initiated on that sa:rnf!

day between 1 6 : 3 0 and 17 : 3 0 at a dosage of 700 IU/kg followed 24

hours later by a resolving inj ection of 1400 IU/kg . Spawning wa,s

detected for TCBC on July 1 6 , 199 3 , approximately 12 hours aftei;

receiving the second injection. Although TC underwent hydration,

she did not spawn .

,,

During the second phase of the consultancy , male and female

broodstock were caught by hook and line and treated as described'

previously. A total of five females were found to possess:

vitellogenic oocytes and induction of spawning was initiated in,

three of the females . HCG was administered at 7 0 0 IU/kg for the,

priming inj ection followed 24 hours later with a resolving,

inj ection of 1400 IU/kg . The other two females did not receive any

HCG. As with the previous spawning trials , all treated females'

spawned naturally approximately 12 hours after receiving a second

dose of HCG. The two untreated females , "TCBC and BC" a lthough

possessing mean oocyte diameters >400 µm, did not spawn { Table 2 ) .

Although all treated females underwent final maturation in

response. to the hormone treatment as indicated by their abdominal:

swelling , only one female ( i . e . , TC , Table 1 ) did not spawn . It

should be noted that she possessed the smallest egg diameters , thus

possibly indicating a critical oocyte diameter o f > 4 0 0 µ111 , for

hormone induced spawning of this species . To validate this

observation , females that possess mean oocyte diameters < 400 µm

should be obtained and further induced spawning trials attempted .

I f such individuals continue to complete f inal maturation but do

not spawn natural ly , the critical oocyte diameter will have been

determined. Future work should also focus on sequentially lower

dosages of HCG until the minimum effective dose can be determined .

Table 2 . Summary o f induction of spawning trials for E . microdon in

August 1993 at MMDC .

IFish

ID

I TC

I TW

I BW

I TCBC

I BC

BW

Oocyte

I {kg) Diameter

( 11m )

I 1 . 27

424

I 1 . 19

414

I 1 . 03

419

I 1 . 3 3

438

I 1 . 34

406

First

Inject.

( IU/ kq)

700

0

second

Inject.

(IU/kq)

1400

.

1400

0

# of Eggs Fert .

Spawned

(%)

1 . 13 x 106

1- . 13 x 106

no sp awn

no spawn

98 . 5

98. 3

70. 1

-

] Hatch

(%)

'i 9 1 . 4

1 91 . 4

91. 5 1

- ,l

i

-- '

'

Even though the priming injections used in the second spawning

trial were only one third the amount used in the first spawning

tria l , a similar response was achieved . This would indicate that

a lower dosage of HCG can be effective while significantly reducing

cost . Until further studies are conducted , it is recommended that

16

7.9 Page 69

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HCG be used exclusively for inducing final maturation and spawning.

The advantages of HCG are its standardized potency and ready

availability . However , it is higher in cost than a lternative

hormones ( i . e . , LHRH-a or pituitary homogenate) . Once the lowest

dosage of HCG that result in acceptable spawns in both consistency

and fertility has been established , alternative hormones can be

tested and the most cost-effective hormone treatment formulated .

It should be noted that when conducting future work with hormone

treatments , all females used should possess mean oocyte diameter s .

o f > 4 0 0 µm s o that a l l females are at a similar state o f maturity

when treatment is initiated.

consuAlntancoybswerasvattihoant

consistent during both phases

each consecutive spawn resulted in

of the

a lower

percentage of fertilized eggs ( 3 0 . 6% and 1 0 . 1% , Tables 1 and 2 ,

respectively) . This was particularly evident during the second

phase when the spawning of individual females was directly

observed, indicating a depletion of sperm in males taking part in

successive spawns . It is therefore recommended that to maximize

the percentage of fertilized eggs from each spawn , the composition

of broodstock be at a 2 : 1 male to female ratio . The males would

participate in only one spawning event .

Table 3 . Summary of hormone treatment for maturation of E . microdon

males .

IFish ID

Trial 1

BW (kg)

Initial State

of Maturity

Dosage of HCG

(IU/kq)

I State of

Maturation

Male 1

Male 2

Male 3

Male 4

Male 5

Trial 2

n.a.

cann . milt

cann. milt

cann . milt

1000

-

i

Male 1

2 . 05

cann . milt

500

Male 2

1 . 46

cann. milt

500

Male 3

1 . 45

cann. milt

500

Male 4

1.24

cann . milt

500

Male 5

1 . 51

cann . milt

500

- n . a . - data not available

cann . milt . = milt obtained with a cannula

milting

All males examined 24 hours after receiving HCG were found to

be in a running ripe condition, i . e . , milt could be easily extruded

by applying s l ight pressure to their abdomen (Table 3 ) • I t is

concluded that the hormone treatment was effective in inducing

spermiation at all dosages tested . Modifications in the dosage of

17

7.10 Page 70

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HCG used in induction of spawning require further investigation to

determine the minimum dosage effective for natural spawning .

Future work should also examine the effectiveness of 1.7 er-

methyltestosterone ( 1.7-MT) to increase the production of sperm .

Chronic 1.7 -MT treatment at a dosage of 5 µg/g body weight has been

demonstrated to be effective in increasing the frequency of spawns

in which· striped mullet males could participate without a decrease

in the percentage of fertilized eggs ( Lee et a l . , 1.992a ) .

Upon completion of the spawning trials , some of the captured

individuals were kept at MMDC and placed in an exhibit for public

display . The remaining individuals were returned to Ngerumkaol

Channel .

E. t'uscoguttatus

A total of nine female and male E . fuscoguttatus were caught

and examined on July 13 , 19 9 3 , and treated as described for E .

microdon with the exception of the dosage of HCG administered . An

ovarian biopsy performed on female "BC" at approximately 04 : 3 0 on

Table 4 . Summary of spawning trial for E . fuscoguttatus .

IFish ID

TC

BC

BW

(kg)

3 . 2 1.

3 . 35

Oocyte

D i ameter

( l-'m )

432

435

First

Inject.

( IU / kq)

700

7 0 0-

Second

Inject.

{ IUl k9:l

1. 4 0 0

# Eggs

Spawned

ovulated

- 1. . 5xl.06

TW

4 . 52

4 1. 1.

700

1. 4 0 0

ovulated

BW

4 . 35

400

250

500

ovulated

TCBC

3 . 83

407

NC

2 . 69

4 1.7

NC

4 . 35

464

250

500

ovulated

0

-

0

-

Maturity

of Testes

Maturity

of

Testes

MALE NC

6 . 1.5 cann . milt

700

I MALE DC

6 . 1.5 cann . mi lt

MALE/MMDC 1.0 . 3 0 negative

700

none

cann . milt = cannulated milt , not running ripe

negative = no maturation

milting

Fert.

(%)

-

8 1. . 0

-

Hat!

(t~

-'

;

52.

...,

-

-·';

-

•.

'

I

ff

July 1.5 , 1.9 9 3 , found her to have ovulated completely.

She was

then strip spawned and the eggs were manually fertil ized at 05 : 05

using the dry method (Appendix 2 , Steps - i-6 ) . The resulting

1.8

8.1 Page 71

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fecundity , fertil ization, and percent hatch of the spawned eggs are

presented in Table 4 . Females identified as TC , TW , BW and TCBC

were allowed to complete final maturation in the same tank .

However , they did not spawn and the ovulated eggs were manually

stripped on the afternoon of July 16 , 19 93 , as the ovulated oocytes

by this time period were overripe and nonviable . From past

experience with striped mullet females , if the overripe oocytes are

not removed from the females they will ultimately die . The two

untreated females , "NC" , possessed vitellogenic oocytes ( i . e . , did.

not ovulate) by the end of the trial.

Males of this species were difficult to obtain from the wild

and only two individuals were caught during the entire consultancy .

Both of these were administered HCG as described for E . miorodon

and were found to respond in similar fashion . In addition , a

single male E . fusooguttatus held captive at MMDC for public

exhibit was examined for milt . The gonadal biopsy, however , was

negative .

Although , all treated females responded by undergoing final

maturation, natural spawning was not achieved in this species . The

strip spawn of the one female resulted in fertilized eggs that

developed and hatched normally indicating that the proper state of

maturity had been achieved . It is recommended that future natural

spawning trials for E . fusooguttatus be conducted in a larger and

deeper holding facility . The same recommendations for future work

with E . miorodon regarding dosage and use of a lternative hormones

apply to E . fusooguttatus .

SPAWNING BEHAVIOR

Hormonal induction of final maturation in E . miorodon resulted

in the spontaneous release of eggs from the females and immediate

fertilization by the males . The changes in behavior during the

course of the hormone treatment , including the spawning event , were

photographed (Appendix 3 , Plate 1-3 ) . Initially, ( i . e . , after the

priming injection) both males and females were relatively inactive

and observed resting on the bottom or in the corners of the

spawning tank . By the second injection, however, the abdomens of

the treated females were noticeably dist�nded with the distention

becoming more apparent as spawning approached . With the increase

in abdominal swelling the cloaca began to protrude . Approximately

five to eight hours after the second inj ection , the males were

noticeably closer to the treated females and were observed engaging

in physical contact in the form of nudging. As each female

approached spawning , she began to swim short distances around the

tank . During these brief periods of swimming activity , the males

would collectively rush and surround her.

This activity

immediately preceded spawning . The spawning event concluded with

males (2-3 individuals) and the female often times breaking the

surface of the water . The eggs were usually released in two or

three abrupt bursts and were collected in an egg col lector coupled

to the drain of the spawning tank (Appendix 3 , Plate 4 ) .

19

8.2 Page 72

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TEMPORAL CHANGES OF GROUPER OOCYTES DURING FINAL MATURATION

ovarian biopsies performed during the induction of final

maturation and spawning for both species of grouper were examined

in order to : 1 ) evaluate whether the state of maturity at which

hormone treatment was induced was appropriate , and 2 ) identify the

stage at which the strip spawn method could be employed. The

temporal changes , in vivo , in the size frequency distribution of

oocytes obtained from E . fuscoguttatus (marked "BC" ) as she

underwent hormonal induction of f inal maturation are presented in

Figure 5 . These changes were observed to be the same for E .

microdon .

Morphological changes , in vivo, in oocytes were also observed

for both species and photographed through a compound microscope

(Appendix 4 , Plates A-D) . In summary , the changes observed in

these groupers were found to be characteristic of those observed

for other fish species , e . g . , striped mullet ( Tamaru et al . ,

1991b) • . From these observations and the .high fertili zation and

hatching of eggs obtained during this consultancy , it can be safely

assumed that oocytes >400 µm in size are receptive to hormonal

induction of spawning.

When utiliz ing the strip-spawn method, a common question is

when is the fish ready to be stripped of eggs? A prerequisite to

stripping is that the female complete ovulation. She can be staged

by conducting an ovarian biopsy and examination of the oocytes . A

photograph of an ovulated oocyte is presented in Appendix 4 , Plate

C . Note the similarity between this oocyte and that of the spawned

egg (Appendix 4 , Plate D) . When the majority (>95%) of retrieved

oocytes have ovulated , the female can be stripped and the eggs

fertilized . The percentage of oocytes that have ovulated can be

easily determined by holding the polyethylene cannula up to a light

source . The ovulated oocytes will resemble a string of small glass

beads and can easily be distinguished from vitellogenic oocytes

(Appendix 4 , Plates A and B) by their transparent appearance .

Future work should continue to focus on achieving natural

spawns from E . fuscoguttatus .

FATTY ACID PROFILES OF SPAWNED EGGS

The. fatty acid profile� of spawned_ eggs from the first

spawning trial of f ive E . mi crodon females and from the single E .

fuscoguttatus spawn are summarized in Table 5 . The analyses were

conducted by Dr . Harry Ako , Department of Environmental

Biochemistry, University of Hawaii at Manca .

For comparison , the fatty acid profiles of spawned eggs from

other marine teleosts are presented in Table 6 . The total fatty

20

8.3 Page 73

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�

24

20

!:!

I

...

F.i.:c • t :C:n:l •Cti.on

x

4 3 6 um

ao

"'

� :L•

!:! :L2

I•

..

0

'"" -

I_

Second xn;i ect�on

x - 5 3 9 um

30

.,

� 25

20

@!

Cl

:15

I

:LO

"

Spawned Eggs

X - 8 2 8 um

0

2•0

34.0 400 ..6 0

520

s e o ' ' o '7 00

750

OOCYTll: :Cl:AMBTBR CUM)

Fiqure s . Changes in the oocyte diameters of " BC" female E .

fuscoguttatus undergoing induction of spawning .

21

8.4 Page 74

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Table 5 . Fatty acid profiles of spawned eggs from E . microdon and

E . fuscoguttatus . Values are the means ± 95% C . I . from tripl icate

determinations . values are presented in mg/ 100 mg dry weight .

Fatty Acid

C14

C16

C16 : 1n-7

C18

C18 : 1n-9

Cl8 : 2n-6

C18 : 3n-3

C18 : 4n-3

C2 0 : 1n-9

C20 : 4n-6

c2 o : 5n-3

C22 : 6n-3

II Total

Epinephelus

mi crodon

0 . 45 ± 0 . 03

4 . 67 ± 0 . 19

0 . 75 ± 0 . 02

1 . 52 ± 0 . 09

2 . 77 ± 0 . 08

0 . 25 ± 0 . 02

0 . 12 ± 0 . 03

0 . 02 ± o . oo

0 . 05 ± 0 . 01

1 . 00

0 . 29

-±±

0 . 04

o . 0"1

2 . 18 ± 0 . 05

14 . 10 ± 0 . 23

Epinephelus

fuscoguttatus

0 . 32 ± 0 . 05

3 . 31 ± 0 . 12

0 . 64 ± 0 . 05

1 . 14 ± 0 . 01

1 . 73 ± 0 . 10

0 . 24 ± 0 . 02

0 . 15 ± 0 . 01

0 . 03 ± o . oo

0 . 06 ± o . oo

0 . 74 ± o . oo

-

0 . 32 ± 0 . 06

2 . 10 ± 0 . 03

11. 10 ± 0 .78

I

Table 6 . Fatty acid profiles of spawned eggs from various marine

teleosts . Values are expressed in terms of mg/ 100 mg dry weight

and are the means from triplicate determinations of a single

samp l e .

Fatty Acid

Mugil

Polyda ctyl us

cephalus

sexfi l i s

Cl4

Cl6

Cl6 : ln-7

Cl8

Cl8 : ln-9

Cl8 : 2n-6

Cl8 : 3n-3

Cl8 : 4n-3

C2 0 ; ln-9

C2 0 : 4n-6

C2 0 : 5n-3

C22 : 6n-3

II Total

0 . 21

1 . 64

3 . 21

0 . 42

4 . 27

2 . 15

0 . 15

0 . 07

0 . 13

0 . 40

0 . 58

1 . 51

I 14 . 80

.

I

1 . 06

4 . 63

3 . 00

1 . 06

5 . 28

1 . 19

0 . 15

0 . 32

o . �7

0 . 60

1 . 41

4 . 47

23 . 4

Chanos

chanos

0 . 10

3 . 57

0 . 36

1 . 13

2 . 59

0 . 36

0 . 08

0 . 02

_0 . 17

0 . 63

0 . 53

3 . 02

12 . 6

Coryphaena

hippui-us

0 . 14

1 . 74

0 . 18

0 . 99

1 . 35

0 . 78

0 . 06

0 . 02

0 . 09

0 . 59

0 . 65

1 . 77

8 . 40

acids range from a high of 2 3 . 4 mg/ 100 mg dry weight found in moi

(Polydactyl us sexfilis) to a low of 8 . 4 mg/ 100 mg dry weight found

in mahi niahi ( Coryphaena hippurus) . · When the nutritional

requirements of a target species are unknown , one strategy to

determine them is to attempt to mimic the fatty acid profile of the

22

8.5 Page 75

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spawned egg� .

from fish in

It should be noted , however , that the spawned eggs

captivity can be profoundly influenced by their

respective diets . Values obtained from the grouper eggs in the

current ·study are from wild. caught individuals and thus can be

used as a first approximation . In addition, the values obtained for

groupers in the current study can serve as a standard for

comparison against spawned eggs obtained from captive broodstock in

the event that egg quality ever becomes an issue .

ACTIVATION OF GROUPER SPERM AT VARIOUS SALINITIES

Hormone treated E . miarodon and E . fusaoguttatus males were

examined on July 16 , 1993 , for possession of milt . A sample of

sperm was collected from a single male of each species with a 5 cc

syringe and capped with a hypodermic needle . The sperm was then

activated at several different salinities to determine the salinity

and respective osmotic pressure at which sperm activate . Solutions

with salinities higher than 35 ppt were prepared by mixing seawater

with Instant Ocean Sea Salt . Salinities below 3 5 ppt were prepared

by serial dilutions of seawater with fresh water . Osmotic pressure

at the respective salinities were calculated using the formula

described by Lee et al . , ( 1992b) :

mOsm/kg = ( 2 9 . 572 * salinity ppt) -8 . 859

The results of the experiment (Table 7) indicate that salinities

below 27 ppt are not conducive for activation of sperm from these

species of groupers .

Table 7 . Activation of E . microdon and E . fusaoguttatus sperm at

various salinities .

ISalinity

(ppt)

5

7

9

12

15

20

27

35

47

62

72

95

Osmotic

Pressure

mosm/kg

139 . 0

198 . 1

257 . 3

346. 0

434 .7

582 . 6

789. 6

1026 .2

1381. 0

1824 . 6

2 12 0 . 3

2800. 5

E.

fusaogu ttatus

-

)

-

- (weak)

+

.

.+

-

+

I E. miarodon

-

- (weak)

+

The data define the critical ranges in salinities in which

fertil ization can take place. The results of the experiment also

23

8.6 Page 76

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provide basic information regarding the osmolarity at which sperm

from both species of grouper are activated .

It has been

demonstrated that sperm from marine fishes are activated in

response to osmotic pressure rather than the presence of specific

ions (Morisawa , 19 8 S ; Lee et a l . , 1992b) . This information was

used to test various solutions as sperm extenders for use in future

work involving strip spawning .

SPERM EXTENDERS

It -i s hypothes ized that sperm extenders at low ( i . e . , S°C)

temperatures work either by suspending the activity of sperm in a

physiologically suitable ( i . e . , osmotically) medium or by providing

an adequate supply of energy to keep the sperm active for a

prolonged period of time. The energy source is usually in the form

of sugars . The mechanism of action varies with the species but can

be initially tested by mixing raw milt with various solutions

containing sugar at varying concentrations .

on August 14 , 19 9 3 , milt from an E . microdon male that had been

inj ected with HCG was obtained by gentle stripping and collection

with a 12 cc syringe . The milt was then immediately capped with a

hypodermic needle . Four solutions ( i . e . , fish ringers , 1% glucose ,

S % glucose , and 10% glucose dissolved in fish ringer s ) were tested

for their effectiveness as sperm extenders . The fish r inger

consisted of

o . 03 S g MgC12

1.3S g

in 100

NaCl , 0 . 0 6 g

ml distilled

KCl , 0 . 02

water and

g

s

oNaIHUC/0m3l,

o . 02 s g cacl2 ,

penicillin , as

described by Chao et al . , ( 1 9 7 S ) . The glucose solutions were

prepared using a-D-glucose dissolved in fish ringers in a

weight/volume ratio . The osmotic pressure (mOsm/kg) of the

extenders was measured using a Wescor S100 C Vapor Pressure

osmometer . Equal volumes of raw milt and the respective s olutions

were mixed

hypodermic

in separate

needles .

12

All

cc hypodermic syringes

samples were stored

and

at

sc0cap. pedSpweirtmh

activation was initiated by adding a drop of seawater to a drop of

milt or milt + extender placed on a microscope s l ide . A coverslip

was then used to combine the two drops . Activity was qualitatively

scored ( ranging from +++ = active to - = no activity , Table 8 ) at

various time intervals as observed under a compound microscope .

Sperm could be activated in all solutions five days after the

initiation of the experiment . By Day 9 of the experiment only

sperm obtained from the raw milt exhibited s igns of activity. The

solutions containing glucose have been reported to extend the life

of sperm from striped mullet up to 21 days when stored at 5°C .

However , the

least S days

fact

when

that

kept

saptersm0c

from grouper

is valuable

remained viable

information for

for at

future

spawning trials . The fertility of the extended sperm still

requires further investigation . Future work should focus on the

use of other reagents (glycerin , DMSO) reportedly effective in

extending the life of sperm in other species .

24

8.7 Page 77

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Table 8 • . summary

E . microdon at 5

°oCf.

various

sperm

extenders .on

maintaining

sperm

of

Treatment

Osmotic

Pressure

mOsmLkg:

Raw Milt

n.a.

Fish Rinqer

420

1% Glucose

462

II 5% Glucose

700

l 10% Glucose

986

n . a . = not available.

8/14 8/15 8/16 8 / 19 8/23

+++

-

+

+++

-

+++

++

+++

++

+

-

- II

EMBRYONIC DEVELOPMENT OF GROUPER LARVAE

The temporal changes in embryonic development for both species

of groupers were obtained by photographing various stages of

development at approximately one hour intervals through a compound

microscope. The stages of development and- time interval s for E .

fuscoguttatus are sumam rized in Table 9 . Photomicrographs for each

stage of development are presented in Appendix 4 , Plate A-P .

Table 9 . Temporal changes in oocytes undergoing final maturation,

and developmental series of eggs from the strip spawn of "BC" E.

fuscogu ttatus at 2 7°C and 34 ppt .

Plate Number

A

B

c

D

E

F

G

H

I

J

K

L

M

N

0

p

Date

7 / 14

7 / 15

7/16

7 / 16

7/16

7 / 16

7 / 17

Time

17 : 04

17 : 4 1

05 : 30

06 : 00

06 : 3 0

06 : 45

07 : 00

09 : 00

10 : 00

10 : 30

11 : 15

12 : 15

16 : 15

22 : 00

24 : 00

Stage of Development

Vitellogenic Oocyte , First Inj ection

Vitellogenic Oocyte , Second Inj ection

Ovulated Oocyte

Spawned Egg i

First Cleavage , Two-cell Stage

Second Cleavage , Four-cell Stage

Third Cleavage , Eight-cell Stage

Multicell Stage

Blastula, Top view

Blastula, Side view

Early Gastrula

GEiaibsi tryruol. aFotrimonation

Semite Stage

Late Embryo , Muscle Twitching

Newly Hatched Embryo

25

8.8 Page 78

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b~j To determine the quality of a spawn , the investigator must

able to distinguish between "good" and "bad" eggs . Cleavag~}

greatly s impl i f ies the identification of "good" eggs . "Bad" o~

unfertilized eggs , however , can be more difficult to detect,i

especially when poor ferti lization is a result of the mal e . Two'

examples of "bad" eggs are presented in Appendix 4 , Plates Q and

Development was found to be similar for both species in regard

to egg size and rate of development . Hatching took place 18 . 5

hours af�er fertilization of �he eggs which j.s consistent with that

reported for this species ( Chao et al . , 1991) . The rate of

development and time of hatching after fertili zation for E .

mi crodon was found to be comparable.

Future studies should focus on the effects of salinity and

temperature on the rate of embryonic development to define the

optimal conditions for hatching.

BGG TRANSPORT

Fert i l ized E . microdon and E . fuscoguttatus eggs were packed

at a density of 5000 eggs/ l and transported via airline to GADTC to

conduct larval rearing trials . This density has been successfully

used to transport striped mullet and milkfish eggs from Hawaii to

Guam . Because the distance between Palau and Guam is much shorter ,

an experiment simulating transport conditions ( Figure 6 ) was

conducted using E . mi crodon eggs to determine i f spawned eggs could

be transported at a higher density without loss of viability .

Fertilized eggs at various densities were stocked into plastic bags

containing one l iter of seawater at a salinity elevated to 4 0 ppt

(by adding Instant Ocean sea Salt) . The bags were f i l led with

oxygen, sealed with rubber bands and placed into a styrofoam cooler

for eight

After the

hours .

elapsed

tAilmle,tr1e0a0tmeegngtssfrwoemreeaccahrrbiaegd

out

were

in triplicate .

placed into 1-

l iter beakers filled with seawater at 34 ppt and allowed to hatch .

The number of hatched larvae was then determined and the data

summarized . The results of the experiment indicate that grouper

eggs can be transported from Palau to Guam at a density as high as

2 0 , 000 eggs / l . Eggs transported at this density would obviously

result in a significant savings in cost . Future work should

include studies to determine if fertilized grouper eggs can be

shipped from Palau to Guam at even higher ( >2 0 , 000 eggs / l )

densities and retain their viability .

LARVAL REARING

Larval rearing includes the production of l ive feeds at

sufficient quantity and quality for larval survival and growth . The

production of live feeds includes phytoplankton and rotifer

culture , copepod production, and the production and enrichment of

Artemia naupl i i . Once l ive feeds can be produced on demand , the

optimum densities of feed and larvae and the temporal changes in

26 ·

8.9 Page 79

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100 -

80 -

-

:i:

u

� 60 -

� - 40 -

ti!

II<

20 -

0

1000/I..

SOOD/I.. 10000/I.. 20000/I.. 100000/I..

EGGS/LITER

Fiqure 6 . Summary of hatched E .microdon larvae stocked at

various egg densities under s imulated transport

conditions .

preference for a particular feed that occur as the larvae grow need

to be established.

One of the reasons that groupers have not enj oyed the success

of othe� marine species unde� culture is �hat grouper larvae are

one of the smallest encountered in mariculture and thus require a

small ( 7 0-100 µm) initial feed. The rel�tionship between spawned

egg diameters and total length of larvae at hatching of several

other fish species under culture is presented in F igure 7 . To

further il lustrate this point , the morphological changes that

occur during the first four days posthatching of E . fuscoguttatus

are pictured in Appendix 5 , Plate 1-6 . The mouth of the grouper

larvae is not fully developed until Day 2 or Day 3 posthatching .

Measurements from four-day-old larvae indicate a mouth s ize of 150

µm when fully extended (Appendix 5, Plate 6 ) . This is smal ler than

the average size of the s-type rotifer ( 17 0 µm, lorica length)

currently cultured at GADTC . Finding a suitable initial feed is a

major hurdle to be overcome in the development of a protocol for

the larval rearing of grouper .

27

8.10 Page 80

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LIVE FEEDS PRODUCTION AT GADTC

Each live feed cultured at GADTC and their methods of cultur~'J

were reviewed and modified as necessary to meet the requirements

anticipated for the hatchery production of grouper . In addition,

oyster trochophores were tested as an initial feed for grouper

larvae .

26

! 18

I

� 10

ba•••

ca:c p .

sole

�turbot 2_ b.:eam

U: g:co er

mili::f ish

mullet

•

t:cout

0

2

4

EGG DIAMETER (mm)

•

salmon

6

Fiqure 7 . Relationship of spawned egg diameter versus total

length of hatched larvae from various species under

culture .

oyster Trochophores

one obj ective of this consultant was to determine the

feasibility of oyster trochophores as an initial food for grouper

larvae at GADTC . cryopreserved oyster trochophores were purchased

from MTL Biotech, Ltd . , Victoria , B . c . , Canada , for this purpose

( see Appendix 6 for more information regarding the preparation and

handling of this product) • In areas such as Guam where oysters are

difficult to obtain and spawn , this product allows for direct use

28

9.1 Page 81

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of this l ive feed with minimum preparation . A sample has been

submitted for fatty acid analysis . The results of the larval

rearing trials using oyster trochophores are presented in a later

section . A photomicrograph of a rotifer and oyster trochophores is

presented in Appendix 6 , Plate 1, providing a size comparison . It

is apparent that the size of the oyster trochophore is suitable for

first-feeding grouper larvae. The densities necessary for adequate

larval survival have yet to be determined . once the optimal

densities have been determined, the results should be evaluated at

commercial scale so that the economic feasibility of using oyster

trochophores as a feed can be established .

Phytoplankton Production

Production of the phytoplankton , Nannochloropsis ocul ata , has

been ongoing at GADTC s ince 19 9 1 . This particular species of

phytoplankton is vital to the development a hatchery technology for

grouper because of its high fat content , in particular , the long

chain polyunsaturated fatty acids C2 0 : 5n-3 and C2 2 : Gn-3 . At

presrnt , the facility can produce 4000 l iters/day at a density >10

x 10 cells/ml , or enough to e;upply their cyrrent needs . However ,

expansion to larger larval rearing systems will require an increase

in volume of production and additional staff .

N . oculata is currently cultured at 2 8 ppt by the batch

culture system utiliz ing a commercial preparation of nutrients

( i . e . , Mass Pack, Florida Aqua Farms , Inc . ) . No improvements are

currently suggested for the production of this species at GADTC.

The only recommendation is that an increase in volume of production

be considered to meet the projected expansion of larval marine f ish

culture . This would require additional tank space and aeration

capacity. GADTC has purchased materials for the construction of 1 0

additional 2 0 , 00 0-liter round f iberglass tanks that are scheduled

to be installed in 19 94 .

Rotifer Production

Currently, the s-type rotifer (Brachionus plicatilis ) is

cultured at GADTC by the batch culture system described by Lubzens

( 19 8 7 ) and referred to here as the "Old Method . 11

The

phytoplankton , N .

Dai ly production

oculata is

ranges from

used

100

teoxc2l0u0sivxel1y06

as a feed source .

rotifers/day. A

modified version of batch culture system called the "New Method"

. was introduced by the con�ultapt to improve daily rotifer

production as well as the nutritional prof ile of the rotifer s . A

comparison of both culture systems is schematically presented in

Figure 8 . Production results from both systems are summarized in

Table 10 . The "New Method" produces a s ignificantly higher number

of rotifers per day . This is accompl ished by increas ing the amount

of food available to the rotifers resulting in an increase in the

rate of reproduction as depicted in the increased egg ratio values .

29

9.2 Page 82

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OLD METHOD

NEW METHOD

l!ITBP 1 .

CLBAl1 T.unt

ADD 500 L:I:Tmtll Pllr.l"OPLAJrl"lNCa

J:NOC:."QLT.A S llOTX� AT 100/JllL

l!ITBP :I. .

CI.BAH TAHll:

AIJO 500 Lr1'DB l'Krl'O�

DIOCQLATS RO"l"J:l'a. AT 100/ML

STBP 2 ,

STBP 3 ,

lmaVU'1' :a:ar cwu.

ADD 1000 LJ:TJ:RS �

U:CHOCULATS llAltV'UTl:Dl Jtar:tll'D.8

S'l'EP 3 ,

HARYU'1' ltO'r:IPBlSl

QUAHT.IPr ltO'l'.IPBRB llAlSlVZ TBD

DIOCULA.TB NEW TAHICa

Pm:m ltO'l'l:PmtS '1'0 1'%SH :t.\\&'YAZ

STBP 3 ,

JQ.JtYB8'1' RO'l':CPBRS

QUAlr:r CPr JtOr:IPBR.1'1 BAJt.V'UTED

DJOCO'LATB HBW TAHltll

:.ml:D ZDr:Cl'DS '1'0 P.E:SK L\\RVAll

Piqure 8 . Schematic representation of the two batch culture

methods for culturing rotifers as GADTC.

Table 10 . summary of rotifer production at GADTC using different

batch culture methods .

Old Method

Trial 1

Daily Production

160 x 106

Trial 2

Daily Production

Eqq Ratio

143 x 106

1.0 %

New Method

Trial 1

Daily Production

2 2 0 x 106

Trial 2

Daily Production

Eqg Ratio

183 x 106

17 . 6%

Because of the increase in the phytoplankton consumed by the

rotifers , the amount of fat present in the rotifers . also increases

30

9.3 Page 83

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(Table 11)

irespective

. The fatty

methods were

acid profiles of rotifers produced by the

analyzed by Dr . Harry Ako of the Department

of Environmental Biochemistry at

Rotifers cultured by the "Old

the University

Method" show

of Hawaii at

fatty acid

Manca .

values

consistent with previous reports (Tamaru et al . , 1 9 9 1 ) . With the

exception of four fatty acids , all others signif icantly ( P< 0 . 05 , t

test) increased when cultured by the "New Method. "

Among the

fatty acids that exhibited an increase are the long chain fatty

acids C2 0 : 5n-3 and C22 : 6n-3 or EPA and DHA, respectively . Both .

have been found to be essential for survival , growth and stress

resistance in larval marine fish ( Sorgeloos et a l . , 1 9 9 1 ) . The

"New Method" thus not only inc:reas�s daily ;-otifer production , but

results in a rotifer with a higher nutritional value as wel l .

A newly developed product from Provesta Corporation , Oklahoma ,

( i . e . , Microfeast 1 00) tested to boost rotifer fatty acid profiles

reportedly results in the highest EPA and DHA values . The values

are presented in Table 11 for comparison. A modification of the

"Old Method" by

20-liter bucket

st.heTahdedciotmiobninao�f itohnisdipfrfoedruscftrwoamsttheest"OedldaMteGtAhDoTdC"

in

by

the addition of O . 6 g/1 x 10 rotifers which were g iven in two

separate feedings on the second day of the culture process . A

side-by-side comparison of all three methods is presented in Table

12 .

Table 11 . Comparison of fatty acid profiles from rotifers produced

at GADTC by different batch culture methods . Values are the mean

± 95% CI from three separate determinations and are expressed in

terms of mg/ 100 mg dry weight .

Fatty Acids

C14

C16

C16 : 1n-7

C18

C18 : 1n-9

C18 : 2n-6

C18 : 3n-3

C18 : 4n-3

C2 0 : 1n-9

C20 : 4n-6

C20 : 5n-3

c22 : 1n-11

C22 : 6n-3

Total Fatty

Acids

Old Method New Method

0 . 15 ± 0 . 14

1 . 64 ± 0.• 18

0 . 48 ± 0 . 05

0 . 36 ± 0 . 10

0 . 48 ± 0 . 09

0 . 33 ± 0 . 04

0 . 02 ± 0 . 02

0 . 10 ± 0 . 01

0 . 36 ± 0 . 02

0 . 76 ± 0 . 05

0 . 03 ± 0 . 01

0 . 48 ± 0 . 04

5 . 22 ± 0 . 57

0 . 31 ± 0 . 16

. 2 . 4 9 . ± 0.. 22

0 . 79 ± 0 . 16

0 . 57 ± 0 . 44

0 . 83 ± 0. 46

0 . 51 ± ! 0 . 25

0 . 04 ± 0 . 02

0 . 01 ± o . oo

0 . 14 ± 0 . 07

0 . 63 ± 0 . 22

1 . 11 ± 0 . 23

0 . 04 ± 0 . 03

0 . 77 ± 0 . 28

8 . 09 ± 0 . 74

Microfeast

100

0 . 17

0 . 16

0.20

0 . 44

1 . 09

2 . 11

0 . 44

0 . 16

0 . 29

0 . 14

1 . 82

0 . 14

1 . 57

I 9 . 82

31

9.4 Page 84

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Table 12 . comparison of production and fertility of rotifer~]

cultured by three different methods .

Old Method

Trial 1

Daily Production

Egg Ratio

3 . 2 x 106

7 . 6%

I~ I Trial 2

Daily Production

Ratio

2 . 2 x 106

23 . 0%

New Method

3 . 5 x 106

2 5 . 3%

2 . 3 x 1 06

38 . 0%

Combination

4 . 3 x 106

16 .8%

2 . 1 x 106

13 . 7%

Results from these small-scale experiments indicate that there·

are no differences in overall production among the three culture

methods . The fatty acid profiles of the rotifers cultured with

Microfeast-100 in combination with phytoplankton are currently

being analyzed . Future work - should focus -on validation of these

initial results at production scale . The results o f those trials

and the fatty acid profiles can then be used to determine the best

method of culturing rotifers for use in the rearing of grouper

larva e .

The size frequency distribution of the s-type rotifers

produced at GADTC ( Figure 9 ) was determined by measuring the lorica

length of rotifers that were not carrying eggs , using a compound

microscope equipped with an ocular micrometer . The s ize of the

mouth of grouper larvae examined on Day 4 posthatching suggests

they can physically eat rotifers less than 150 µm. This would mean

that only 19% ( i . e . , shaded area of Figure 9 ) of the rotifers

currently cultured are small enough for first-feeding grouper

larvae . The practice of s ieving rotifers through a 150 µm nytex

screen has been found to be extremely wasteful and labor intensive .

In addition , the screened rotifers can increase in size within six

hours which would make this practice almost pointles s .

An

alternative is to establish a population of smaller s ize rotifer at

GADTC. A super small strain of rotifer ( i . e , SS-type , average

lorica length 142 µm) has reportedly been used successfully in the

rearing of E . fuscoguttatus larvae. A starter culture was provided

by Dr . Atsushi Hagiwara , Nagasaki University , Japan . Due to a

misunderstanding of schedules , this particular starter culture did

not survive the trip from Japan . It is recommended that further

attempts be made to establish this strain of rotifers at GADTC for

use in the larval rearing of grouper .

Harvest of Copepods

Copepods have been reported to be an important larval fish

food if they can be cultured or harvested in sufficient numbers

( Fukusho , 1 9 9 1 ) . S ince the concrete raceways at GADTC that hold

32

9.5 Page 85

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I

126

137

147 158 168 179 189

LORICA LENGTH (UM)

2 0 0 2 1 1 .2 2 1

Figure 9 . Size frequency distribution of s-type rotifers being

cultured at GADTC . Shaded area represents size

classes that are acceptable to grouper larvae .

shrimp (Penaeus stylirostris) broodstock were observed to have a

large number of copepods , a study was done to determine their

relative abundance. The harvest was conducted at night using a

siphon hose to filter the raceway water though an aquarium scoop

net and collect the copepods in a 60 µm mesh bag ( i . e . , rotifer

collecting net) • The collecting net was suspended in a plastic tub

filled with seawater . A flashlight was placed j ust above the

siphon hose to attract the copepods . The s iphoning was conducted

for one hour on four consecutive evenings . The raceway conditions

were altered previous to two of the evening collection effort s .

One day the tank raceway was cleaned o f debris and on another day

a prophylactic formalin/ copper treatment for "black gill" had been

added to the water . The results are summarized in Table 13 .

Approximately 8.5 , 000 copepods were harvested per hour of collection

on the two evenings when there was no disruption to the raceway .

However , the numbers of copepods that could be harvested fell

33

9.6 Page 86

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dramatically when the broodstock tanks were cleaned and/or the

broodstock administered treatment . Collection of copepods was als_oi

attempted during daylight hours but resulted the harvest of very

few ( < 1000 /hour) individuals .

Table 13 . summary of harvesting copepods at night from Raceway #2

at GADTC .

II Date

July 2 0

July 2 1

July 22

July 23

# Harvested/Hour

Conditions

II

8 6 , 111 -

8 4 , 63 0

2 , 700

8 , 000

..

- Normal

Normal

Raceway Bottom S iphoned

Formalin£Co~er Treatment

~

The number of copepods harvested were significant enough to be -

used in the larval rearing trials for grouper, and one of the !

initial harvests was used to inoculate the extensive larval-rearing

tank ( see section Extensive Larval Rearing) •

Presently , an

analysis of the fatty acid composition of the collected copepods is

being conducted.

By harvesting the copepods as described, the younger stages of

the copepods were also captured in the process . In addition , many

of the adult copepods were found to be carrying eggs (Appendix 6 ,

Plate J ) , and thus are a natural source of seed for young copepods

when stocked into larval-rearing tanks . The young copepods are in

the 7 0-100 µm size range which is suitable for an initial feed for

grouper larvae . A comparison of a rotifer and a copepodite is

presented in Appendix 6 , Plate 4 .

Artemia Nauplii Production

Production of Artemia nauplii- is cons�antly ongoing at GADTC

for use in the rearing of shrimp postlarvae and larval catfish . An

example of a newly hatched Artemia nauplii i s presented in Appendix

6 , Plate 2 . All of the feed organisms were photographed at the

same magnification to il lustrate the size s imilarities and

differences among them . It has been reported that Artemia cysts

are commercially available from three major sources : San Francisco

Bay, The Great Salt Lakes in Utah and Saskatchewan in Canada

( Sorgeloos et al . , 1 991 ) . As cysts from all three sources are

characteristically deficient in the long chain polyunsaturated

fatty acids , samples of newly hatched and enriched (with Protein

Selca at J O O ppm) nauplii were obtained and are currently being

analyzed. A comparison of the fatty acid profiles from newly

hatched and Artemia nauplii enriched with two types of boosters are

presented in Table 14 ( obtained from Tamaru et al . , 19 9 J b ) . The two

enrichment media significantly elevate the total fat content of the

naupl i i , in particular, both C2 0 : 5n-J and C2 2 : 6n-J ( i . e . , EPA and

DHA , respectively) were significantly elevated .

34

9.7 Page 87

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� ,able 14 . Fatty

. temia naup l i i .

acid profiles of newly hatched and o;mr ched

Values are the means from triplicate

. eterminations and are expressed in terms of mg/ 1 0 0 mg dry weight .

data was obtained from Tamaru et al . , 1993b .

Fatty Acid

Newly Hatched

Artemia

Naup l i i

C14

C16

C16 : ln-7

C18

C18 : ln-9

C18 : 2n-6

C18 : 3n-3

C18 : 4n-3

C2 0 : ln-:9

C20 : 4n-6

C2 0 : 5n-3

c2 2 : 1n-11

C2 2 : 6n-3

TOTAL

n . d . = not detected

0 . 06

0 . 82

0 .32

0 . 53

1 . 52

0 . 49

1 . 94

0 . 28

0. 05

0 . 17

0 . 36

n.d.

6 . 55

Enriched

Artemia

(Menhaden Oil)

300 m

0 . 36

1 . 77

0 . 75

0 . 77

2 . 38

0 . 64

2 . 57

0 . 47

0 . 12-

0 .20

1 . 02

0 . 02

0 . 50

11 . 55

Enr i ched

Artemia

( Super Selco)

100 m

1 . 13

1 . 68

0 . 40

0 . 80

2 . 81

0 . 68

2 . 58

0 . 18

0 . 23

0 . 14

1 . 28

0 . 06

0 . 69

11 . 66

This data and the report by Lin et a l . , ( 19 9 1 ) suggest that

Artemia nauplii used in the rearing of grouper larvae should be

enriched . Therefore, the staff at GADTC should become familiar

with and include procedures for boosting the fatty acids of Artemia

nauplii as part of their daily activities .

One of the current activities at GADTC is the hatchery

production of P . stylirostris j uveniles for

Guam. Nauplii production ( i . e . , 100-300

the

x1

0s3h/ rdaimyp)

industry in

from mated

females occurs daily but rearing is not conducted for all spawns

due to the lack of rearing space . Likewise, the naup l i i are only

reared in response to demand . _ In short , a potentially large number

of nauplii that are equivalent in size to that of Artemia nauplii

are being disposed of . Samples of nauplii were obtained and the

fatty acid analyses are in progress. The nutritional analysis is

being conducted to evaluate the potential use of the excess naupl i i

a s a feed for grouper larvae.

35

9.8 Page 88

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Sea

Sea urchin eggs have long been used in the study of

development because of their availabil ity and the ease by

spawning can be manipulated . Sea urchin larvae can also serve

· an alternative initial feed to· oyster trochophores because of

size ( 7 0- 1 0 0 µm) . During this consultancy , several locations,

around Guam were surveyed to determine areas where this species can

be obtained . Two locations on the west shores of Guam ( i . e . , Tumon

Bay and Asan Point) were devoid of this species . However , this

species of sea urchin was found in the holding faci l ities at the

University of Guam Marine Laboratory on the east coast of the

island. Interviews with staff indicate an abundance of this

species j ust offshore of the laboratory .

A procedural guide to the induction of spawning this species

is provided in Appendix 7 , courtesy of Dr . Samuel Haley , Department

of Zoology , University of Hawaii at Manca . It is recommended that

a broodstock of T . gratilla be established at GADTC and the

reproductive season be defined both in captivity and in the wild.

EFFECT OF LIGHT ON NEWLY HATCHED LARVAE

It has been reported that grouper larvae are extremely

sensitive to l ight , particularly from the time of hatching to

feeding ( Lin et al . , 1991) • One rearing trial o f E . microdon

larvae during the first phase of the consultancy was conducted in

3 0 0 0- liter fiberglass tanks situated in a back room of the hatchery

· at GADTC-. Although this area - is the darkest area in the hatchery ,

total mortality was observed prior to Day 3 posthatching ( i . e . ,

prior to initial feeding) . A similar observation was made at MMDC

where the newly hatched larvae were held in glass aquaria .

These observations prompted an experiment during the second

phase of the consultancy . At the MMDC facility , spawned E .

microdon eggs were stocked at a density o f 1 0 0 - 1 5 0 eggs/ l and

al lowed to hatch in two glass aquaria equipped with continuous

aeration . One aquarium was covered with black plastic sheet while

the other was exposed to ambient light conditions . Larval survival

was determined volumetrically and survival was recorded at various

intervals ( Figure 1 0 ) . As observed previously , the newly hatched

larvae of E . microdon appeared to be sensitive to l ight and

covering of the larval-rearing tanks during the first three days

posthatching must be undertaken with all future larval rearing

trials to ensure surviva l .

FIRST FEEDING EXPERIMENT

Small-scale ( 15-liter) larval rearing experiments were

conducted during both phases of the consultancy to evaluate the use

of available live food organisms for the initial feeding of grouper

larvae . During the first phase of the consultancy , f ifteen 15-

liter plastic tanks were stocked with 3 0 0 larvae/tank ( i . e . , 2 0

36

9.9 Page 89

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100

h/FFYl LIGHT

80

- DARK

60

40 -

20

0

24

48

INCUBATION (HOURS )

Figure 10 .

Effect of l ight on survival of E . microdon

larva e .

larvae / l ) at a salinity of 3 4 ppt . Each tank was provided aeration

via a single

of 300-500 x

a1i05

stone and

cells/ml .

stocked with phytoplankton at a density

Each day , 25% of the volume of water

was siphoned from each tank and replaced with phytoplankton . All

tanks were covered with a shade cloth throughout the course of the

experiment . Five d ifferent feeding treatments were conducted and

triplicated. The experiment was carried out for seven days

posthatching . The treatments were :

TRIAL 1

Treatment #1 oyster trochophores ( 2 0 /ml)

Treatment #2 Oyster trochophores ( 1 0/ml) + Rotifers ( 1 0 /ml )

Treatment #3 Rotifers ( 4 0/ml ) .

Treatment #4 Rotifers ( 4 0/ml) + ML-100 Artificial feed

Treatment #5 No feed ( Control )

37

9.10 Page 90

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High mortalities were observed between Day 2 and Day

posthatching . By the end of the experiment only three larv

survived . Two larvae were found in a tank receiving Treatment

and one larvae remained in a tank receiving Treatment # 1 .

of the large number of mortalities that occurred prior to fir.

feeding , the results are inconclusive . However , that·

larvae were present in treatments containing oyster trochophore

provides indication of the importance of this product as an

feed .

,

The rearing experiment during the second phase of

consultancy was conducted using the same rearing tanks and

. the same conditions except that the exterior of each tank wa.

painted black to prevent exposure to light . The experiment desig.' ,

was as follows :

2

Treatment #1 oyster trochophores only ( 2 0 /ml)

Treatment #2 Oyster trochophores ( 10/ml) + Rotifers

Treatment #3 Rotifers only ( 4 0/ml)

Treatment #4 No feed ( Control )

( 3 0 /ml)

The second rearing trial also sustained high mortal iti

during the initial three days posthatching and the experiment

terminated on Day 5 . The experiment was terminated since the

larvae in the control tanks ( i . e . , no feed) died and the results of

the feed could thus be evaluated ( Figure 11 ) . overal l survival was

very poor ( i . e . , < 1 . 0 % ) and no significant differences could be

detected among treatments (analysis of variance) . Although

is no statistical validity to the results , once again the highest

survival was observed in treatments employing oyster trochophores .

Future work should focus on varying the rotifer/trochophore ratios

at different larval densities to obtain the optimal ratios of feed

to larvae . Small-scale tri�ls snould be_ covered with a black

plastic sheet during the first three days posthatching .

It should be emphasized that these trials were conducted at

laboratory sca le . Extrapolation of the results ' to commercial scale

indicate that the use of oyster trochophores would not be

economically feasible, thus warranting a review of alternative

feeds of similar size ( e . g . , sea urchin, trochus , giant clam, and

mussel larvae) during future research activities .

INTENSIVE LARVAL REARING

Intensive larval-rearing trials were conducted in 3 000-liter

·fiberglass tanks during both phases of the consultancy . During the

first phase , two tanks were stocked with spawned eggs from E .

microdon and two tanks were stocked with eggs from E . fuscoguttatus

at a density of 2 0 eggs / l . The salinity of the water ranged

between 3 2 and 3 4 ppt and moderate aeration was provided to each

tank . The tanks were filled with only 1000 liters of water because

38

10.1 Page 91

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o.a

0.6

,,

�

H 0.4

�

(ll

0.2

0

NO FEED

OYSTER

OYSTER/ROTIFER

TREATMENTS

ROTIFER

Fiqure 1 1 .

summary of small-scale larval rearing trial of

E . microdon .

of the rationing of the oyster trochophores .

The hatching

percentage was estimated to be 20% , resulting in an initial larval

density of 4 larvae/ l . All tanks were stocked with oyster

trochophores ( 10/ml ) , s-type r'?tifers ( 3 0-4:0/ml ) , and phytoplankton

at a density of 3 0 0-500 x 1 0 cells/ml from Day 2 posthatching .

Initially, only the tanks containing eggs from E . fuscogu ttatus

were provided with shade clotfi . Tlie feeding regimen was to remain

the same until Day 12 posthatching at which t ime Artemia naupl i i

would be introduced at an initial density of 0 . 0 1/ml and increased

depending upon the amount of consumption by the larvae .

Major mortality of E . microdon larvae was observed by Day 3

posthatching and the rearing trial was terminated. The mortality

occurred prior to the feeding of larvae and the lack of a shade

cloth cover was presumed to be the cause .

The initial larval survival of E . fuscogu ttatus remained

relatively high over the first f ive days posthatching . Major

mortality occurred on Day 6 posthatching and the rearing trial was

39

10.2 Page 92

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terminated on Day 7 . The lack of oyster trochophores was presume .

to be the major cause in this case. Project funding allowed

enough trochophores to feed the larvae only until Day

posthatching .

.

During the second phase of the consultancy , four rearing

were stocked with eggs from E . miarodon at a density of 4 0

The tanks were again filled at a low level of 1000 l iters to

the oyster trochophores and initial feeding was delayed until

3 posthatching. The four tanks were divided into two

treatments ( i . e . , two received oyster trochophores and rotifers

densities of 10 trochophores/ml : 3 0 rotifers/ml and two received

rotifers . only at 4 0 rotifers/ml ) . All tanks were covered

shade cloth and black plastic sheeting continuously during

initial three days posthatching . Addition of phytoplankton

daily maintenance was carried out the same as described in

first rearing trial . Larval survival in all tanks was

improved during the first five days posthatching in comparison

the first trial , where total mortality occurred by Day 3 . Heavy

mortality in the second trial occurred on Day 6 and the experiment

was terminated . The preliminary results indicate that the feeding

ratio of trochophores : rotifers needs further investigation . Based

on the trends observed in the small-scale rearing trials , future

trials should employ a higher initial stocking density o f

trochophores ( i . e . , at recommended density of 2 0 trochophores/ml : 2 0

rotifers/ml ) and initial feeding should begin on Day 2

posthatching .

EXTENSIVE LARVAL REARING

One extensive larval-rearing trial was conducted in a 2 0 , 000-

l iter fiberglass tank during the initial phase of the consultancy .

Tank preparation began eight days prior to the stocking of E .

fusaogu

initial

ttatus eggs

density of

.

1

-P5hyxto1p06lacnekltlosn/mwlasansdtoaclkleodweidnttoo

the tank at an

grow . On the

second day after the introduction of phytoplankton , 7 0 , 0 0 0 copepods

collected from Raceway #2 were stocked into the tank . Rotifers

were then stocked into the tank on the fourth day after

introduction of the phytoplankton . Rotifers were added at an

initial density of 5 rotifers/ml and allowed to grow over the next

three days to attain a density of 15-20 rotifers/ml when the eggs

were stocked . Egg stocking density was 2 eggs / l . Although the

rearing tank was covered with a shade cloth, heavy rains lowered

the salinity to 2 7 ppt at the time of stocking . By first feeding ,

rotifer densities remained at 2 0 rotifers/ml and estimates of

copepod densities indicated that their population had at least

doubled during the same time period . on the last day of the first

phase of the consultancy ( Day 10 posthatching) , several hundred 10-

day-old _grouper - larvae could .be s(!en in ·th_:e tank, apparently and

growing in s i z e . GADTC staff attempted to rear the larvae through

to j uvenile stages ( i . e . , 25-30 . days posthatching) . Estimating

survival in this tank was very difficult but using the 2 0 % hatching

40

10.3 Page 93

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rate observed in the intensive rearing tanks , an estimated 8 0 0 0

larvae would have been present at hatching . A 1 0 % survival would

indicate approximately 8 0 0 larvae , which is a close approximation

of the amounts present on Day 1 0 posthatching . Larvae were

detected unti l Day 15 posthatching , after which no larvae could be

found .

Upon arrival at GADTC for the second phase of the

consultancy , the consultant found a large number of medusae in the

larval-rearing tank at a density of 5-10/ l . The presence of

medusae is the probable cause of mortality in this trial as they

are carnivorous and feed on larvae as well as live feed organisms .

Interviews with the staff at GADTC indicate the occurrence of

medusae during other rearing trials at GADTe and this appears to be

an ongoing problem. An examination of the entire system, and

especially the main seawater source is warranted to determine the

origin of the medusae . One known location ( Bauerlein, pers . comm . )

of the medusae is in the shrimp broodstock raceway that was the

source of copepods collected during the project. Unfortunately,

this fact was not known to the consultant prior to the discovery of

medusae in the extensive tank . It is recommended that future

rearing trials employ f i lters ( 5-10 µm) on all incoming water

sources to prevent this occurrence . Formalin treatments to rid

medusae in existing stock tanks were found fatal to the l ive feeds

and potentially to the larvae, as described in the fol lowing

section .

Prior to the discovery of medusae, it is interesting to note

that the larvae in this extensive rearing trial did not receive any

oyster trochophores , yet it remained the most successful rearing

trial . An examination of the copepods in the tank and those that

were collected from Raceway #2 revealed the presence of a large

number of copepodites that were similar in size ( i . e . , 7 0 - 1 0 0 µm)

to oyster trochophores . It is presumed that the larvae were

feeding on these organisms . A second observation is that the

larvae were doing well at a reduced salinity. Investigations of

the effects of salinity on larval growth. and- survival vary with the

species . There are reportedly no effects of salinity on larval

growth and survival for striped mullet larvae (Murashige et al . ,

19 9 1 ) . In contrast, larval rearing of , milkfish is optimal at

salinities between 2 0 and 25 ppt (unpublished data ) . Future work

should focus on the optimal salinity for the larval rearing o f

grouper .

During the second phase of the consultancy , another extensive

larval-rearing trial was conducted in a different 2 0 , 0 00-liter

tank . Tank preparation was conducted as described previously .

However , strong rains reduced the salinity to 25 ppt when spawned

eggs of E . microdon were stocked at a density of 2 0 eggs / l . No

hatching was observed during this rearing trial . It is recommended

that future extensive rearing trials be undertaken in tanks covered

with a shade cloth attached as close to the water level as

possible. In addition, the entire tank area must be covered with

a waterproof tarp to keep out the rain . It is crucial that the

coverings ( i . e . , tarp and shade cloth) be securely fastened to keep

41

10.4 Page 94

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the low light environment constant as well as to withstand

winds�anthdirrdainextthenatsivoecculrarvinalG-ruaemar. ing "t;rial was attempted

100 m concrete pond stocked at a density of 2 eggs / l . The

was prepared in similar fashion to the extensive

tank . However , because of its size, no waterproof covering

be affixed to this facil ity and a decrease in salinity occurred

to rain. This trial also resulted in a poor hatch , as no

could be seen in the pond after stocking .

An additional problem with the larger extensive system was

establishment of phytoplankton. The water in the tank needs to

nutrified and careful monitoring of the phytoplankton must

carried out . variabi lity in the available sunlight wil l lead

variations in cell density and ultimate changes in pH . Future

should focus on gaining experience in managing pond conditions

mfoaoiid:f-aoirngeadniasrmes

.

a

Critical

salinity of

operating

3 2-35 ppt ,

parameters that must

algal densities of

x 1 0 cel ls/ml , rotifers at 5-10 /ml , and the presence o f copepods'�

· EFFECT OF FORMALIN ON SURVIVAL OF �IVE FEEDS

The presence of the medusae ( See Appendix 6 , Plate 5 ) in a,

larval rearing trial is an extremely serious condition as there is

no known remedy to this situation without mortality to the larvae.

One treatment known by the consultant to disinfect eggs was

evaluated for effectiveness in eliminating the medusae found in the·

extensive rearing tank at GADTC. The treatment consists of

immersing the eggs into a 100 ppm formalin bath for one hour .

Spawned eggs from a variety of species are not effected by

concentrations as high as 1 000 ppm (unpublished data) , but it

remained to be seen whether the live feed organisms could tolerate

such a treatment . Medusae , rotifers , copepods , and Artemia nauplii

were placed into 1-l iter beakers and subjected to various

concentrations of formalin in seawater . After one hour the number

of surviving individuals were quantified either volumetrically or

by direct count . The results ( Figure 12 ) indicate that a dosage of

5 0 ppm is fatal to the medusae . However , it is apparent that

rotifers and copepods are also as sensitive to the treatment and

thus the treatment cannot be used in the event that an outbreak of

the medusae occur during the early stages of a larval rearing

trial . Artemia nauplii were found to be unaffected by formalin at

concentrations as high as 1 000 ppm .. The. use- of formalin during the

latter stages of a rearing tria l , when this feed is used

exclusively therefore does warrant further investigation . The

sensitivity of the larvae to formalin at various stages of

development would provide an indication of whether this treatment

can be used at a l l .

42

10.5 Page 95

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70 -

co -

50 -

••

..

••

10

•

70 -

··-

50-

·· -

�

dP

�

30 -

··-

10

�•

H

••

�

00

.. -

1•

MEDUSA

COPEPOI>S

•

100-

··-

co-

·· -

20-

0

l.O

50

l.00

500

FORMALXN DOSAG� ( PPM)

l. 0 0 0

Fiqure 12 .

Effect of various concentrations of formalin

on l ive feeds and medusae .

43

10.6 Page 96

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OTHER GROUPER SPECIES

one of the tasks of this project was to assess the

reproductive seasons of various species of groupers . Several

species under consideration for culture in addition to E . microdon

and E . fuscoguttatus were collected . Pictures of E . microdon , E .

fuscogu ttatus and some of the other groupers species encountered

during the consultancy are presented in Appendix B , Plates 1-6 .

When possible , biological data from gonadal biopsies and length and

weight measurements were obtained . A summary of data obtained for

Plectropomus areolatus and Cephalopholis miniata fol lows .

areolatus

Five P . areol atus individuals were captured by hook and line

on July 13 , 1993 , and examined for their state of maturity (Table

1 5 ) . Previous data regarding the spawning season for this species

( FitzGerald et al . , 1991) and the results of the gonadal biopsies

indicate that the peak in the spawning season had already passed by

the initial phase of this consultancy . After the individual s were

examined they were returned to the spawning channel .

Table 15 . Sumam ry of data obtained by gonadal biopsies of P .

areol atus captured on July 13 , 1993 .

Number

Sex

1

2 . 42

unknown

2

2 . 78

male

3

1 .66

unknown

4

2 . 52

male

5

1 . 42

female

cann

pv =

.

sperm =

previ tel

cannulated sperm

logenic ooc}rtes -

State of

-

cann . sperm

-

cann . sperm

pv

miniata

Individuals . of this species were also caught by hook and line .

The habitat of this species differed from the other groupers caught

during the consultancy . Whereas E . microdon and E . fuscogu ttatus

were caught in the channel areas and at relatively deep depths , c .

miniata were caught inside of the barrier reef and appeared to

inhabit solitary outcroppings of coral .

As many as 3 -10

individual s were obtained from a single location . Attempts were

made to capture individuals of this species in July and August . A

summary of the state of maturity of the captured fish is presented

in Table 16 . Gonadal maturation was assessed from individual s that

had died during transport to the laboratory by excision of their

gonads and a wet mount prepared from a small section of excised

gonad .

The wet mounts were then viewed under a compound

44

10.7 Page 97

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microscope . Individuals scored as negative (Table 1 6 ) were a l ive

and biopsied us ing a cannula but resulted in no gonada l sample

being obtained . Three stages of maturity , ( i . e . , mature testes ,

transitional male , immature ovary)

are presented in

photomicrographs of wet mounts presented in Appendix 9 , Plates 1-3 .

sperm from the male caught in July was activated when exposed

to seawater.

Females were found to range in maturity from

possessing only previtellogenic oocytes , to being fully gravid with

a few ovulated eggs . The presence of mature males , and females at

various stages of maturity would indicate the existence of some

reproductive activity during the months of July and August .

Table 1 6 . summary of gonadal biopsies obtained from C . miniata .

Number Date

Total

Length

(cm1

Body

Weight

(q)

Sex

-

State of

Maturity

1

7/17/93

20.5

2

7/17/93

26.2

3

7 / 17/93

28 . 5

4

8 / 14 / 93

23 . 0

5

8 / 14 / 9 3

21.5

6

8/14/83

23 . 0

7

8 / 14/93

23 . 0

8

8 / 14 / 9 3

28. 6

9

8 / 14 / 93

34 . 0

10

8/14/83

25. 5

11

8/14/93

28 . 2

12

8 / 14 / 9 3

25.1

13

8/14/93

30.5

14

8 / 14 / 9 3

30.5

15

8 / 14 / 8 3

27 . 9

16

8 / 14 / 93

32 . 1

183

452

516

175

122

181

151

330

540

301

435

277

470

320

370

490

female

male/female

male

female

male

negative

pv + vit

trans itional

cann. sperm

vit

pv + vit

pv

cann. sperm

cann . sperm

cann. sperm

cann . sperm

n.a.

cann. sperm - cannulated sperm

pv = previtellogenic oocytes

vit = vitellogenic oocytes

transitional = individual undergoing transition in sex

n . a·. = not available

-

·

The length versus weight relationship of the C . miniata

individuals l isted in Table 16 is summarized in Figure 13 . The

data was subj ected to regression analysis and the following linear

model was found to provide the best fit of the data :

BW = 3 1 . 3 � * (TL) - 506 . 1 6

r = o . 76

p < 0 . 001

The sex of the individuals is also presented in the graph and

while all of the smaller individuals were females , the larger

45

10.8 Page 98

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600

•

MALE

+

PENALE

•

500

<>

NEGATIVE

-

C!I

�

� 400

HC!I ·

�

>i 3 0 0

§

tQ

!; TRANSITIONAL

l«>DEL

•

BW • 3 1 . 33 (TL) - 5 0 6 . 16

200

r2 • 0 . 7 6

p < 0 . 001

+

20

22

24

26

28

30

32

34

TOTAL LENGTH (CM)

Piqure 13 .

Length x weight relationship of C. miniata

captured in July and August 1993 .

individuals were males or of undetermined sex .

From the

distribution of the sexes , it would appear that the maj ority of

individuals scored as negative were males . One individual was of

a trans itional sex and marks the size at which transition from

female to male occurs .

A fortunate occurrence was the capture o f two females during

August that were fully gravid. A comparison o f oocyte size

frequency distribution is shown in Figure 14 . one female possessed

a few oocytes that had clearly ovulated , indicating that she had

also spawned recently . The other female that did not have any

ovulated oocytes , however , also possessed a larger number of

oocytes > 4 6 0 µm that might indicate the oocyte diameter most

receptive to hormonal stimulation of final maturation and spawning.

46

10.9 Page 99

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,. ...

20

:LC

.

0 :L a

GI

I•

4

0

:>-&

"-"'

i :>-0

.-

El ..

I ...

a-

0

aao

360 6 0 0 ••o sao • • o s•o '7 0 0 7 & o

a:zo ••o

Figure 1 4 .

S ize frequency distribution of oocytes from

two female c . miniata caught on August 16 ,

1993 .

TRANSPORT OF ADULT GROUPERS TO GADTC

E. microdon

During the first phase of the consultancy , a s imulated

transport . experiment was conducted with two adult E . mi crodon

males . The individual s , 0 . 9 1 and 0 . 6 6 kg in body weight , were

placed in separate J O-gallon glass aS\\,uariums with water

temperatures gradually lowered to 2 5 and 22 c , respectively . At

these temperatures the fish were easy to handle and did not thrash

when placed into plastic bags ( 3 -ml in thickness) f i l led with 12

liters of seawater . The bags were filled with oxygen , sealed with

rubber bands and placed in styrofoam coolers containing a smal l

amount of ice placed in the bottom. The duration of the experiment

was 12 hours, or the actual transport time if the shipment was to

take place by air freight . At the end of the experiment , the fish

47

10.10 Page 100

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were removed from the bag and allowed to acclimate in a 1000-li:t>'•

tank equipped with running seawater and continuous aeration . wa,.-c.

temperatures in the transport bags dropped to

respectively , by the end of the experiment .

23

No

.

5

m

and

orta

l

i

t1i9'.<t°:,:

occurred .

During the second phase of the consultancy , six adu•ll..t

individuals { 3 males and 3 females) were transported successful}~!

to GADTC . However , one female died after the first injection '.a

hormone in an attempt to induce spawning. Another female and mair.

died when a female tried to eat a male of comparable size (Sell

Appendix 1 0 , Page 1 0 ) . They were found locked together at tlr•

bottom o f the tank . Twelve-inch PVC fittings were placed into th""1i

tt•a holding tanks to provide hiding places for the groupers . All

received antibiotic treatments in response to fin rot most like.l,~

due to trauma received during capture and transport activiti�s

The antibiotic treatment consisted ·of Prefuran ( 5 ppm for one hou·.

repeated over a five-day period . At last observation on August

19 9 3 , all individuals were eating and appeared healthy .

E. fuscoguttatus

Three adult female E . fuscoguttatus individual s , 2 . 6 9 , 3. 21,

and 3 . 3 5 kg in body weight , respectively were shipped from Palau to1

Guam . The preparation was the same as that in the transport;.

experiment using E . microdon . When the shipping boxes arrived i)1,

Guam , the boxes were opened at the airport . Water temperatures!

were exceeding low, ( i . e . , ranging from 9-13°C ) and the fish were·

massaged to regain mobility. One individual died within hours and

the remaining two individuals died two days later. Although the

experiment ended in mortality, a protocol was established . With

modifications in the placement and amount of ice , adult E . ·

fuscoguttatus groupers can be shipped from Palau to establish a

broodstock at GADTC .

Cephalopholis miniata

To establish a broodstock of this species at GADTC, 44

individuals captured by hook ·and l"ine were -shipped using the same

methods as described .

Upon arrival at GADTC these individuals

received Prefuran treatments and mortalities (n=4 ) were due only to

infections that resulted from capture and transport activities . At

last observation all remaining fish were eating and healthy .

SUMRMA Y AND RECOMMENDATIONS

Although all the obj ectives and then some were accomplished

during this consultancy, a hatchery technology for the selected

grouper species has yet to be fully established at GADTC and

validated ·at commercial scale. One major problem that must be

addressed is the presence of medusae in the rearing tanks at GADTC .

The source ( s ) need to be located and corrective action taken in

48

11.1 Page 101

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to minimize

system in

the

chances

future . ·

of

medusae

-

entering

-

into

the

larval

The following section represents recommendations for areas

require additional work in order to establish a protocol for

he artificial pr_opagation of groupers studied during this

- onsultancy . They are :

reduction

•

Establish captive broodstock of groupers targeted for

domestication

•

Define the spawning season of selected groupers both from

the wild and in captivity based upon gonadal maturity

•

For each species , determine the s ize at first maturity

and size at which the transition from female to male

occurs

•

Determine the stage of ovarian maturation most receptive

to hormonal induction of spawning

•

Establish a broodstock that spawns naturally ( i . e . , with

no hormone treatment )

•

Determine the minimum effective dosage of HCG for

induction of final maturation and spawning

•

Determine the effectiveness of alternative hormones

( i . e . , carp pituitary homogenate, LHRH-a) for inducing

final maturation and spawning

•

Determine the effectiveness of 17-MT on induction of

testicular maturation in captive broodstock

•

Determine the effectiveness of 17-MT on induction of sex

reversal of captive broodstock

•

Determine the effects of chronic LHRH-a treatment on

ovarian maturation

•

Determine the optimal temperature : salinity combinations

for hatching of grouper eggs

Larval

•

.

Determine the l ight intensity of which newly hatched

grouper larvae are tolerant

•

Determine optimal initial larva l : feed ( i . e . , oyster

trochophores , rotifers) densities at first feeding

49

11.2 Page 102

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•

Establish a population of sea urchins at GADTC

determine the availabil ity of sea urchin

throughout the year

•

Determine the suitability of alternative initial

feeds ( i . e . , sea urchin , trochus , musse l , giant

larva e )

•

Establish and mass culture the smaller strain ( i . e ,

type rotifer) at GADTC - for -use- in larval rearing

groupers

•

Determine feeding preference of grouper larvae

stages of development

•

Determine the optimal sal inity for larval culture

•

Determine the effects of enriched versus nonenriched

Artemia nauplii on larval growth , survival and stress

resistance

•

Determine the effects of different levels of C20 : 5n-3 and

C2 2 : 6n-3 on larval survival , growth and stress

•

Incorporate the results of the experiments into a

rearing protocol and validate at commercial scale

•

Adapt the larval rearing protocol to other

species

•

Gain experience in manipulating extensive larval systems

to remain in the critical opera.ting parameters ( i . e . ,

safinity 3 2 - 3 5 ppt , phytoplankton dens ities of 3 0 0-500 x

1 0 cel ls/ml , rotifer densities of 10-2 0 /ml , and the

presence of copepods

•

Determine the optimal larval stocking density and food

density in extensive ( i . e . , 2 0 , 000-l) rearing systems

50

11.3 Page 103

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LITERATURE CITED

Anonymous . 1989 . Fishery statistics bulletin for south China Sea

area. 1 9 8 7 . Southeast Asian Fisheries Development Center ,

Thailand . 162 pp .

chao , N . -H, H . -P . Chen and I . -c . Liao . 19 7 5 . Study on cryogenic

preservation of grey mullet sperm . Aquaculture , 5 : 3 8 9 -4 0 6 .

Chao , T . M . , L . -c . , Lim and L . T . Khoo . 19 9 1 . Studies on the

breeding of brown marbled grouper Epinephelus fuscoguttatus

( Forsskal) in Singapore . Paper presented at : Finf ish Hatchery

in Asia 1 9 1 Conference, Tungkang , Taiwan , December 17-19 ,

19 9 1 . 2 4 pp .

FitzGerald , W . Jr . , M . Bauerlein and T . Behrenfeld .

1991 .

Prel iminary spawning and culture of groupers in Palau and

Guam .

Final Report, - Pacific . Aquaculture Association .

Honolulu , Hawaii USA, pp . 1-53 .

Fukusho , K .

Review of the research status of zoop lankton

production in Japan . In: W . Fulks and K . L . Main ( Eds . ) ,

Rotifer and Microalgae Culture systems , Proceedings of u . s .

Asia Workshop . Oceanic Institute , Hawaii, U . S . A . pp . 5 5 -6 0 .

Johannes , R . E . 198 1 . Words of the Lagoon : Fishing and Marine

Lore in the Palau District of Micrones ia. University of

California Press , Berkeley CA . 2 4 5 pp .

Kitalong , A . and E . Oiterong .

1991.

Review of the grouper

fishery for two predominant species : Epinephelus fuscoguttatus

and Plectropomus leopardus . Marine Resources Division , Koror ,

Palau . 10 pp .

Kohno , H . , M . Durray and P . Sunyoto . 19 9 0 . A field guide to

groupers of Southeast Asia . 2 4 Pusat Penelitian dan

Pengembangan PHP/KAN/PT . No . 14 / 19 9 0 Jakarta , 2 6 pp .

Lee, c . -s . , C . S . Tamaru , C . D . Kel ley and J . E . Banno . 1 9 8 6 . Induced

spawning of milkfish , Chanos chanos , by a single application

of LHRH-analogue . Aquaculture , 58 : 8 7-98 .

Lee ,

c . -s . , C . S . Tamaru , C . D . Kel ley , G . T . Miyamoto and A .

Moriwake . 1 9 9 2 a . The minimum effective dosage of 17 a

methyltestosterone for induction of testicular maturation in

striped mullet , Mugil cephalus L . Aquaculture , 104 : 1 8 3 - 19 1 .

Lee.

c . -s . , c . s . Tamaru , C . D . Kelley , A . Moriwake and G . T .

Miyamoto . 1 9 9 2 b . The effect of salinity on the induction of

spawning and fertilization in the striped mullet , Mugil

cephalus. Aquaculture , 102 : 2 8 9 -2 9 6 .

51

11.4 Page 104

▲back to top

Lubzens , E .

1 9 8 7 . Rais ing rotifers for use in aquaculture .

Hydrobiologia , 1 4 7 : 2 4 5 -2 5 5 .

Murashige , R . , P . Bass , L . Wallace , A . Molnar , B . Eastham , V .

Sato , c . S . Tamaru and c . -s . Lee . 19 9 1 . The effect of

salinity on the survival and growth of striped mul let, Mugj,J.

cephalus, larvae in the hatchery . Aquaculture , 9 6 : 2 4 9-254 .

Morisawa , M . 1 9 8 5 . Initiation of sperm motil ity at spawning in

teleosts . Zoological Science, 2 : 605-615 .

Shehadeh , Z . H . , c . -M . Kuo and K . Milisen. 19 7 3 . Validation of

an in vivo method for monitoring ovarian development in the

grey mullet (Mugil cephalus) . J . Fish. Biol . , 5 : 4 8 9 -4 9 6 .

Sorgeloos , P . , P . Lavens , Ph . Leger and w . Tackaert . State of

the art in larviculture of fish and shel lfish . 1 9 9 1a . In: P .

Lavens , P . Sorgeloos , E . Jaspers , and F . Ollevier ( Eds . ) .

Larvi 1 9 1 - Fish & Crustacean Larviculture Symposium, European

Aquaculture Society , Special Publication No . 15 , Gent ,

Belgium . pp . 3 - 5 .

Sorgeloos , P . P . Lavens , Ph. Leger and w . Tackaert . 1 9 9 l b . The

use o f Artemia in marine fish culture . Paper presented at :

Finfish Hatchery in Asia 1 9 1 Conference , Tungkang, Taiwan .

Dec.. 17-19 , 1 9 9 1 . 12 pp..

Tamaru , c . s . , c . -s . Lee and H . Ako . 1 9 9 1a . Improving the larval

rearing of striped mullet , (Mugil cephalus) by manipulating

quantity and quality of the rotifer, Brachionus plicatilis.

In: w . Fulks and K . L . Main ( Eds . ) , Rotifer and microalgae

culture systems . Proceedings of a u . s . - Asia workshop . The

Oceanic Institute , Hawaii , U . S . A . pp . 8 9-103 .

Tamaru , c . s . , C . D . Kelley, c . -s . Lee, K . Aida , I . Hanyu and F .

Goetz . 1 9 9 l b . steroid profiles during maturation and induced

spawning of the striped mullet , Mugil cephalus L • •

Aquaculture , 9 5 : 1 4 9-168 .

Tamaru, c . s . , F . Cholik, J . c . -M . , Kuo and w . FitzGerald , Jr .

199 3 a . status on the culture o f milkfish ( Chanos chanos ) ,

striped mullet (Mugil cephalus) and grouper (Epinephelus sp . ) .

Paper presented at World Aquaculture 1 9 3 , Torremolinos , Spain,

May 2 6-2 8 , 19 9 3 .

European Aquaculture Society , Special

Publication No . 19 . Oostende , Belgium. pp 2 9 6 .

Tamaru , c . s . , w . J . FitzGerald and v . s . Sato . 1 9 9 3 b . In: Christine

Carlstrom-Trick ( Ed . ) , Hatchery manual for the artificial

propagation of striped mullet (Mugil cephalus ) .

Guam

Aquaculture Development and Training Center . 165 pp .

( In

press ) .

52

11.5 Page 105

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APPENDICES

53

11.6 Page 106

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APPENDIX 1 .

PICTORIAL ESSAY OF THE METHODS OF CAPTURE , TRANSPORT ,

INDUCTION OF SPAWNING, AND LARVAL REARING TANKS

54

11.7 Page 107

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Step 1 .

Adult . .groupers

obtained by hook

line .

are

and

Step 2 .

Captured individuals are examined on

board to assess their state of maturity .

55

11.8 Page 108

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Step 3 .

Suitable individuals are placed in a live

bait well for transport .

Step 4 . Unloading adult groupers .

56

11.9 Page 109

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-- !IA!,."!c•.'!'--

Step 5 .

cavity

l . ...

.• -

Step 6 . Obtaining body weight .

57

11.10 Page 110

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step 7 . Examining tlie ' maeurity or adult males .

Step 8 .

Conducting an ovarian biopsy with a

polyethylene cannul a .

58

12.1 Page 111

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Step 9 .

Preparation of -hormone

for the induction of

spawning .

Step 1 0 . Administering hormone to a mature female .

59

12.2 Page 112

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step 1 1 . Fertilized eggs from E . microdon .

�·

·�

--.:.._ - - -

. //.-'-/'

I

.. ..•

.

. f·.

.

..

� . ·-� • ·

J° .'

step 12 . Newly hatched grouper larvae .

60

12.3 Page 113

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step 13 . Preparation for shipment of grouper eggs

to GADTC.

step 14 . Setup for sma ll-scale larval rearing

experiment at GADTC .

61

12.4 Page 114

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( -t:t---

step 15 .

larval rearing tank at GADTC .

62

12.5 Page 115

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APPBND:IX 2 .

PROCEDURE FOR STRIP SPAWNING AND "DRY METHOD" OF FERTILIZING EGGS

63

12.6 Page 116

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Step 1 .

Removal of ovulated oocytes from female

by applying pressure to the abdomen .

step 2 .

D istr ibute col lected sperm with syringe .

Note there is no seawater .

64

12.7 Page 117

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step 3 . Mix eggs and sperm with your hands .

Step 4 . Activate sperm by adding seawater .

65

12.8 Page 118

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step s .

Mix eggs and sperm to maximize

fert i l i z a t i o n .

step 6 .

Skim the floating ( i . e . , fertil ized) eggs

with a beaker .

66

12.9 Page 119

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APPDII>I X 3

SPAWNING BEHAVIOR OF EPINEPHELUS HICRODON

67

12.10 Page 120

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Plate 1 .

and treated E .

receiving the

the distended

Plate 2 .

Swimming of female in the spawning tank

as spawning approaches .

Note the

proximity of the males .

68

13.1 Page 121

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Plate 3 . Natural spawning of E . mi crodon

Plate 4 . Egg col lector for spawned eggs of E .

mi crodon

69

13.2 Page 122

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APPBl>lIIX 4

TEMPORAL CHANGES OF OOCYTES DURING FINAL MATURATION,

SPAWNING , AND EMBRYONIC DEVELOPMENT FOR EPINEPHELUS FUSCOGUTTATUS

70

13.3 Page 123

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A

B

.�·

(

.--'

13.4 Page 124

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~

,.� <

D

I. .

·�

'.

• _..t' .

K

M

!N

~

"

13.5 Page 125

▲back to top

•.i: ~ ,

,'·\\ . - ·. --. I _.. ·

t' ' ' __.,..- I

I

. /.

/ ----4

//

(

I

-

,✓---..,,

..-

.

,-.....

/ .,.....~

:

(

Ji

\\ .ti'.:.1-. .

Plate Q . Example of a "bad"

spawned grouper egg.

�

•

.·�. /�

(I

P late R . Examp le of " bad" spawned

grouper egg .

I

•

/

0

P late S . Fertilized "good " eggs .

73

13.6 Page 126

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APPENDIX 5

MORPHOLOGICAL CHANGES OF EPINEPHEWS FUSCOGU'l"I'ATUS LARVAE

74

13.7 Page 127

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-·_;-.;:-~'~<>'-~

----------·,···- ----·--

--- - -- ---- ------------,~- -·

Plate l . Newly hatched E . fuscoguttatus larvae .

-----------------------------· ·- ·- ..... .

Plate 2 . E . fuscoguttatus larvae

posthatching .

seven

hours

.7 5

13.8 Page 128

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Plate 3 . E . fuscoguttatus

posthatching .

larvae ,

24 hours

Plate 4 . E . fuscoguttatus

posthatching.

larvae ,

48 hours

76

13.9 Page 129

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.

Plate s .

Four day posthatched E . fuscoguttatus

larvae from extensive larval rearing

tank .

Plate 6 .

Closeup of head region of 4 day old E .

fuscoguttatus larvae . Mouth opening is

150 µ.m .

77

13.10 Page 130

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llPDmIX 6

OYSTER TROCHOPHORES AND A COMPARISON OF ROTIFERS

TO TROCHOPHORES AND COPEPODITES

78

14.1 Page 131

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' ' TrochoFeed-Back

Be " sides saving us

having the 18-llter

container with frozen

on hand all the

time has made feeding schedules much more

flexible. Now I canfeed as llttle or as much as I want,

whenever I want, and since the need for

trochophores changes depending on wWch ani-

mals rm tryingtoraise,

that flexibility ls Im

portant"

.'

. .. . .

• '·'

"lbls past spawning

season we fed your

Trochofeed to larval

sableflsh (black cod).

and not only were the

stomachs of the larval

flsh

full

of

rrochophores, It was

also evident that the

rrochophores were in

an advanced stage of

degradation from di

gestion"

"We have been fortu

nate enough to be able

to experiment with a

wide variety of differ

ent food types and

sizes.Your Trochofeed

product has proven to

be very promising as a

first food offering for

the fry in their early

stages of develop

ment"

:� .

'

---·- · - ··--··----

"A prey preference

study showed (that) red drum selected

rrochophores over rotifers during the first two days

of feeding"

"On analyzing your oyster rrochophores with about

1 5% 20:5n3 (EPA) and 15% 22:6n3 (DHA) it was

evident your product provided many of the nutri

tional requirements ror initial feeding of larval fish''

M

L

BIOTECH LTD

Tro eh oFe e d

For more information

on TrochoFeed, contact

MTL Biotech Limited

P.O. Box 5760, Station B

Victoria, B.C. Canada V8R 658

Tel: (604) 598-60 1 9

Fax: (604) 598-2047

Cellular: (604) 744-9850

14.2 Page 132

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TrochoFeed. . .

A new, natural first feed for larval

marine fish

Cl What is TrochoFeed?

TrochoFeed is a suspension of lMng trochophore-stage

Pacific oyster larvae in seawater, cryopreserved in

liquid nltrogen until the moment they're needed to feed

your fish Trochophores are free-swimgmni and: about

SOµ In diameter - about aquarter the size of the average

frlosthiferT,roanchdotfheeerdeifsorthe eanflirdset aalnfdirostnfleye"dinfsotralnatrlvivalemfeareidn"e

for marine larval rearing, and we have an impressive Ust

of satlsfled users.

0 Availability: The unique

advantage of TrochoFeed

TrochoFeed is available every day of the year, thanks to

MTL Bio tech Limited' s unique technology for

cryopreservlng the trochophores In bulk. We freeze

trochophores during oyster spawnlng season, store them

in liquid nitrogen and then ship them to you, froz enInbulk,

whenever you want them. Frozen-thawed trochophores

are alive and identical to fresh one: fish cannot tell the

difference.

0 High Food Value

The most importanthighlyunsaturated fatty acids, 20:Sn3

haTnarsdocn2ho2ot:F6benee3dg,.uanAr; eswheaeccoshnhptiperneTstreionsctlhoaowtF1eb5ede%ctoaoufystoeotuashlaeftaltlthtfyeoarpcmeiaadktsiooInnf

its nutritional value.

0 What does TrochoFeed look

like ? How is it used?

Trocho(eed is packaged In plastic straws that must be

stored in liquid nitrogen until you are ready to feed (we can

recommend a suitable liquid nltrogen container). Straws

contain between 5 million and 100 million trochophores

each. so you can choose which to tha\\.V depending on your

daily needs. Thawing is a simple procedure, requiring only

a few litres of sea water and a 20µ screen You can feed

the trocbophores over a wide temperature range: Arctic

cod eat them as well as red drum do.

Live Larval Feeds (to scale)

Cl Why do larval fish need

TrochoFeed?

It's well known that live first feeds out-perform artificial

diets, but they are labour-intensive to culture or collect.

Oyster trochophores ha\\'e been kno\\vn for years to be

an excellent flrst feed for larval marine fish; until now,

however. the difficulty of obtaining them has

then1 an in1pructical diet

0 Quality assurance and

environmental safety

Trochophores are produced from certified, dlsease

free selected broodstock oysters and are culntred

under controlled conditions In our facility. Users in

areas where Pacific oyster is an "e.\\'.otic" species need

not fear that uneaten trochophores in effluent water

will become estab-

lished locally: our

cryopreservation pro

cess ensures that the

trochophores cannot

complete normal em

bryonlc development.

0 Shipping

TrochoFeed

YToroucrhooFredeedr

of frozen

is shipped

to you, by air cargo, In

a pre-chilled "dry ship

ptheer".froSziemnplTyrotcrahnoFsfeeerd

t o your own storage

container and send the

shipper back to us. If

you do not have a stor

age container, MTL

Biotechlimited canrent

you one.

0 Ordering

Contact MTL Biotech

Llmited's sales depart

ment at:

MTL BIOTECH LTD

P.O. Box 5760, Station B

14.3 Page 133

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M

L

BIOTECH LTD

Date: 13 July 1993

. Dept. of Commerce

Guam Aquaculture Development Center

GITC Building, #601

Tamuning, GUAM

Attn: Mike Bauerlein/ Mark Norman

Tel: 671-646-5841; 734-3011

Purchase Order:

Via: Canadian Airlines

Item

Batch

Quantity Unit price

Trochofeed

14 Aug (green) 6 X 5 million

102 (blue)

1.5 X 13 million

101 (blue)

0.5 X 15 million

109 (blue)

1 X 35 million

103 (blue)

1 X 15 million

5.00

DISCOUNTS AVAILABLE ON LARGER ORDERS

Shipping

collect

Please pay:

Cost

150.00

100.00

N/ C

175.00

75.00

500.00

P.O. Box 5760, STATION 8, VICTORIA, 8.C., CANADA, VSR 658

"§f TEL: (604) 589-60 1 9 FAX: (604) 598-2047

••••••

ll • • • • •

14.4 Page 134

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HOW TO THAW AND RECOVER FROZEN TROCHOFEED

Important safety preca11tio11

Always wear protective eyewear when handli11g cryopreserved Trocl1ofeed. Cn;oge11ic sample

holders, illcl11di11g vials, t11bes a11d straws, ca11 shatter. Cryoge11ic storage vessels, including

dewars a11d refrigerators, are supplied with mat111facturl'T's safety g11ideli11es, wlzic/1 should

alwa s be allowed.

1.

Selecting material

Select a straw of cryopreserved trochophores and place it immediately into the plastic

thawing bag provided. The bag is for co11vet1ience i11 tlzawi11g, a11d for your 1irotectiou i11 ll1e

uin11t1oik2el-.l3f

eve11t of straw breakage. Holding the outside of the bag, immediately snap the

inch segments. If you're not using the whole straw, replace the unused portion

straw

in the

liquid nitrogen storage container immediately. DO NOT ALLOW UNUSED PORTION TO

WARM UP, AS DAMAGE TO THE REMAINING MATERIAL WILL OCCUR.

2.

Thawing

Add warm seawater (25-28C) to the bag without delay, using about 300 ml per straw. Shake

genlly, squeezing the Trochofeed out with your fingers to achieve a rapid thaw. The straw

pieces must be submerged lo get the right thaw rate. Remove empty straw pieces.

3.

Recovery period

Filler the thawed trochophores over a 20µ Nitex screen, wash with a few squirts of 25-28C

seawater. then rinse them into a container of 25-28C seawater for an approximately 1-2 hour

recovery period. Density during the recovery period should be ro11gl1ly 400 ml waler per million

trochophores (2,500 trochophores/ ml). After one hour, harvest the trochophorcs on the 20µ

screen as before, then rinse them directly into the rearing tanks for feeding.

· s;

Counting trochophores

Your packing list or invoice tells you roughly how many trochophores are contained in each

straw. If you need more accurate counts, dilute the washed trochophores to an easily counted

density (10,000 trochophores/ ml works well) and count using a Sedgwick-Rafter cell at 40X

magnification.

6.

Thaw too many?

Unused trochophores will keep overnight in a refrigerator (4-SC).

81

14.5 Page 135

▲back to top

•

••

•

••

. ..

••

• ••

• • ••

•

Plate 1 . A photomicrograph taken at 4 0x comparing

a rotifer and oyster trochophores .

Plate 2 .

Newly hatched

naupli i .

....- - - - - -

Artemia

s -a.

14.6 Page 136

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',,,""

.I.

~,

Plate 3 .

Adult herpactocoid copepods from shrimp

broodstock tanks . Note female carrying

eggs .

. ,.- ., ·

-~-·~ ·"'"'

"

'~q&p ~-. .

Plate 4 . A photomicrogaph of a rotifer and

copepodite . - 4ox ; -

83

14.7 Page 137

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. ,.

-

..,

Plate s . Example of ·medusa present in the

extensive larval rearing tank .

14.8 Page 138

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APPDIDrlC 7

SPAWNING PROTOCOL FOR THE SEA URCHIN, Tripneustes gratilla

SS,:

14.9 Page 139

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Exercise 2

Sea Urchin Fertilization

Introduction

Sea urchin gametes have been used extensively

in studies of fertilization because they are easily

obtainable in large quantities and develop in vitro.

Much of what we know about the physiology and

biochemistry of early development has come from

studying sea urchin embryos. The early literature

was well sumamzried by Harvey (1956); more

recentreviewsbyGiudice (1973) and Stearns (1974)

provide insight into an array of research efforts

involving these animals. Modem research with

these animals involves primarily molecular biol

ogy.

This exercise will provide you with an oppor

tunity to learn how to acquire, examine, and

manipulate sea . urchin gametes to produce

embryos of various stages for subsequent study.

Procuring and Storing Gametes

Objective

D Know how to induce spawning, and

how to properly wash and store gametes.

Sexes can be distinguished only by examining

the reproductive produc!S; milky semen and yel

low (or orange), granular eggs. Gametes may be

obtained by several methods:

• an animal may be cut open and the gonads

removed and minced in sea water;

• spawning may occur spontaneously in the

laboratory (often due to stress);

• spawning may be induced by electric shock,

or injection of 0.55 M KO solution into the

coelomic cavity. Both induction methods in

duce smooth muscle contraction, causing

gametes to ooze from the five gonopores on

the aboral surface.

In this course, spawning will be induced by in

jection of 0.55 M KO solution into the coelomic

cavity. With a hypodermic needle mounted on a

syringe, puncture the soft perioral membrane,

pointing slightly away from the oral-aboral axis

andinto the coelomic cavity. Use 1-5cc ofsolution,

depending on the animal's size and initial

response. To avoid contamination of gametes, use a

separate syringe and needle for each animal, or

rinse the instruments well in tap water after each

use.

Examine the gametes shed at the aboral surface

and determine the sex of the animal; semen is

creamy white, whereas eggs are finely granular

and yellow or orange. Females should be inverted

over a beaker of filtered sea water (FSW) such that

the gonopores are submerged and the eggs stream

out without exposure to air. Collect semen "dry"

(without dilution) by inverting the spawning male

over a dry petri dish. Remove any excess fluid that

drips from the animal into the semen by pipet.

Cover the dish to prevent desiccation until the

sperm are to pe used.

Eggs should be washed promptly because

coelomic fluid hinders fertilization. When the

female no longer sheds gametes, remove her and

gently swirl the beaker to suspend its contents.

Permit the heavier "crud" (bits of spine, tube feet,

etc.) to settle for about 30 seconds and then decant

the suspended eggs into another beaker. The eggs

86

14.10 Page 140

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2.2 Experimental Embryology

will settle to the bottom; the supernatant, called

egg water, should be decanted (save for later use)

and replaced with clean sea water. This washing

process should be repeated at least twice.

eggs Hint Small quantities of

may be washed

by moving in a circular pattern on a table top

a small petri dish containing an egg suspen

sion. Eggs collect in the middle of the dish

where they may be removed by pipet to a dish

eggs of clean sea water. Alternatively, small quan

tities of

may be suspended in sea water

in centrifuge tubes and quickly spun down by

hand centrifugation. TI;e wash water is then

easily decanted and replaced.

� Undiluted ("dry") sperm maybe k t in a closed

container in the-refrigerator (about 5 C) and used

successfully up to 48 hours after collection. Washed

eggs (if not extremely crowded) may be stored cold

eggs up to about 6 hours without appreciable deteriora

tion. Thereafter, the percent of

fertilizable

declines markedly.

Examining Gametes

Objective

D Learn to determine the fertility of a

sample of gametes.

eggs Sea urchin

are usually about 70-100 µm in

diameter and are surrounded by a 50 µm-thick

jelly coat. Immature oocytes have a conspicuous

large nucleus (germinal vesicle). Most of the eggs

(ova) will have completed both maturation

(meiotic) divisions and may have polar bodies ad

hering to the animal pole (difficult to detect). The

nucleus of the mature ovum is small, but con

spicuous.

Spermatozoa consist of a cone-shaped head

about 2 µm long and 05 µm in diameter at the base,

where several mitochondria fol"I}I the midpiece. A

50 µm- long tail projects from the midpiece. Sper

matozoa are relatively inactive in the undiluted

("dry") state, but become active immediately upon

dilution with sea water and are soon exhausted.

Because of this activating dilution effect one

egg.s should always use a fresh (less than 10 minutesold)

dilution of semen when fertilizing

Once

diluted in sea water, spermatozoa are fertile only

about 20-30 minutes. (Low pH retards activation;

high pH accelerates it.)

Obtain some gametesand examine them closely

eggs before mixing sperm and

.

• Are the sperm active?

• Are the eggs uniform in size, round, and free

of contaminants?

• What proportion of the eggs are mature? Do

they touch each other? Why?

eggs • Are any of the

fertilized prematurely?

time These questions should be considered each

you procure gametes for use.

Fertilization

egsg When sperm and

are brought together in

ocuc rs. the right conditions fertilization

The most

immediately recognizable changes occur at the egg

surface; darkfield microscopy reveals there an in

conspicuous alteration of the color of the difcfra

tion rings. Within seconds of sperm entry the egg

produces a fertilization membrane which rises

than above the egg surface; it is more prominent in some

species (e.g., Pseudoboletia)

in others (e.g., Trip

neustes).

sene A small protrusion (the fertilization cone) may

be

where the sperm head enters the egg

cytoplasm, but only from a (relatively rare) side

view. The male pronucleus moves to the female

pronucleus and the nuclear membrane of each dis

appears. The appearance of the cytoplasm slowly

changes as the mitotic cleavage spindle forms and

karyokinesis (nuclear division) progresses. Ul

timately, cytokinesis (cytoplasmic division) com

mences and very quickly the single-celled zygote

divides into two blastomeres.

15.1 Page 141

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Sea Urchin Fertilization 2.3

Achieving Optimum Fertilization

Objective

0 Learn to recognize fertilization changes

in gametes and how to maximize yields

of viable embryos.

Determine the dilution of semen which gives

optimum fertilization by adding equal numbers of

eggs to a series of dishes containing serially diluted

sperm. To do this, add equal volumes (e.g., 2 ml) of

filtered sea water to a row of labeled plastic petri

dishes. Make a stock suspension of sperm by ad

ding one drop of "dry" semen to SO ml of FSW in a

beaker. With a pipet, transfera volume (2 ml) of this

stock suspension into an equal volume of FSW in

the first dish to produce a \\i dilution. Then transfer

half of this dilution into the next dish of FSW to

give a \\14 dilution, and so on. To each dish add an

equal volume• of eggs (e.g., 1 drop) and examine

for evidence of fertilization (elevation of fertiliza

tion membranes). There should be too many

sperm in the first dilution (leading to polyspermy

and abnormal development) and too few in the last

(decreased fertilizationrate). lf not, extend thedilu

tion series.

The number of sperm in the stock suspension

can be estimated by measuring its optical density

with the colorimeter and comparing the result with

a standard curve. With this information you can

estimate how many sperm are required to fertilize

an egg.

Using a format similar to Table 2.1, record the

eggs percentage of mature

fertilized in each sperm

eggs dilution, based on elevation of fertilization

membranes. Count at least 100

in several

microscopic fields of view. Cover the dishes to

eggs these prevent desiccation and count the percentage of

cleaved

later. How do

counts compare

with those based upon fertilization membrane

elevation? (lf you really know how to recognize

will fertilization membranes, the two percentages for

each dilution

be the same.)

Note: What you seke from this exercise is the

sperm concentration which yields optimum fer-

Table 2.L Optimum sperm dilution for fertilization in Tripneustes gTatilla.

Sperm Dilution

1

Drops "Dry" Semen

per ml FSW

1/50

% Mature Eggs

Fertilized

% Mature Eggs

Cleaved

1/2

1 / 1 00

1/4

1/200

1/8

1 /400

1 /16

1 /800

1 /32

1 / 1 600

1 /64

1 /3200

1 / 128

1 /6400

"' Theexactnumberof eggs isunimportan� butprecision (repeatability) is important. As astart,hold a pasteur pipetfilledwith egg

this suspensionverticalyl until the eggscollect into the tapered portion, then deliver one dropof this compactsuspension into the water

in the dish. (How would you determine the mean number of eggs delivered by method? What effect does repeated washing

of the eggs have upon this technique?)

87

15.2 Page 142

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2.4 Experimental Embryology

tilization; i.e., the fewest sperm required to fertiliz.e

all of the mature eggs.

Timing Normal Development

Objective

D Produce a timetable for normal develop

ment at standard laboratory conditions.

In order to use embryos for experimentation

later in this course, you need to predict the time of

appearance of a particular stage of development

following fertilization. This will require that you

produce a developmental timetable such as that

shown in Table 2.2.

Rate of development varies with such factors as

temperature, pH, salinity, and even individual

variability. For thiscourse,standardconditions will

be room temperature (20°-22°C ) and filtered sea

water (pH 7.8-8.3; 35 °Ioo salinity).

Trme to achieve a particular stage of develop

ment is recorded when 50% of the sample is at that

stage. This will require your attention following

fertilization to note when the first individuals enter

into a particular stage, and constant monitoring

(and counting) until 50% have done so.

Observing Fertilization

egsg While you are waitingforcleavage to begin, put

some washed, unfertiliz.ed

on a slide and ex

amine them in detail. To prevent crushing the eggs

with the coverslip, prop it up with pieces of broken

coverslip, grains of sand, a hair, or some clay or

wax on the corners (Nfooted coverslip"). Examine

spermatozoa in the same manen r. Add some

diluted sperm to one side of the egg preparation

and watch the sperm swim to the eggs and activate

them. Make sure you can recognize fertilization

membrane elevation.

Table 2.2. Tunetable for normal early development of Tripneustes gratilla.

Location: _ _ _ _ _ _ _ _ _ _ _ _ _ __ Temperature_ _ _ _ _ _ _ _ _ _ _ __

Stage

Gametes mixed

Fertilization membrane

Pronuclei fusing

2-cell stage

4- cell stage

8-cell stage

16-<:ell stage

Blastula

Hatching

Gastrula

Prism

Pluteus

Datetrime

TllDe Alter Fertilization

to 50% at Stage

15.3 Page 143

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Sea Urchin Fertilization 2.5

Specific Interacting Substances of

Eggs and Sperm

Objective

D Demonstrate that eggs and sperm have

chemical properties which contribute to

specific interaction during fertilization.

seaF. R. Lillie (1912) demonstrated that ripe eggs of

urchins release a substance, fertilizin, which

activates and agglutinates spermatozoa of the same

species. Fertilizin was believed to interact with a

sperm substance, anti£ertilizin, in a manner im

portant to the natural process of fertilization. The

following exercises demonstrate some of the

properties of these substances and (hopefully) will

stimulate curiosity about the process of gamete

activation.

A. Preparation of Egg Water Fertilizin.

(Vasseur, 1948)

1 . Wash unfertilized eggs one time in a small

volume of FSW.

2. Extract the egg jelly coat by treating the eggs

for 15-3- 0 mins in an equal volume of acid

SW (2 ml N HO + 100 ml FSW, pH 5.5-5- .8).

3. Sediment the eggs with the hand centrifuge

and decant the jelly coat extract into another

beaker. Save the eggs in FSW.

4. Adjust the extract to pH 8.0 with lN NaOH.

B. Preparation of Sperm Water Antifertilizin.

(choose one method)

1 . Dilute "dry" sperm with an equal volume of

FSW. Heat 10 mins at 60°C, then cool and

centrifuge or millipore filter to remove

sperm. (Frank, 1939).

2. Dilute "dry" sperm in an equal volume of

acid SW and freeze. Thaw and centrifuge or

millipore filter to remove sperm. Adjust pH

to 8.0 with lN NaOH (Tyler, 1939).

C. Agglutination and Activation Properties of

Fertilizin. (Lillie, 1912)

1 . Prepare a slide with a footed coverslip. Add

a drop of diluted sperm to one side, and a

drop of egg water to the other side of the

coverslip. Observe what happens as the two

materials mix and interact.

2. How might this property function in normal

fertilization?

D. Testing Agglutinated Sperm Capacity for

Fertilization. (Lillie, 1913)

1. Mix 2 drops of diluted sperm (1 drop "dry"

sperm in 50 mis FSW) into 10 drops of egg

water (egg jelly extract, from Al and allow to

stand for 10 mins.

2. Set up and fertilize two dishes of normal

eggs.

a. Dish #1 fertilized with 1 drop of the above

agglutinated sperm.

b. Dish #2 fertilized with 1 drop of normal

diluted sperm (1 drop "dry" in 50 ml

FSW).

3. Observe percent fertilization in each dish

and compare.

4. What role in normal fertilization might be

played by this property?

E. Agglutination of Egg Jelly by Antifertilizin.

(Frank, 1939)

1 . Place 10 drops of sperm water antifertilizin

(from B, above) in a small dish.

2. Add 1 drop of normal eggs (with jelly coats).

Let stand for 10 mins.

3. Place a sample of the eggs on a footed

coverslip mount and examine under oblique

lighting for a precipitation ring at the outer

edge of the jelly. Add a small drop of

Toluidine Blue solution to accent the ring.

4. How do you account for this result?

88

15.4 Page 144

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2.6 Experimental Embryology

F. Agglutination of Naked Eggs with Antifer

tilizin.

(Frank, 1939)

1. Place 10 drops of sperm water antifertilizin

(from B, above) in a small dish.

2. Add 1 drop of naked eggs (jelly coat

mins removed, from A, above). Let stand for 10

.

3. Place a sample of the eggs on a footed

coverslip mount and examine.

4. How might this property function in normal

fortilization?

G. Testing the Fertilizability of Naked Eggs.

(Lillie, 1915)

1. Prepare two dishes of eggs:

a. Dish #1: normal egsg (washed once).

b. Dish #2: naked eggs (jelly coats removed).

2. Fertilize each dish with normal diluted

sperm and compare the results.

3. What might you conclude about the role of

the jelly coat in the normal course of events

in fertilization?

References

Frank, J. A. 1939.Someproperties ofsperm extracts ,

and their relationship to the fertilization reaction;

in Arbacia punctu/ata.

76: 190-216.

Biol.

Bull.

(Woods

Hole)

.,

Giudice, G. 1973. Develapmenta/ Biology of the Sea

Urchin Embryo. Academic Press, N.Y.

Harvey, E. B. 1956. The American Arbacia and

Other Sea Urchins. Princeton Univ. Press.

Lillie, F. R. 1912. The production of sperm isoag

glutinins by ova. Science 36:527-530.

Lillie, F. R. 1913. Studies of fertilization. V. The

behavior of the spermatozoa of Nereis and Ar

bacia with special reference to egg extractives. J.

Exptl. Zoo!. 14: 515-574.

Lillie, F. R. 1915. Sperm agglutination and fertiliza

tion. Biol. Bull. (Woods Hole) 28: 18-33.

Tyler, A. 1939. Extraction of an egg membrane lysin

from sperm of the giant keyhole limpet

(Megathura crenulata). Proc. Natl. Acad. Sci.

(US.) 25: 317-323.

sea Vasseur, E. 1948. Chemical studies on the jelly coat

of the

urchin egg . Acta Chem. Scand. 2:

900-913.

15.5 Page 145

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APPDIDJ:X 8

VARIOUS GROUPER SPECIES ENCOUNTERED DURING THE CONSULTANCY

89

15.6 Page 146

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1....: ;

- . : ~ ,:-".

.. _,., :

- . - • �-:;. •• ' ' i

!C

•• :-

-,. .

Plate 1 . Representative of Epinephelus mi crodon .

Plate 2 . Rep r e s entat ive

of

Epi neph e l u s

fuscoguttatus .

90

15.7 Page 147

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Plate 3 . Representative of Plectropomus aerol atus .

Plate 4 . Representative of Plectropomus leopardus .

91

15.8 Page 148

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,. - '· �·

, _'; .

-. _ / :.. .

Plate s . Example of Cephalopholis miniata .

'.

1-

f..-

~~/: ·~ ·~--!, ...

... '~ ~..,

7' .

_! _,!

r---�

..

Plate 6 . Examp le of Aethaloperca rogaa .

92.

15.9 Page 149

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APPDIDIX 9

GONADAL TISSUE FROM CEPHALOPHOLIS MINIATA

93

15.10 Page 150

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':'--

.i':

..

..�., · -.

·; -,z- -.. -· :�;;,�... :!i_i-·

.-;._ .....·..- .~--'

~,;,

-~ .· ·. ~:.:.. -·

--;j- �: . . ,.. .

·-r

. ·.;:...

:

II-"._

... -· .. -~- -

_

r

~

•...• -·..•

;;......,__ :'

--. . .,,.-

,. �- � ; ::· .

. :�·/:'.}\\-�;;.-i::��· ·..-:: ..-;:-=-r-.-.,·... ·-!" ....i·- -....

r ;_-,-):..·,·:.:... ..

...·-"!

i

. ·.. ·:�

.�

··.:.'"

199 3 . Plate 1 . Wet mount of ovarian tissue from c .

miniata caught July 15 ,

4 0x

Plate 2 . Wet mount of gonadal tissue from a

"transitional male of C . miniata . 4 0 x .

9Ll .

16.1 Page 151

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�· . . .

.. :-

..·'.: .

· . ' -.

0 . ·: :·/

. . ..

•

-

... ....

· .. .

'---� . ..

Plate 3 . Wet mount o f testicular tissue from c .

miniata caught July 15 , 1993 . 4 0x .

9S

16.2 Page 152

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APPENDIX 10

PROCEDURES FOR THE ARTIFICIAL PROPAGATION OF GROUPERS

IN MICRONESIA

96

16.3 Page 153

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PROCEDURAL GUIDE

PROCEDURES FOR THE ARTIFICIAL PROPAGATION OF GROUPERS

IN MICRONESIA

Prepared By : Clyde s . Tamaru , Ph . D .

Edited By : Christine Carlstrom-Trick

Hawaii C ' s Aquaculture Consultant Services

1157 Lunaapono Place

Kailua, Hawaii 9673 4

September 28 , 1993 -

16.4 Page 154

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TABLE OF CONTENTS

. INTRODUCTION

.

..

PURPOSE • • •

.

..

... . .

l3 .·

3''

ESTABLISHING A CAPTIVE BROODSTOCK

. Capture . . . . . . . . .

Air Transport of Live Fish

. .. . .. . .. . .

.

3·

4

5

. . . REPRODUCTION

•

. . . Broodstock Maintenance

Tank Des ign • • •

Tank Maintenance

Water Quality •

. . . . Observation • •

. . Feeding • • • •

. . . . Examining Broodstock

. . . Induction of Spawning

Strip Spawning . • • •

. . Incubation and Hatching

Egg Transport

•

.

•

.. . .

.

-

.

..

.

7

9

11

13

.

15

16

PRODUCTION OF LIVE FEEDS

. Phytoplankton culture

. . Rotifer Culture

•

. . . . . Harvesting of Copepods

. . Oyster Trochophores

. Artemia Nauplii

•

. Hatching of Artemia

. . Enrichment of Artemia

.

.. .. . . . . .

..

17

20

23

24

26

. . . . . . . LARVAL REARING

•

. Tank Preparation • • • • •

. Intensive Larval Rearing

. . . . . . Extensive Larval Rearing

. . . . . . . Feeding Regimen

•

-

•

.-

•

. . . . . . Intensive Larval Rearing

. . . . Extensive Larval Rearing

. Water Exchange

. . . . . . . Daily Routine •

. . . . . . . . . . . Sorting

27

28

29

30

32

33

. Stress Test

Harvesting •

..

34

36

. . PARASITES AND DISEASE

First Aid

Caligus sp . or sea Lice

..

.

36

37

38

. . . . . LITERATURE CITED

...

.

39

16.5 Page 155

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INTRODUCTION

Groupers are a preferred species for fishermen because of

their high market value in local and fore ign markets as well as

their suitability as a food source. Groupers are genera lly an easy

catch in areas where overfishing has not depleted the stocks ,

because the fish are curious by nature and almost any bait will

satisfy their voracious appetites (Myers , 199 1 ) . Efforts to

conserve native stocks represents a loss of -revenue and food on the

table, and in several areas of Micrones ia , specific grouper

populations have all but disappeared. In Guam , for example ,

preferred species such as Epinephelus fuscogu ttatus , E . mi crodon ,

and Cephal opholis miniata are a rare catch .

The artificial propagation of groupers represents a viable

means to circumvent this di lemma by the generation of income from

the sale of hatchery-produced juveni les , growout culture , and the

augmentation of natural stocks via stock enhancement .

Development of grouper hatchery technology , in general , has

been l imited because of the biological peculiarities of grouper s .

Grouper hatchery technology i n Micronesia i s hindered by the lack

of facilities at which to carry out hatchery production or , in the

case of Guam, depleted stocks in surrounding waters where

facilities exist.

PURPOSE

The intent of this manual is to provide the basic procedures

for developing a hatchery technology for groupers . Much of the

rationale for many of the procedures is based upon preliminary work

on Epinephelus microdon and E . fuscoguttatus species performed for

this consultancy . The reader is · asked to refer to appropriate

sections in the precursory Final Report and appendices for purposes

of il lustration . However , some of the procedures are similar to

those employed for the culture of striped mullet (Mugil cephalus)

and milkfish ( Chanos chanos ) and thus have been incorporated from

Tamaru et a l . , ( 19 9 3b) .

This guide is geared for the recruitment of groupers from

Palau and their propagation at the Guam Aquaculture Development and

Training Center . It cannot be considered a complete hatchery

manual , as many of the procedures are still being refined and

requ ire validation at commercial scale. This procedural guide

does , however , provide a basis from which a complete technical

manual can later be contrived .

ESTABLISHING A CAPTIVE BROODSTOCK

The first step in developing a hatchery technology for

groupers is to obtain live adult individuals ( i . e . , 25-50) of the

desired species . The number is dependent upon factors such a s ,

the available holding facilities and manpower , the activity

3

16.6 Page 156

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obj ectives ( e , g , . research or production ) , proj ected hatchery

outputs , and availabil ity of feed .

capture

� Local fishermen in Palau have long known that various spec es

of groupers are easiest to catch during annual spawning

aggregations (Johannes , 1981) • Spawning aggregations o f 11temekai11

(e.g. ,

E . microdon , E . fuscoguttatus ) , and 11tiau11 ( e . g . ,

Plectropomus areol atus , P . leopardus) take place in various

passages throughout the barrier reefs known only by the f ishermen

( FitzGerald et al . , 1991) . Their knowledge extends from annual

spawning occurrences to the times of the month ( i . e . , lunar cycles)

and day ( i . e . , tides) when fishing is best. They also know in

great detail what baits are best for a particular species .

Therefore , it is highly advisable to employ the services of a local

fisherman to aid in locating a desired species of grouper in the

j

waters surrounding Palau.

;

One particular channel (Ngerumkao l ) has historically been a

site of spawning aggregations _ and �herefore_heavily fished . Since

198 0 , the area has been . declared a sanctuary to help protect

against overfishing . Fishing for broodstock is permitted , however ,

national and state collecting permits are required .

A pictorial essay of the procedures for catching groupers and

their subsequent treatment is presented in the Final Report ,

Appendix 1 , Plates 1-5 .

Materials Required :

1 . boat with live bait well

2 . hand lines ( 1 0 0 yards of 50-130 lb test ) , assorted hooks

( size 20-36) , and lead weights*

3 . bait ( e . g . , squid , sardines , tuna , reef fish) *

4 . bait knife and cutting board

5 . large scoop net

6 . 12 ·cc syringe with 2 0-gauge hypodermic needle

7 . polyethylene cannula ( 0 . 86 mm i . d . , 1 . 52 mm o . d . )

8 . tape measure

9 . holding tanks ( 1000 to 4000 liters)

10 . seawater and aeration systems

1 1 . Prefuran (Argent Chemical , Redmond , Washington , USA)

* = varies with the species is to be captured

Step 1 . Determine the 'l"arget· Species: A decision should be

made prior to fishing as to what species is to be captured .

This will determine what time of the year to fish and the

fishing gear and bait to be used .

For examp l e , 11t iau11

aggregate in the channels between March and June and require

moderate size fishing gear . "Temekai " aggregate between June

and August and require larger fishing gear and bait ( i . e . ,

reef fish and/or tuna ) . Cephalophilis miniata can be caught

4

16.7 Page 157

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year-round inside of the barrier reef with smaller fishing

gear and prefer sardines .

step 2 .

Removal of Air From the Coelo1lil c cavity:

Most

species of groupers are caught in relatively deep water . When

brought on board a boat their coelomic cavity will normally

fill with air and swell to compensate for the decrease in

pressure as they rise to the surface. After the captured

individuals have been gently "unhooked" and placed in the bait.

well the air must be removed from the cavity of those floating

upside down with a swollen abdomen . Hold the fish abdomen

side up with the head downward. Use the syringe to puncture

the side of the abdomen as close to the anus as possible. Be

sure to remove the plunger so that the air can escape . After

the trapped air has escaped ( i . e , one to two minutes ) , remove

the syringe and release the fish back into the bait well .

This activity can be repeated , if necessary .

step 3 . Return to Hat�hery_: When _the desired number of

individuals are obtained from the ocean , they should be taken

to the hatchery as soon as possible. During the return trip,

the fish should be checked frequently and the water in the

bait well changed every 2 0 minutes or so . Groupers tend to

regurgitate bait and sometimes the entire stomach contents

after capture and when placed in the bait well . Remove any

debris vomited by the captured individuals to prevent fouling

of the water and reingestion .

Step 4 . Back at the Hatchery : At the hatchery , place all

captured individuals in holding tanks containing seawater and

supplied with continuous aeration. Continue to remove air

from the coelomic cavity of affected individuals as necessary .

Allow broodstock to recuperate for at least 24 hours before

handling again.

step 5 .

Antibiotic Treatment :

The captured fish will

unavoidably receive bruises and/or abrasions from the capture

process and should be treated at the hatchery with an

antibiot ic .

is presented

Tihnethcoempselectteiopnro"cFeidrustr�. Afiodr"

antibiotic treatment

under Parasites and

Disease . Captured individuals can be fed from the second day

after being captured uniess spawning or shipping activities

must be undertaken immediately after recuperation from

capture .

Air Transport of Live Fish

Establishing a grouper broodstock outside of Palau requires

transportation by air to their final destination . The following

sections describe the methods for preparing the broodstock for long

distance transport .

5

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Materials Required:

1 . scoop nets

2 . seawater and aeration systems

3 . oxygen cylinder with two stage regulator

4 . plastic buckets

5 . 2 8 11 x 16" 3 -ml in thickness plastic bags

6 . strapping tape

7 . rubber bands or electrical tape

8 . 5-liter plastic pitcher

9 . gang valve and airstones

10 . ice

1 1 . 100-liter plastic container

12 . scale ( 5 0 kg capacity)

13 . styrofoam shipping boxes

14 . airline shipping bags

step 1 . Preparation for Transport : Preparation for transport

can take several days . Transportation of live fish by air has

restrictions imposed by the airlines . Before the shipping

date , obtain all necessary documents ( e . g . , health

certificate , agriculture inspection forms , certificate of

origin, custom declarations , method of payment) . If any one

of these is not in order , the entire shipment can be detained

upon departure or arrival at the destination . Obtain a fl ight

schedule and make shipping reservations well in advance . The

shipment can be sent as cargo on a domestic airline or handled

by a freight forwarder . The time necessary for packing and

travel to the airport should be considered . It is important

that the individuals to be shipped NOT BE FED at least 4 8

hours prior t o transport .

Step 2 . Water Temperature : on the day of shipping, lower the

water level of the tank containing the individuals to be

shipped as much as possible. Be sure the tank continues to

receive aeration. Place bags of ice directly into the water

to lower the water temperature between 22 and 25°C . Fill a

100-1 plastic container with seawater and lower to the same

water temperature using bags of ice . The seawater in this

container is used to fill the transport bags .

Step 3 . Packing: All transport boxes must be clearly

labelled with the address at final destination and the name

and phone number of a contact person . Place two plastic bags

( one inside of the other) into the styrofoam shipping box.

Depending on the size of the individual ( s ) to be shipped ,

measure enough chilled water to completely cover the fish and

place it into the transport bag . The entire transport box

including its contents cannot exceed 45 lbs when shipped by

domestic airline. Place the desired number of individuals

into the transport box and fill the bags approximately 2 / 3

full with oxygen. Seal the bags with rubber bands or

6

16.9 Page 159

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electrical tape to insure a tight sea l . Close the transport

box and seal with strapping tape . Place the entire transport

box into a large plastic bag obtained from the airl ines and

seal . Take boxes to the airport and ship . If using the

services of a freight forwarder , · the packing must be

coordinated with the forwarding service pick-up time which is

usually two to three hours before departure of the flight.

Step s . Acclimation : When the fish have arrived at their .

final destination remove the fish from the boxes and leave

them in the plastic bags . Float them in the receiving tanks

for approximately ten minutes before emptying the fish and

shipping water directly into the receiving tank .

The

receiving tank should be provided continuous aerat ion and the

seawater exchanged at a rate of 100%/day . The transported

fish should be observed in the receiving tank for at least 4 8

hours before removal to another tank • Food can be offered

after the fish have acclimated . Small amounts should be

o ffered and personnel should watch to see that it is consumed.

Any uneaten food should be removed. UNEATEN FOOD SHOULD NEVER

BE LEFT TO ACCUMULATE ON THE BOTTOM OF THE TANK for it will

decompose and foul the water . The fouled water could result

in bacterial growth on the fish which could be fatal without

treatment .

REPRODUCTION

To understand the reproductive process in fishes and the

rationale for techniques developed for controll ing maturation and

spawning , hatchery personnel should have some comprehension of the

hypothalmo-pituitary-gonadal

(HPG)

axi s .

A schematic

representation of the axis and the levels at which it is inf luenced

by environmental and hormonal parameters is presented in F igure 1 .

Broodsto·ck Maintenance

The main task of the hatchery operator is to alleviate any

stress that would prevent the captive broodstock from their normal

reproductive processes . Excessive handling , . overcrowding, diet , or

changes in tank conditions ( temperature , salinity pH , or disso lved

oxygen) are stressors that , if not alleviated can inhibit growth

and reproduction (Bil lard et al . , 198 1 ) . Daily maintenance

activities are monitoring water qual ity , feeding , tank maintenance

and observation of the broodstock. These activities are construed

as tedious but are essential for a healthy and productive

broodstock •

... Tank Des ign

.f Natural spawning of groupers as been achieved in earthen

bottom ponds , concrete tanks ( 100 m ) and net cages ( 5x5x3 m) for

7

16.10 Page 160

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HYPOTHALMO - PITUITARY - GONADAL AXIS

(HPG AXIS )

NERVOUS SYSTEM

- TEMPERATURE

--------------- DAYLENGTH

GONADAL

STEROIDS

PITUITARY

'

GONADS

GONADOTROPIN

RELEASING

HORMONE

GONADOTROPIN

( e . g . , HCG)

l'iqure 1 . Schematic representation of the HPG axis and the

levels of action for various hormones . From Tamaru

et al . , ( 1993b) . ·

E . salmonoides , E . fario and E . fuscogu ttatus, respectively (Tamaru

et

of

al. ,

<- 1

199Ja ) 2 when stocked at a 1 : 1 male/female ratio at a density

fish/m • For the purposes of this procedural guide , the

existing tanks at GADTC will be used as referenc e . Seawater

entering into the tank should be filtered by gravel or a f i lter bag

( 1 0 µm size) . The seawater system should have the capacity to

achieve a sustained flow rate sufficient for a minimum of 100%

water exchange/day . Since groupers are known to be territorial in

the wild, the broodstock tank should contain hiding places , which

. can be made from PVC pipe or concrete blocks . To prevent excessive

algal growth , the tanks should be covered with a shade cloth.

Inordinate algal growth is conducive to the harboring and outbreak

of several pathogens . It is common practice in mariculture to

place herbivores into broodstock tanks to naturally crop the growth

of macroalgae . However , because of the voracious carnivorous

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feeding habits of groupers , large milkfish ( i . e . , 2-3 kg body

weight) are recommended.

� Tank Maintenance

Siphoning of the tank bottom should be done on a regular basis

to remove excess food and debris and to prevent the tank from

becoming anaerobic. At least once a year the entire pond should be

emptied and al lowed to sun dry before use again.

� water Quality

The most important factors affecting water quality are water

temperature, dissolved oxygen , and salinity and they should be

monitored daily . A thermometer ( ° C) , dissolved oxygen ( DO) probe

and a refractometer are used to measure these parameters ,

respectively . The normal practice is to take daily measurements in

tthheatmtohren.idnagtaancdanafbteerenaosoinly.

Readings should be logged in

suinmarized and scrutinized for

a manner

changes .

Deviations outside of "normal" values require corrective action.

� Observation

An important part of the daily routine is to observe the

broodstock for changes in appetite or behavior at the hatchery .

This requires the personnel responsible for the care of the

broodstock to be familiar with the swimming and feeding patterns of

the fish . Loss of appetite and erratic swimming behavior are

indications of the presence of parasites and/or poor water quality

and if left unattended could result in a broodstock catastrophe .

� Feeding

At present , there is no formulated feed for use in grouper

culture . Trash fish fed exclusively at a rate of 2 -5% body

weight/day and distributed between two or three separate feedings

has resulted in natural spawning of captive broodstock in Taiwan

and - Singapore (Tamaru et al . , 1993a) . The feed should be

distributed as evenly as possible . As mentioned previously,

groupers are extremely territorial and during feeding the larger

males will hoard all of the food i1' presented in only one area of

the tank . Time should be taken during the feeding process to

insure that all individuals get food to reduce the probability of

cannibalism ( Figure 2 ) .

Examining Broodstock

The monitoring of broodstock is an essential part in

establishing the spawning season and assessing the general health

of the fish. Broodstock examinations should take place at monthly

intervals . It is recommended that all broodstock individuals be

identified with a PIT tag ( Identification Devices , Boulder ,

9

17.2 Page 162

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3 inches into the body cavity . Apply suction to retrieve a

sample of the gonads and place sample into a 5% forma lin in

seawater solution by blowing through the cannula . Label each

sample vial with the date of biopsy and the tag number of the

fish.

Step 3 . Physical Examination : Each fish should be examined

for the presence of external parasites , discoloration , bruises

or abras ions .

If any abnorma lities are detected the

individual should be p laced in a separate tank for treatment .

The treatment tank should have running seawater and continuous

aeration . Repeat steps _2 and 3 until_ all of the broodstock

have been examined . If parasites are detected the entire

broodstock must be treated , including the tank .

Step 4 . Examination of Samples: Each gonadal sample should be

viewed under a compound microscope equipped with an ocular

micrometer and the state of maturity recorded for each

individual examined . If vitellogenic oocytes are observed ,

the average oocyte diameter from a minimum of 50 oocytes

should be obtained .

Step 5 . Su1alm1 rizing the Data : Temporal changes in maturation

and growth should be summarized and plotted to determine the

spawning season based upon the presence of vitel logenic

oocytes and spermatozoa . This data can then be correlated to

temperature and daylength to provide indicators as to what

environmental cues are mediating the reproductive process in

groupers . Oocyte growth data from individual females must

also be summarized to obtain the rate of oocyte growth . This

data is invaluable for anticipating when the females will

reach the state of maturity at which induction of spawning can

commence .

Induction ot spawninq

The hormonal induction of final maturation and spawning for

groupers has been successfully achieved with human chorionic

gonadotropin (HCG) . This gonadotropin acts directly on the ovary .

Alternative hormones used in mariculture are gonadotropin releasing

hormones and their more potent synthetic analogues .

These

stimulate the pituitary into releasing its own gonadotropin ( Figure

1 ) . In either case, the ovary must be in an appropriate state of

maturity and this must be assessed with each fish prior to

administering the hormonal treatment .

The procedure for induction of spawning is as follows :

Materials Required :

1 . 1-cc tuberculin syringe

2 . 23 gauge hypodermic needle

3 . HCG in 1 0 , 0 0 0 IU/vials

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17.3 Page 163

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Figure 2 . Mortalities of adult E . mi crodon due to

cannibalism.

Colorado_, USA) •

musculature and

This particular tag

when activated with a

irseai.dmeprl

anted into

transmits

the

a 10

dorsal

digit

code. This is a means of permanent identification to aid in the

evaluation and monitoring of the broodstock .

Materials Required :

1 . PIT tag reader and tags

2 . scale { 1 0 kg capacity)

3 . measuring board

4 . polyethylene cannula ( 0 . 8 6 mm i . d . , 1 . 52 mm o . d . )

5 . large scoop net

6 . solution of 5 % formalin in seawater!

7 . 10-ml sample vials with screw caps

8 . 1 0 00-l iter holding tank

9 . aeration and seawater

Step 1. . Length and Weight Determination : Lower the volume of

the broodstock holding tank .

Groupers can then be

individually caught with scoop nets .

Obtain the

identification number of each fish and place on the scale to

determine the body weight . Remove the individual from the

scale and place on a measuring board to obtain total length .

Record the data on an appropriate data sheet .

Step 2 . Gonadal Biopsy: Hold the individual upside down and

insert a polyethylene cannula into the cloaca approximately 2-

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17.4 Page 164

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4 . physiological saline

5 . polyethylene cannula ( 0 . 8 6 mm i . d . , 1 . 52 mm o . d . )

6 . large scoop net

7 . scale ( 10 kg capacity)

8 . 4 0 0 0 -l iter tank

9 . seawater and aeration systems

1 0 . solution o f 5 % formalin in seawater

11 . 10-ml sample vials with screw caps

12 . compound microscope with- ocular micrometer

13 . depression s lide

14 . disposable pasteur pipettes with bulbs

15 . egg collector

16 . calculator

17 . aquarium scoop net

18 . hand tally

step 1 . Obtaining an ovarian Biopsy: Restrain a female with

a scoop net and insert the polyethylene cannula through the

cloaca and into the . oviduct approximately 2-3 inches . By

applying suction an ovarian sample can be retrieved . Blow out

the sample into a vial containing a solution of 5% formalin in

seawater . Label each vial with the PIT tag number of the

individual and the date of biopsy .

step 2 . Hean Oocyte Diameter: The mean oocyte diameter is

determined by measuring the diameters of 50-100 oocytes with

a compound microscope equipped with an ocular micrometer . The

mean oocyte diameter serves as a measure of the maturity of

the female . Only females found to possess a mean oocyte

diameter > 400 µm should be used in spawning trials .

step 3 . Assessing Testicular Maturation : The state of

maturity of male individuals to be used in the spawning trials

also must be assessed. This can be done by restraining the

individual in a scoop net and applying s light pressure to the

abdomen . If no milt is expressed , the cannula is used as

described for females . If milt can be obtained by either

method , the male can participate in spawning trials .

Step 4 . Administering the Priming Injection : HCG is supplied

in glass vials and in lyophilized form and must be dissolved

in physiological ( O . 7 % ) saline prior to administr.ation. Using

a 1-cc tuberculin syringe capped with a 2 3 gauge hypodermic

needle place 2 ml of physiological saline into the vial

containing 1 0 , 0 0 0 IU of HCG . This will result in a solution

that contains 5 0 0 0 IU/ml = 5 IU/µl . Obtain the weight of the

selected female or male and administer a priming inj ection

that is equivalent to 7 0 0 and 500 IU/kg body weight for female

and male , respectively . A good location to administer the

hormone is three scale rows below the dorsal f in . The normal

practice is to administer the hormone between 16 : 0 0 and 17 : 00

12 .

17.5 Page 165

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hours . Place the treated individuals into a 4 000- l iter

spawning tank equipped with running seawater and continuous

aeration .

Step 5 . Administering the Resolving Injection : Twenty four

hours later , the second ( i . e . , resolving inj ection) is

administered to the female using the procedure described

previously . In this case, however , the dosage of the second

injection is 1 4 0 0 IU/kg body weight . Males need not be given .

a second inj ection . Return the treated individual s to the

spawning tank and setup a col lecting net within the drainage

system as pictured in the Final Report, Appendix 3 , Plate 4 .

Step 6. Spawning: It is preferable to let the female spawn

the ovulated oocytes and . the males fertilize the spawned eggs

naturally .

In the case of E.microdon , this occurred

approximately 8-12 hours after administration of the second

injection. Spawned eggs are then collected using an egg

collector ( in the Final Report , Appendix 3 , Plate 4 ) or by

scooping the eggs with a fine mesh aquarium scoop net . Place

the collected eggs into a 100-liter polycarbonate tank f i lled

with seawater and aerate .

Step 7 . Quantify the Number of Spawned Eggs and Percent

Fertilization : Obtain a samp le of eggs from the 1 0 0 - l iter

polycarbonate tank us ing a 10 0-ml beaker and count the number

of eggs / 1 0 0 ml . Repeat this three times to determine the

average number of eggs/ 100 ml and extrapolate to 1 0 0 liters to

determine the total number of spawned eggs . From a random

sample of approximately 100 eggs , determine the percent

fertilization using a compound microscope . Fertili zed eggs

can be distinguished from unfertilized eggs by their cleaving

cells ( F inal Report , Appendix 4 , Plates E-H) . Using the

average number of eggs / 100 ml , the appropriate number of eggs

can be removed volumetrically.

Strip Sp!lwning

Strip spawning is a method used to ' produce fertili zed eggs in

the event that spawning does not occur natura lly . It is not a

preferred method because of the physical strain placed on both

males and females , as well as the incons istent results regarding

fertilization .

The inconsistency results because of the

variability in synchroniz ing the mixing of ovulated oocytes with

milt . It is , however , a means of producing fertilized eggs that

should be part of the hatchery technology . A pictorial essay of

this procedure is found in the Final Report, Appendix 2 .

Materials Required:

1 . 25- l iter plastic basin

2 . 5-liter plastic pitcher

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17.6 Page 166

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3 . 2 0-liter plastic bucket

4 . sea salt

5 . refractometer

6 . seawater and aeration systems

7 . 12 -cc syringe

8 . polyethylene cannula

9 . large scoop net

1 0 . paper towels

1 1 . 1-liter glass beaker

12 . 100-liter polycarbonate tank

13 . compound microscope

14 . depression s lide

15 . hand tally

1 6 . filter bag ( 10 µm)

Step 1 . Obtaining Hil t From Hales : Catch a hormone treated

male with the scoop net and hold the individual upside down .

Use a paper towel to wipe the cloaca dry . Apply s light

pressure to the abdomen and milt will be extruded from the

cloaca . As the milt is extruded use a 12-cc syringe to

collect approximately 5-10 ml of milt . Cap the syringe with

a hypodermic needle.

Step 2 . Staging the Female : During final maturation the

oocytes wi ll undergo ovulation and hydration and the abdomen

of the female will noticeably swell (Final Report , Appendix 3 ,

Plates 1 and 2 ) . Approximately 12 hours after the second

injection, an ovarian biopsy can be performed as described

previous ly . When a female has completely ovulated , her

oocytes can be stripped . An example of an ovulated oocyte is

presented in the Final Report , Appendix 4 , Plate c . A quick

and simple means of determining whether the eggs have ovulated

is to hold the cannula containing the ovarian biopsy up to a

light source . Ovulated oocytes will have the appearance of a

string of glass beads .

Step 3 . Stripping and Fertilizing Eggs : When the female has

completed ovulated hold her firmly above a dry plastic basin

and apply pressure to her abdomen ( Final Report , Appendix 2 ,

Step 1 ) . You may need to push several times to remove the

majority of eggs . When the majority of eggs have been removed

the female can be returned to the holding tank . Disperse the

milt from the syringe over the stripped eggs ( F inal Report ,

Appendix 2 , Step 2 ) . Gently mix the eggs and milt with your

hand ( F inal Report , Appendix 2 , Step 3 ) . Gently pour f iltered

seawater ( 3 5 ppt) from the 5-liter pitcher into the plastic

basin to activate the sperm , and allow fertilization to take

place . Slowly fill the basin with seawater using the 2 0-liter

bucket and gently mix the eggs and seawater with your hand

( Fina-1 Report , Appendix 2 , Step 4 -and 5) . Approximately ten

minutes after adding the seawater the fertilized eggs will

14

17.7 Page 167

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begin to float to the surface and can be removed by skimming

with the 1-liter glass beaker (Final Report , Appendix 2 , Step

6 ) . Place the collected eggs into a 100-liter po lycarbonate

tank f i l led with f iltered seawater and aerate.

Step 4 . Quantifying the NUJlrlbe of Spawned Eggs and Percent

Fertilization : Same as described in Steps 6 and 7 ,

respectively, in the previous section .

Incubation and Batching

Incubation period extends from the time of fertilization until

hatching. This t ime interval for E . microdon and E . fuscoguttatus

is approximately 18 1 / 2 hours at 27°C . The main activity during

this period is the development of the grouper embryos . The time

frame and stages of ontogeny are presented in Table 9 and Appendix

4 , Plates D-P , respectively , of the Final Report .

Materials Required :

1 . 1000-liter f iberglass tank equipped with center standpipe

2 . 2 5 0 µm nytex screened cap

3 . aeration and seawater systems

4 . 10 µm f i lter bags

step 1 . Stocking of Spawned Eggs : The-incubation tank should

be fil led with f iltered seawater ( 3 5 ppt ) and provided with

continuous aeration . The center standpipe should be capped

with the 2 5 0 µm nytex screen and seawater provided at an

exchange rate of 100%/day. Remove the appropriate v�lume from

the 100-liter polycarbonate tank to result in 5 x 1 0 eggs and

place them into the incubation tank . Allow the eggs to

develop until they are ready to be stocked ( i . e . , somite

stage , Final Report , Appendix 4 , Plate N) .

Step 2 . Separation of Eggs : During the incubation process

unfertil ized or non-developing eggs ( Final Report , Appendix 4 ,

Plates Q and R) wi ll turn opaque and become negatively buoyant

at salinities between 35-40 ppt., When the embryos have

reached somite stage , stop the flow of water and aeration .

Allow the contents in the incubation tank to stand for

approximately 15-20 minutes at which time the develop ing eggs

wi ll float to the surface . Skim the floating eggs and place

them into the 100-liter polycarbonate tank .

Step 3 . Quantifying the Eggs : When the majority of floating

eggs have been obtained , fill the 100-liter polycarbonate tank

with seawater till the 100-liter mark and obtain the total

number and density of grouper eggs as described previously.

15

17.8 Page 168

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Step 4 . Stocking the Eggs : The eggs in the polycarbonate

tank are now ready for stocking into larval-rearing tanks .

The desired number of eggs for each rearing tank can be

removed volumetr ically .

Bqq Transport

Transportation of fertilized eggs is normally not part of

hatchery operations s ince the broodstock and larval-rearinq

facilities are usually at the same location . However , during the

initial phases of any hatchery development , the locale where

broodstock are accessible and the location of larval-rear ing

facilities may be different , as is the case with the grouper work

currently ongoing at MMDC and GADTC . Thus , until a captive

broodstock is established, larval-rearing activities (at GADTC) are

dependent on the transport of fertilized eggs from the site where

spawning can be achieved ( at MMDC) .

When the hatchery technology for grouper is fully developed,

shipment of eggs from an "egg production center" to various

hatcheries may become an actual scenario as seen with the hatchery

production of milkfish in Taiwan . Therefore a procedural guide for

the transport of fertilized eggs is warranted.

Materials Required :

1 . oxygen bottle with two stage regulator

2 . styrofoam transport boxes and plastic bags

3 . sea salt

4 . 1 0 0-liter plastic trash can

5 . fine mesh aquarium net

6 . 5- liter plastic pitcher

7 . refractometer

8 . hand tally

9 . strapping tape

1 0 . electrical tape

11 . 100-ml beaker

12 . filter bag ( 10 m)

Step 1 . Adjusting Sal.inity of Water for Transport :

The

transport of fertilized eggs at a salinity of 4 0 ppt aids in

keeping the eggs suspended in the water column . A supply of

4 0 ppt seawater solution is prepared in a 100-liter container

by adding sea salt to filtered seawater . Dissolve the sea

salt and measure the sal inity with a refractometer until the

desired salinity is obtained .

step 2 . Stocking of Eggs : A specific number of eggs to be

shipped should be determined. Place a corresponding volume of

4 0 ppt seawater into plastic transport bags . The size of the

bags and volumes wi ll vary depending on the quantity of eggs

to be shipped . NOTE: TRANSPORT BOXES AND THEIR CONTENTS MUST

NOT EXCEED 45 LBS IF SENT BY AIR . After obtaining the egg

16

17.9 Page 169

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density as described in

eggs required to achieve

the previous section,

a stocking density of

the

10-

2n0umxbe1r03o/ f1

can be removed volumetrically. These are then s ieved through

a fine mesh aquarium net and the eggs are then placed into

each transport bag .

Step 3 . Packaging of Eggs : Each of the plastic bags is then

filled with oxygen and sealed with rubberbands ( Final Report ,

Appendix 1 , Plate 1 3 ) . If transport is to take place by air .

it is advisable to use electrical tape to insure a tight sea l .

Place a l l bags into transport boxes and seal with strapping

tape .

step 4 . Acclimation : When the eggs have arrived at the

hatchery , float the unopened bags containing the eggs in the

receiving tanks to al low the eggs to acclimate. After

approximately 15 minutes open the bags and place the entire

contents directly into the tanks .

PRODUCTION OF LIVE FEEDS

Production of live feeds is a major activity for the larval

culture of any species and must be coordinated with the

availability of fertilized eggs . Live feeds production for grouper

larvae includes the culture of phytoplankton and rotifers , the

hatching and enrichment of Artemia riaupli i , the harvesting Of

copepods and the processing of oyster trochophores .

Phytoplankton Culture

Nannochloropsis oculata is the algal species recommended for

use in the larval rearing of grouper . It is used primarily as a

feed for culturing rotifers , one of the initial feeds of grouper

larvae . However , its presence in the rearing tanks during larval

culture of striped mullet has also been shown to be beneficial to

overall growth and survival ( Tamaru et a l . , 1993b) . The main

reason that this particular algal species is used in rearing fish

larvae , in general , is its nutritional value (Watanabe et al . ,

1 9 8 3 ) . In particular, this species has been shown to contain

elevated levels of long chain polyunsaturated fatty acids ( PUFAs )

which are essential for a large number of fishes . It has also been

demonstrated that the nutritional quality of the algae fed to the

rotifers directly effects the nutritional value of the rotifers

(Tamaru et al . , 1 9 9 1 ) .

The culture of this species of algae is carried out through a

series of batch cultures where each series represents an

amplif ication of its volume . A schematic representation of the

process is presented in Figure 3 .

The production of cultures indoors progresses through three

stages : 1 ) stock culture , 2 ) flask culture , and 3 ) carboy and/or

cylinder· culture . In the indoor production scheme , the flask and

17

17.10 Page 170

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STEI? 1 .

STOCK CULTURE

STBP 2 ,

PI.ASK CULTURE

STBP 3 .

CAR.BOY CULTURE

STEI? 4 .

LIGHT

SOORC&

V0LCMB

(LITEU)

.

STOCXIliG

DENSITY

(at.W/MLJ

on:u..,_,

0 . 01

ElAR.VBST

DKNSITY

(a1.la/lel.l

tMU.t.ZOllSl

1.8

20 - 3 0 100 - 150

20

20 - 30

70 - 80

400

3 -5

20 - 25

STBI? S .

TANK CCI.TORE

2000

3 -5

20 - 25

Piqura 3 . Flow chart of the process for production of

phytoplankton. From Tamaru et al . , ( 1993b) .

carboy cultures represent starting and intermediate stages in the

culture of algae , respectively. Carboy and cylinder cultures are

used as inocula for outdoor cultures .

- Outdoor production of algae consists of two stages : 1) 4 0 0-

liter tanks which are inoculated from carboys or a cylinder from

the indoor algae room, and 2 ) 2 0 00-liter tanks which are inoculated

from the 4 0 0-liter tanks after they attain harvest density . At

full production , two of the second stage ( i . e . , 2 0 00-liter) culture

tanks are required each day to support rotifer culture and larval

rearing activities . The procedures for production and monitoring

of indoor cultures have been described in detail in Tamaru et a l . ,

( 1993b) . The procedure for outdoor cultures is as fol lows :

Materials Required :

1 . six x 400- liter fiberglass tanks

2 . - - -14 x 2 000-liter fiberglass tanks

3 . aeration system

4 . two submersible pumps

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18.1 Page 171

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5 . garden hoses

6 . top loading balance ( 10 kg capacity)

7 . 20-liter plastic buckets

8 . compound microscope

9 . hemacytometer

1 0 . hand tally or multicell counter

1 1 . chemicals ( i . e . , nutrients , sodium hypochlorite , sodium

thiosulfate)

12 . pipettes

13 . storage room

Step 1 .

Intermediate culture Preparation :

A 4 0 0-liter

starter culture tank should be chlorinated between use and

scrubbed clean . Fill with filtered water at a salinity of 25-

28 ppt and chlorinate with a 5 . 25% sodium hypochlorite

solution . Commercial bleach solutions , such as Clorox or

Purex , are genera l ly available as 5 . 2 5% sodium hypochlorite

solutions . Aerate for 24 hours .

Dechlorinate with the

addition of sodium thiosulfate .

The proportions of

hypochlorite and sodium thiosulfate needed for various tank

sizes are presented in Table 1 .

Table 1 . Amounts of hypochlorite and sodium thiosul-fate for

various tanks sizes .

I TANK

VOLUME

(liters)

II

500

II 1000

II 2 0 0 0

II 5000

5 . 25% NaOCL

SOLUTION

(ml)

1500

3000

6000

15000

SODIUM

THIOSULFATE

(grams)

I

75

II

150

~

300

II

750

II

. Step 2 . Intermediate CUltur� : Nutr�ents ( e . g . , Mass Pack,

Florida Aqua Farms Inc . , Florida , USA) can then be added

according to the instructions with ' an inoculum from the algae

room ( 2 0-liter carboy) . After addition of the iroculum , .the

initial stocking density should be 3 to 5 x 10 cells/ml .

Monitor cel l growth , and use as an inoculum

cultures when densities of 2 0 to 25 x

f1o0r6

the production

cells/ml are

attained .

Step 3 • Production CUI tures : Culture tanks should be scrubbed

clean and rinsed between each use . Tanks holding the final

production stage for algae ( i . e . , � 2 0 0 0 liters) DO NOT need

to dechlorinated . Fill tanks with water at a salinity of 25-

28 ppt and inoculate with phytoplankton from the intermediate

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18.2 Page 172

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tanks to obtain an initial density of 3-5 x 106 cells/ml .

Nutrify as described previous·ly and monitor cel l growth .

Step 4 . Harvest : When cell densities reach 2 0 to 2 5 x 106

cells/ml , the culture is ready to be used as food for rotifer

culture or as background algae for use in the larval rearing

tanks . When the culture is completely harvested the process

is started again.

Rotifer culture

The rotifer Brachionus plicatilis has been recognized as

pivotal for successful culture of a variety of marine fish larvae

( Lubzens , 19 8 7 ; Lubzens et al . , 1989) . Rotifers are small ( i . e . ,

14 0-2 3 0 µm) multicel led organisms that fi lter food particles out of

the water column by means of a cil iated corona located on the

anterior portion of their body .

The main reasons for its

popularity are its small size , to lerance to a wide range of

salinities , fast reproduction rate , abil ity to be cultured at very

high densities ( i . e . , > 1000 rotifers/ml ) , slow swimming speed , and

tendency to stay suspended in the water column ( Fulks and Main,

1991) .

Rotifers can be grown on a variety of feeds , but the most

. common practice is the use of phytoplankton cultures . As stated

previously , the recommended phytoplankton is N . oculata because of

its higher fat content . The method for its culture is as follows :

Materials Required:

1 . six 1000-liter f iberglass tanks equipped with center drain

2 . 60 µm nytex bag

3 . 100-liter plastic basin

4 . dissecting microscope well counting dish

6 . Veeder-Root multicell counter ( 5 well )

7 . 1-ml rotifer counting pipette

8 . magnifying eyepiece ( 10 x)

9 . data sheets

10 . Lugol ' s solution

1 1 . airstones and airl ine tubing

12 . 50 0-ml graduate cylinder

13 . 3-liter graduate pitcher

14 . 20-liter plastic buckets (marked with 1-liter increments)

15 . calculator

1 6 . Nannochl oropsis oculata

17 . freshwater source

.18 . seawater source

19 . refractometer

All rotifer culture tanks should be elevated about three feet

above the ground or near a drainage ditch in which a collection net

can be

�Tnis simplifies col lection of rotifers from the

tanks which is done by draining the culture through a col lecting

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18.3 Page 173

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net. The method described assumes enough rotifers are available to

serve as an inoculum . Each step is schematically presented in

Figure 4 .

FLOW CHART OF ROTIFER PRODUCTI ON

BATCH CULTURE SYSTEM FOR 1 0 0 0 LITER TANK

STEP 1 .

CIJWf ""'""

ADD 500 � PHYTOPLANX"l'Otf

� llO'rXnM Ar 100/NL

STEP 3 .

� aar:c.,..

QCAl1%1l"' 'Y ROTI.-S HMl'YUTm

IHOCt;JIT.A J: liSW TAND

PlBDl JlOTJ:!"BltB TO l"ISH LlUlVJ\\l!:

STEP 2 .

JQ2VZS'l' ROTil'IZB

l":CI.I. TMDC WITH 1000 Lrl'JIRS �Ea'l'OPt.AHX'I'ON

ltl:l::CHOCULA.TS llO'l'I7BJUI

Figure 4 . F low chart for the batch culture of rotifers .

Step 1 . Tanlc Preparation and Inoculation : A) Clean the inside

of three 1000-liter tanks by scrubbing with a scrub brush and

hxos10in6 gcewliltsh/mflr)esfhrowmattehre.

Add 5 0 0 liters of N .

outside phytoplankton

oculata ( � 10

cultures into

each of the three tanks .

Check the salinity with a

refractometer and adjust sal inity with fresh water or more

phytoplankton culture to attain a salinity ranging between 2 0

and 2 8 ppt . Inoculate the three tanks with enough rotifers to

achieve an initial density of 100 ind . /ml . Aerate the tanks

and allow the culture to grow for 24 hours .

Step 2 . Second Day: After 2 4 hours , turn off the aeration to

the tanks and allow the contents to remain undisturbed for

approximately 15 minutes . Harvest all of the rotifers from a

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18.4 Page 174

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single tank by draining its contents through the 6 0 µm nytex

collection net . Be sure that the net is suspended in a

plastic tub fil led with seawater to lessen the shock of

harvesting . Fill the tank with 1000 l iters of phytoplankton

and re inoculate the harvested rotifers . Aerate ' and allow the

culture to grow for an additional 24 hours . Repeat this

activity for the other tanks .

Step 3 . Harvesting and Quanti.fying o.f Roti.fers : A) After the

2 4 hours has elapsed turn of the aeration and allow the

culture to stand for approximately 15 minutes , to allow the

large particles and debris to settle to the bottom of the

tank . Drain the contents of the tank into the rotifer

collecting net as described previously. When the entire

culture has been drained into the collecting net , place the

collected rotifers into a 2 0-liter plastic bucket containing

3 to 5 liters of N . oaulata . Place one airstone and aerate

the contents . Repeat this exercise for the other two tanks

and place collected rotifers into the same bucket.

B) Bring the total volume of the bucket up to the 2 0 liter

mark by adding N . oaulata . Quantify the number of rotifers

harvested by first making a 1 : 250 dilution of the contents in

the bucket u:3 in� a 5 00 ml gr�duate cylinder fil led with

seawater . This is done by removing 2 ml of seawater from the

5 0 0 ml graduate cylinder using the rotifer counting pipette

and replacing it with 2 ml obtained from the harvested

rotifers in the bucket . Take a 1 ml sample using the rotifer

counting pipette from the graduate cylinder and count the

number of rotifers in the pipette with the 10x magnifier .

Rep�at this five times _ and . take th� average to give the

density of rotifers /ml . Multiply the average value by 2 5 0 to

give the density of rotifers/ml in the bucket . The total

number of rotifers can be obtained by multiplying the density

of rotifers in the bucket by 2 0 , 0 0 0 .

C ) Repeat Step 1 for all of the tanks and inoculate by

removing the appropriate volume from the bucket to result in

a stocking density of 1 0 0 rotifers/ml . The remaining rotifers

are available as food for the grouper larvae . Be sure to keep

the rotifers aerated before stocking into the larval rearing

tanks .

The cycle from inoculation to harvest for three tanks takes

. place over a 4 8 -hour period. A continuous daily production of

rotifers is achieved by treating a second set of three tanks in the

same manner , however , the inoculation step is offset by one day

from the other set of three tanks . Thus , on any given day three

tanks can be harvested and the other set of three tanks are midway

to being - harvested .

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Barvestinq of Copepods

GADTC is fortunate in that the larqe shrimp broodstock

raceways contain a considerable number of copepods . The only

drawback is that their availability is affected considerably with

the normal tank maintenance for the shrimp broodstock .

The

collection of copepods is most efficient during the night when

their phototactic characteristic is an advantage for their harvest .

Materials Required:

1 . 6 0 µm collecting net

2 . plastic tub

3 . 1 inch I . D . s iphon hose

4 . flashlight

5 . medium mesh aquarium net

6 . 5-liter pitcher

7 . dissectinq scope

s . Lugol ' s solution

9 . 10-well depression slide

1 0 . hand tally

11. seawater and aeration systems

Step 1 . Harvesting of Copepods : Turn off the aeration system

to the broodstock tanks and suspend a flashlight above the

surface of the water . Place one end of the siphon hose in the

water where the beam of light penetrates the surface . Place

the other end of the s iphon hose where the effluent will flow

throuqh a medium meshed ( 5 00 µm) aquarium net and f inally

through a 60 µm nytex col lecting net . The collecting net

should be suspended in a plastic tub filled with seawater .

After the copepods have been harvested be sure to turn on the

aeration system for the shrimp broodstock tanks .

Step 2 . Quantifying the Harvested Copepods : Concentrate the

collected copepods in the col-lecting net and place into a S

liter plastic pitcher . Fill the pitcher to the 5 liter mark .

Aerate the contents of the pitcher and retrieve a 10-ml

aliquot . Place the 10-ml sample into a watch glass and add

one to two drops of Lugol ' s solution. Gently rotate the watch

glass to mix the Lugol ' s solution, inactivate the copepods ,

and concentrate them into the center of the watch glass .

Using a disposable pasteur pipette , distribute the

concentrated copepods into a 10-well depression s lide, place

under a dissecting microscope and count the number of copepods

in each well . Continue until all of the copepods are counted .

Repeat this activity three times and obtain the average number

of copepods / 10 ml . Extrapolate ( i . e . , multiply by 5 0 0 0 ) to

obtain the total number in the 5-liter pitcher . The copepods

can then be stocked into their respective rearing tank by

removing the appropriate volume .

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oyster Trochophoras

Oyster trochophores can be used as an initial feed for grouper

larvae and are purchased from MTL Biotech Ltd . , Vancouver , B . c . ,

Canada . The trochophores are cryogenetically preserved and need

to be reactivated prior to use as feed . The trochophores are

shipped prepackaged in plastic straws in a "dry shipper" containing

liquid nitrogen . The straws containing the trochophores can be

held in the shipping container for at least a week . The straws are

then removed as needed and the trochophores are rej uvenated . The

procedure is presented in the Final Report , Appendix 6 .

Art:emia Nauplii

It was recognized very early by a number of investigators that

freshly hatched Artemia nauplii are a high value feed for fish fry.

Because of their size , Artemia nauplii also represent , in many

cases , the only available food source for the early stages of fish

and crustacean larvae (Bardach et al . , 19 7 2 ) • Although critical to

larval culture , they have some drawbacks . The first is that the

dry weight and caloric content of nauplii can decrease as much as

25 % within 2 4 hours after hatching when kept at 25°C . This means

the nauplii must be fed to the larvae as soon as possible after

hatching or be stored at low temperatures to decrease their rate of

metabolism . The second is that they have been found to be

nutritionally deficient, especially in the long chain PUFAs . This

shortcoming, however , can be corrected by enriching the Artemia

nauplii .

� Hatching of Artemia

Artemia cysts can be purchased from a variety of sources ,

however , the majority ( i . e . , 8 0 % ) of the cysts that are sold

commercially originate from the Great Salt Lakes in Utah , USA

( Sorgeloos et al . , 199 1 ) . The operator should be aware that there

is considerable variability in the hatching percentage of cysts

from different origins as well as from different batches from the

same strain (Sorgeloos et al . , 1986) . A check of the hatching

percentage should be conducted periodically . This is done by

incubating a known amount of cysts ( i . e . , 1 gram of cysts equals

approximately 250 , 000 cysts) · and hatching them . By estimating the

total number o f nauplii and dividing by the number of cysts

incubated , the hatching percentage can be calculated .

The cysts are packaged in vacuum packed cans that must be

.refrigerated after opening. Hatching of Artemia is a relatively

simple process but there are some parameters that should be

followed . A constant water temperature ( 2 5 to 28°C ) , salinity ( 15

to 3 5 ppt) , pH � 8 . 0 , saturated DO levels accomplished by vigorous

aeration , and strong illumination ( 2 , 000 lux) are essential to

initiate the hatching response ( Sorgeloos et a l . , 1 9 8 6 ) .

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Materials Required :

1 . two 100-liter fiberglass cyl inders with funnel shaped opaque

bottom and drain

2 . 50-liter glass aquarium

3 . black plastic sheet

4 . 12 11 glass tubing ( 1 / 8 11 diameter) .

5 . 10 1 airline tubing ( 1/ 8 " diameter)

6 . flashlight

7 . 20-liter plastic buckets marked with 1 liter graduations

8 . aeration and seawater system

9 . top loading balance ( 10 kg capacity)

10 . submersible heaters

1 1 . collection net 1 0 0 µm*

12 . 100-liter plastic basin

1 3 . tygon tubing 111 diameter

14 . 500-liter graduate cyl inder

15 . 1-ml glass pipette

1 6 . lOx ocular magnifier

17 . l ights ( 10 0 watt)

* = rotifer collecting net may be substituted

Step 1 . Incubation of cysts : Fill f iberglass cylinders with

seawater and aerate vigorous ly . Place heaters in cylinders

and set temperature at 2 8 ° C .

Measure amount ( grams) of

Artemia cysts required and add to the cylinders . (This is

assuming that the hatching percentage of the Artemia cysts is

already known) . An alternative method to measuring cysts by

weight is to measure them by volume . Convert # of grams of

cysts = # of ml of cysts . Aerate the cysts vigorously and

turn on the lights above cylinders and il luminate . Incubate

overnight .

Step 2 . Separation of Empty cysts and Nauplii : After Artemia

have hatched ( at this stage the Artemia are called nauplii)

turn off aeration and remove heater and airstone . I l luminate

the bottom of the tank with a strong l ight .

Wait

approximately 10 minutes for the empty cysts to float to the

surface of the water . Support collecting net in 1 0 0 - l iter

plastic tub securely and fill tub �ith seawater . separate the

cysts from the hatched Artemia by ' draining the cyl inder into

the collecting net . Be sure to close the drain before the

cysts are emptied .

step 3 . Collection of Nauplii : Fill aquarium with seawater

leaving enough room to accommodate the collected naupl i i .

Remove the collecting net from the plastic tub and rinse

collected nauplii with running seawater and place contents of

the collecting net into the aquarium . Do not aerate . Cover

the aquarium with a black plastic sheet leaving a space at the

bottom. Turn on flashlight and il luminate the bottom of the

aquarium . Wait approximately five minutes and s iphon nauplii

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18.8 Page 178

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from the

connected

bottom of the aquarium

with airline tubing as

using the

a siphon

glass

hose .

tubinq

Let the

·

·'

nauplii drain into a 2 0 -l iter . plastic bucket . CAUTION : DO

LET NAUPLII COLLECT ON THE BOTTOM OF THE AQUARIUM FOR LONGER ,

THAN 15 MINUTES .

Step 4 . Quantifying Nauplii Harvested: Bring the volume of

the siphoned nauplii in the plastic bucket up to the 2 0 liter

mark and aerate vigorous ly. Fill graduate cylinder with

seawater to the 5 00 ml mark . Remove 2 ml of seawater with the

1-ml pipette and replace with 2 ml of nauplii from the plastic

bucket . Make sure that the nauplii are aerated ( i . e . , mixed)

vigorously. Mix the contents of the graduate cylinder and

remove 1 ml aliquot with the pipette and count the number of

nauplii in the pipette using the 1ox magnifier . Repeat five

times to determine the average number of naupl i i per ml .

Multiply this value by 2 50 to estimate the number of

nauplii/ml in the plastic bucket . Multiply this number by

2 0 , 0 0 0 ml to determine the total number of nauplii available.

The amount of nauplii required for feeding or for enrichment

can be removed volumetrically.

� Enrichment o f Artemia

Most strains of Artemia are la.eking in _some of the nutritional

elements required by certain species of fish , in particular the

long chain omega-3 polyunsaturated fatty acids ( PUFAs ) as presented

by sorgeloos et al . , ( 19 9 1 ) . This deficiency , however , can be

surmounted by an "enrichment" process in which the Artemia nauplii

are fed a source rich in PUFAs to improve their nutritional

composition . The enrichment procedure for Artemia naupl i i using

Protein Selca is presented below .

Materials Required:

1 . blender

2 . 2 0-liter bucket

3 . Protein Selca (Artemia Systems ,

4 . Artemia naup l i i

5 . 2 0 0 µm collecting net

6 . seawater and aeration system

Belgium)

Step 1 . Obtain Arte.mia Nauplii : A rough estimate of the

amount of nauplii required for the following days feeding

should be obtained . Place the nauplii into the bucket with

seawater at a density of 3 0 0-4 0 0 /ml . To insure that the

total days ration of nauplii is enriched , you may need to

increase the volume or number of buckets to be used in the

enrichment process . Aerate the nauplii vigorous ly.

Step 2 .

Preparation of Protein Selca :

Weigh out the

appropriate amount of Protein Selca required to result in a

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3 0 0 ppm final concentration in the enrichment container .

Place the Protein Selca into the blender and add fresh water .

Be sure to take into account the volume of freshwater being

used in mixing. Blend the mixture until it has a creamy

appearance and add the blended mixture to the Artemia nauplii .

Incubate the nauplii with the enrichment media overnight .

step 3 .

Collection of Enriched Nauplii :

The following

morning col lect the enriched nauplii with the collecting net

and rinse with seawater . Place collected nauplii into a 20-

liter bucket f i lled with seawater and aerate . Quantify the

density of nauplii and remove the appropriate amount for

feeding to the larvae .

Step 4 . Storage of Enriched . Nauplii: Enrich and process a

large volume of nauplii as described .

stored at 5°C and used for up to a week .

These can then be

The nauplii must be

aerated during the storage process .

LARVAL REARING

Larval rearing is viewed as the most difficult aspect in

developing a technology for the artificial propagation of grouper .

The major reason is the small size of grouper larvae. Conventional

methods such as using rotifers as an initial feed have been found

to be ineffective and alternative sources ( e . g . , oyster

trochophores , mussel larvae , and sea urchin larvae) have been

employed to bridge the period from first feeding until the larvae

are large enough to accept rotifers ( i . e . , 7-10 days posthatching) .

A second major impediment has been the reported cannibalism that

occurs between 25 and 3 5 days posthatching .

At present there are two methods ( extensive and intensive) for

the rearing of grouper larvae .

The extensive system is

characterized by being conducted outdoors and in relatively large

tanks or even ponds . Stocking densities are low ( 1-10 larvae / l )

and the feed for the larvae i s dependent on the productivity o f the

ponds . _Results are highly v�riabie , as ex_pected for large-scale

cultures occurring outdoors . However , the extensive method has one

major advantage in that it does not require as much labor . The

intensive system differs in that re�ing is done indoors and in

relatively smaller tanks ( 3-10 x 10 liters) stocked with high

densities of larvae ( 3 0-40 larvae / l ) . The advantages are that the

system is much easier to manage and the results are more

consistent . However , it is labor intensive , especially during the

period where cannibalism occurs and larvae have to be continually

sorted .

Tank Preparation

As stated previously , one of the major differences between the

intensive and extensive methods is the tank size and that the

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extensive culture system is dependent on the productivity of the

tank . The preparation of rearing tanks is presented below.

• Intensive Larval Rearing

Materials Required :

1 . 2 0 0 0 to 1 0 , 000-liter round fiberglass larval rearing tanks

2 . 2 11 center drain with stand pipe and coupling

3 . 250 µm nytex screened drain cap*

4 . 550 µm nytex screened drain cap*

5 . 12 11 inner diameter ring s iphon drain ( 2 5 0 µm mesh) *

6 . 1 2 11 inner diameter ring siphon drain ( 5 5 0 µm mesh) *

7 . blower with aeration system

8 . freshwater and seawater supply

9 . 5 ' of 1 / 8 11 I . D . glass tubing

1 0 . 10 ' of 1 / 8 11 airline tubing

1 1 . 5 1 of 1/4 11 I . D . glass tubing

12 . 10 ' of 5 / 1 6 11 tygon tubing

13 . 10 1 of 1 11 I . D . tygon tubing

14 . airline tubing , airstones , and airline gang valves

15 . black plastic sheet ·

1 6 . shade cloth

17 . 10 µm filter bags

* = items that must be made

NOTE : THE QUANTITY OF ITEMS 2 to 6 ARE DEPENDENT UPON THE NUMBER OF

REARING TANKS TO BE EMPLOYED .

The recommended larval rearing tank should be circular in

shape , constructed out of fiberglass with a black epoxy coated

interior , equipped with a center drain in which a two inch center

standpipe can be fitted . It is preferred that the bottoms have a

sl ight slope ( i . e . , 3 ° towards the center drain) , however , this

feature is not essential . The interior walls should be smooth .

The center standpipe should be equipped with a coupling in which a

short drain pipe with the sides cut out and screened with 250 µm

can be inserted . As the larvae grow older and the rate of water

exchange increases , this can be replaced with a similar cap with

5 00 µm screening .

The number or size of tanks can vary and is largely dependent

on spacial restrictions . However , ths number of rearing tanks

employed in a rearing trial should be based upon the availabil ity

of oyster trochophores , phytoplankton and rotifer production

outputs . Because of the large number of larvae resulting from a

. single spawn , there will be a _desi�e to . sto9k all of the available

larva e .

step 1 . Tank Preparation : The larval rearing tanks and their

components should be air dried when not in use . They must be

assembled on the day of the second inj ection of the spawning

sequence described previously. Rearing tanks should be

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scrubbed and rinsed with fresh water (avoid using detergents

or chlorine) and filled with filtered seawater ( i . e . , � 3 2

ppt ) . The center drain should be capped with a 2 5 0 µm nytex

cap and an airstone (preferably ring type) placed near the

center drain. At least four additional airstones should be

placed in separate quarters of the rearing tank to help keep

the eggs suspended in the water column until they have

hatched .

Step 2 . stage of Development for Stocking: When the embryos

are in somite stage, ( Final Report , Appendix 4 , Plate N) , they

can be stocked into the larval-rearing tanks at an initial

density of 3 0-40 larvae/ l . After the eggs have been stocked ,

the rearing tanks should be covered with a black plastic sheet

to prevent the hatched larvae from being exposed to sunlight .

This should be maintained for at least three days posthatching

at which time the larval tanks can be covered with a shade

cloth.

� Extensive Larval Rearing

Preparation of the extensive rearing tank should commence

approximately seven to eight days prior to receiving eggs and the

operator must exercise proper p lanning in order to manage this type

of rearing system effectively .

Materials Required:

1 . 2 0 , 0 00-liter fiberglass rearing tank ( s )

2 . aeration and seawater system

3 . shade cloth

4 . black plastic sheet

5 . waterproof tarp

6 . 4 11 center stand pipe

7 . 4 11 cap with 25 0 µm nytex screen

8 . filter bag ( 10 µm)

9 . 2 5 0 µm nytex bag

- The tank should be located outdoors in a location where it can

be easily stocked with phytoplankton . �he inner sides of the tank

should be black and smooth . Prior to ' a rearing trial , the tank

should be scrubbed and rinsed with fresh water and allowed to air

dry .

Step 1 . Tank Preparation : Seven to eight days prior to

receiving eggs , fill the tank with f iltered seawater ( 3 2-3 5

ppt ) to a depth of approximately o�e meter . Place an aeration

ring or distr ibute airstones ( 1/m ) and aerate the water in

the tank . Above the tank ( i . e . , one meter) suspend a

waterproof tarp to prevent rain water from entering during

preparation of the tank and the larval rearing trial . Be sure

that the tarp is properly secured .

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19.2 Page 182

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Step 2 . Inoculation of Phytoplankton : After f i l l ing the tank

with seawater , inofulate it with phytoplankton to attain a

density of 3 0 0 x 10 cells/ml . Be sure that the phytoplankton

is also filtered us ing the fi lter bag . During the following

days add phytoplankton as needed to maintain the initial

density .

Step 3 .

Inoculation of Copepods :

� On the same day o

inoculating phytoplankton , stock the tank with 8 0 x 10

copepods . Be sure that the copepods have been f iltered

through a 2 5 0 µm mesh net . Allow the copepods to multiply

directly in the tank .

Step 4 . Inoculation of Rotifers : Three to four days prior to

stocking the eggs inoculate the tank with rotifers at a

density of 5 rotifers/ml .

Step 5 . Stocking of Eggs : When eggs become available they

should be stocked into the rearing tank at a density of 10

eggs / l and allowed to hatch . The shade cloth should be

stretched over the entire surface of the tank and firmly

secured . The black plastic sheet should then be placed on top

of the shade cloth and also secured . Allow three or four

locations where the shade cloth can be easily lifted for daily

monitoring activities .

Peedinq Reqim.en

� Intensive Larval Rearing

A schematic representation of a generalized feeding regimen

employed in the intensive rearing of grouper larvae ( Tamaru et a l . ,

1993a) is presented in Figure 5 . It depicts the various feed

organisms reported in the literature and when they should be

introduced to the grouper larvae as feed .

Step 1 . Inoculation of Phytoplankton :

Phytoplankton is

introduced into the larval-rearing tank on the second day

posthatching to achiev�

between 3 0 0 and 5 0 0 x 10

an - initial - cell density ranging

cells/ml . · Additional phytoplankton

should be added to the rearing tank daily as needed to

maintain the initial cell densities over the next 25 days of

the larval rearing trial .

Step 2 . Introduction of oyster Trochophores : Beginning on the

second day posthatching, oyster trochophores should be

introduced into the larval-rearing tank to attain a density of

20 individuals/ml . This density should be maintained until

day 1 0 posthatching . As seen in Figure 5 , SS-type rotifers ,

mussel- larvae, or sea urchin larvae can be used as substitutes

if available .

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19.3 Page 183

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I l 11 1· I I I I I I I 11 I I I I I I I I 1- I I l I ·1 I I I 11 I I I I I I I I 11 I I I I I I I I

Nannochlo� ops is oculata

111111111111111 II 11111 t1111111111111111111111 I11111111

MINCED FISH/ SHRIMP

ENRICHED AR'I'EMIA NAUPLI I /COPEPODS

S - TYPE ROTIFER

S S - TYPE ROTIFER

OYSTER TROCOPHORES /MUSSEL LARVAE/SEA URCHIN EGGS

r 1 , ,. 1 1 1 1 1 1 1 1 1 1 , 1 1 1 1 1 , 1 1- 1 1 1 1 ·1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 , 1 1

0

10

20

30

40

50

DAYS AFTER HATCHING

Figure s . Feeding schedule for groupers . From Tamaru et al . ,

1993a.

Step 3 .

Introduction of Rotifers :

On the second day

posthatching , s-type rotifers can be introduced into the

rearing tank . An initial density of 10-20 rotifers/ml is

recommended. Rotifers are added as needed on a daily basis to

maintain the initial stocking density .

Step 4 . Introduction of Artemia Nauplii : Between Day 10 and

12 posthatching enriched Artemia nauplii can be provided to

the larvae.

An initial density of 0 . 0 1 nauplii/ml is

recommended . At the end of each day, it should be determined

whether all of the nauplii have been consumed . This will

determine whether to increas� the next day ' s ration. If all

of the nauplii have been consumed , the number of nauplii

provided the following day should be doubled. This process is

repeated until a density of 2 or 3 nauplii /ml is attained .

This density of nauplii should remain constant until the

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larvae are harvested . It should be noted that the total daily

ration should be separated into at least three different

feedings .

step s . Introduction of Copepods: copepods , if available

should also be introduced into the rearing tank using the same

protocol . Because of the limited availability of copepods ,

the total harvested should be evenly divided amongst the

larval-rearing tanks .

Step 6 . Minced Fish/Shrimp : By Day 3 0 posthatching , larvae

should be large enough to consume minced fish and/or shrimp

flesh . It should to be chopped to a suitable size before

being fed to the larvae . Provide the grouper larvae this feed

ad libitum and spread the feedings over the course of the day .

Be sure they are not overfed so that unconsumed feed does not

accumulate on the bottom of the tank .

� Extensive Larval Rearing

The main difference between extensive and intensive larval

rearing systems is that no oyster trochophores ( or other small

feeds) are added to the extensive rearing tanks . The feeding

regimen employed basically fol lows the same time frame as the

intensive method except that the amounts of feed are not as high.

Large amounts of feed are impractical because of the s ize of the

rearing tank . Ad libitum feeding is practiced for all of the major

feed items depending on the amount of larvae and the number of

copepods growing in the tank .

water Exchange

The same time frame for water exchange can be followed for

both the intensive and extensive system . Again , the major

difference will be the amount . Initially , no water exchange should

occur during the first ten days posthatching. For the intens ive

system, a 2 0 % rate of exchange can be initiated on Day 11

posthatching . The amount of exchange is gradually doubled with

each successive day until a rate of 100% /day is attained . This

rate is then maintained for the duration of the tria l .

In the extensive system , it is again emphasized that the

productivity of the pond provides the major source of the feed for

the grouper larvae . An excessive rate of water exchange wil l

result i n flushing out the available feed . The operator will have

to continually use his judgement on a daily basis as to whether to

increase of decrease the rate of exchange . Beginning on Day 11

posthatching an initial 10% rate of exchange is recommended. Again

the rate is doubled on a daily basis until a rate 75% /day is

achieved. - From- this point on the 75% daily - exchange rate should be

maintained until the end of the tria l .

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Daily Routine

Daily monitoring of the larval-rearing systems is critical to

the success of the rearing tria l .

Materials Required :

1 . DO meter

2 . refractometer

3 • thermometer

4 . 1-ml rotifer counting pipette ·

5 . 10x magnifier

6 . data sheet

1 . s iphon hose ( airline tubing and 1 / 2 "

s . 5 1 glass tubing ( 1 / 8 11 and 1 / 2 " i . d . )

i.d. )

Step 1 . Water Quality: Temperature , DO , and salinity should

beexa.mmienaesdurfeodr

daily .

trends .

Record ('lata so .:that

it can

be easily

Step 2 . Rotifer Measurements: The rotifer dens ities in the

tank should be measured in the morning and afternoon by taking

five 1-ml aliquots at various locations in the tank .

Calculate the average rotifer density. These values dictate

whether addition of rotifers is warranted as well as whether

the rate of water exchange is excessive . Adjustments can be

made on a daily basis .

Step 3 . Tanlc Maintenance : Although not the most pleasant

activity during larval rearing but necessary none the less , is

the siphoning of the tank bottom to remove dead larvae ,

uneaten food and accumulated debris . The tank bottom should

NEVER be allowed to have an excessive amount of accumulated

debris . This activity may prove to be more difficult with the

outdoor tanks .

Trends in water quality measurements , feed levels , and

conditions of the tank bottom must be summarized daily to assess

the progress of the larvae. Based on the observations , adjustments

can be made to minimize mortalities .

.

-

.

Sorting

A high degree of cannibalism during the rearing o f grouper

larvae has been reported to take place after Day 2 5 posthatching

and mortalities can reach as high as 10%/day, especially with a

rearing trial that results in a wide size distribution o f larvae .

This usually occurs in rearing trials with high stocking densities .

For all practical purposes, sorting is restricted to intensive

rearing systems because of the size of tanks as well as the

densities encountered . Currently , the only means of reducing

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losses as a result of cannibalism is to sort the larvae by size .

This is an extremely labor intensive but necessary task which must

be repeated at regular intervals (every two or three days) during

the course of the rearing trial . The procedure for sorting is as

follows :

Materials Required :

1 . fine mesh barrier net

2 . 2 0-liter plastic buckets

3 . 5-1 plastic buckets

4 . aquarium dip nets

Step 1 . Reduce the Volume of the Rearing Tank: All rearing

tanks should have the facility to reduce its volume without

loss of the larvae . This is accomplished by s lowly opening

the main drainage system of the rearing tank and/or using ring

siphons .

Step 2 .

Concentrating the Larvae:

Using the fine mesh

barrier net, the larvae can be corralled into one area of the

rearing tank . The larger individuals will usually be able to

avoid the net and this is a means of separating the size

classes .

Step 3 . Removal of the Larvae : Determine which of the size

groups are easiest to .be removed from the . rearing tank .

Although fewer in number , the larger individuals are often

more difficult to catch . In all case s , the larvae are

susceptible to handling and precautions should be taken so

that the larvae are not suspended out of water . The smaller

individuals are removed by scooping with buckets and stocked

into a separate rearing tank . Repeat until the sorting is

completed . An alternative procedure is to purchase size

sorters . The entire larval community can be placed directly

within the sorters and the smaller individuals will swim

between the bars of the sorters leaving behind the larger

individual s . The larger individuals can then be removed and

placed into a separate tank .

Stress Test

The stress test was developed to assess the health of hatchery

produced larvae and j uveniles and is currently an accepted quality

control measure for both fish and crustaceans ( Dhert et al . , 19 9 2 ) •

. In the past , growth and survival were the only means of assessment .

However , applying stress at various stages of larval development

has become a major criteria used in evaluating the condition of the

larvae during the rearing trials and/or

are able· to withstand harvestlng . ·

dete-rmining

when

the

larvae

- The stressors that are employed can be characterized into two

categories : physical and physiologica l . Resistance to capture and

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being suspended in air for a predetermined duration of time are

examples of physical stressors and exposure to various sal inities

at predetermined t ime intervals is an example of a physiological

stressor . It has been demonstrated that stress resistance can be

used to detect differences in treatment effects where no

differences can be found by survival or growth (Tamaru et al . ,

1993b) . In addition, there appears to be a direct link between the

stress resistance exhibited by larvae and the nutritional value of

their feed ( Tackaert et a l . , 19 8 9 , Dhert et a l . , 1992 ; Tamaru et

al. t 1993b) •

The methodology for using physical stress as a means to

determin� the time of harvestlng . f�r stripe<! mullet (Tamaru et al . ,

1993b) is described below and can be adapted for use with grouper .

Materials Required:

1 . 2 0-liter buckets , two for each tank to be tested

2 . air stones and airl ine tubing

3 . aeration and seawater system

4 . fine mesh aquarium dip nets

5 . hand tally

step 1 . Application of Physical Stress : Fill the buckets with

seawater and aerate the water in each . Catch approximately

25-50 individuals from the rearing tank with the scoop net and

hold the larvae suspended in the air for 15 seconds . After

the time has elapsed place the fish into one of the buckets .

Repeat the exercise for the duplicate bucket . Keep the

collected fish in the buckets for one hour .

Step 2 . Recording Mortalities : After one hour has elapsed,

count the dead fish and record the number . count the number

of fish still alive and return them to their respective

rearing tanks .

Step 3 . Estimation of survival : Total -the number of dead and

surviving fish in each bucket. Divide the number of surviving

fish by the total number of fish collected and multiply by 100

to give a percent survival . A summary of the calculation

follows :

Bucket 1

(A)

(# of fish dead)

+ ( # of fish alive)

--------------------

Total # of fish

( B ) ( # of fish al ive + Total # of fish) x 100 = %

survival for Bucket 1

Repeat for Bucket 2 .

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Average the percent survival from both buckets and record the

data . When percent survival of the stress test is above 90% ,

the larvae are ready for harvest .

Harvesting

The major activity at the t ime of harvest is the

quantif ication of the surviving larvae . If the numbers are not

excessive this can be achieved by direct count . However , if the

numbers of surviving larvae are high , estimations need to be used .

Materials Required :

1 . 1 x 0 . 5 m fine mesh net stretched between two poles

2 . hand tally

3 . four 20-liter buckets marked with 1 liter increments

4 . airstones and airline tubing

5 . aquarium scoop nets

6 . seawater and aeration systems

7 . 100-ml plastic cups

8 . four 2 11 diameter ring siphons ( 5 0 0 µm)

Step 1 . Preparation for Harvest : Drain the rearing tank to 1 / 3

its volume a s described previously and concentrate the fish

us ing the large net .

Step 2 . Preparation of Holding Buckets : F i l l the 2 0 -l iter

buckets with 10 liters of seawater and aerate .

Step 3 . Preparation of Standard : Count out 1000 fry into one

2 0- liter bucket and mark it as the standard . Use the ring

siphons to remove excess water and bring the volume back to 10

liters . NOTE : THE DENSITY OF THE STANDARD BUCKET CAN BE

CHANGED AT THE MANAGER ' S DISCRETION . Place an airstone and

aerate .

Step 4 . Quantifying the Fry: Place another 2 0 - l iter bucket

next to the standard bucket and add fry until the density

appears to be the same . Repeat this sequence and record the

number of buckets removed from the tank . Multiply this number

by 1000 to obtain the total number of fish harvested .

PARASITES AND DISEASE

Any organism in captivity undergoes a certain degree of stress

.due to the artificial conditions ( e . g . , food, space , water quality)

under which it is forced to live . Accidental occurrences such as

power outages or malfunctioning of equipment can cause acute stress

( e . g . , low DO leve ls , high ammonia) . The organisms can die quickly

and often in large numbers unless promptly relieved of the adverse

conditions . A more subtle and often undetectable kind of stress

is low level chronic stress that often results from neglect and

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poor vigilance. This type of stress results in poor performance

( i . e . , growth , surviva l , reproduction) at all phases of the

operations (Bil lard et a l . , 198 1 ) . It must be emphasized that poor

performance alone is not the only outcome of chronic stress . such

conditions , if not remedied can lower the resistance of the

organism and sets the stage for infection by pathogens .

First Aid

The administration of first aid is not directly related to

disease , although it is an activity in which therapeutic treatments

are conducted . These procedures are the same as those used in the

treatment of infections and for that reason are introduced here .

First aid is usually required for larger individuals such as

broodstock and j uveniles .

Fish obtained from the wild or fish

that have undergone physical handl ing ( i . e . , gonadal biopsy ,

induction of spawning) often suffer from acute stress and bodily,

inj ury ( e . g . , loss of scales , abrasions and cuts ) . If left .

unattended the cuts and abrasions can become infected and

ultimately may lead to death. First aid treatment can reduce these

unnecessary losses .

Materials Required :

1 . top loading balance

2 . 500 to 1000-liter tank

3 . seawater , fresh water and aeration system

4 . 5-liter bucket or pitcher

5 . Prefuran (Argent Chemical Redmond Washington ,

USA)

Step 1 . Treatment Tank : A separate tank with a continuous

supply of seawater , fresh water , and aeration should be

designated specifically for treating fish . The tank and the

water systems should have the capacity to accommodate a rate

of water exchange of 300 to 4 0 0%/day.

step 2 . Reagent : Antibacterial treatment is recommended if

the wounds are large or appear to be infected .

The

recommended antibiotic is Prefuran which is a broad spectrum

antibacter ial effective against both gram positive/ negative

bacteria .

It is often used as an antiseptic and/or

disinfectant and is also reported to be active against

coccidia type infections . It is usually administered as a

bath ( 5 ppm) for one hour followed by flushing to remove the

chemical. The treatment is repeated for five day s . Fol low

the instructions that come with the reagent and calculate the

amount of Prefuran required to result in a final concentration

of the active ingredient of 5 ppm .

Step 3 . Treatment : First turn off the water supply to the

treatment tank . Dissolve the amount of Prefuran needed to

treat the volume of water in . the tank.. and distr ibute evenly

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into the treatment tank . Keep the aeration on and leave the

fish in the treatment for one hour . After the time has

elapsed turn the water on to flush the tank . Repeat steps 2

and 3 for five consecutive days . During this period of time

the fish should be fed ad libitum , never allowing excess feed

to accumulate in the tank .

Caligus sp . or sea Lice

Parasitic copepods of Caligus sp . were present on groupers

captured from the wild. They are relatively large and can be seen

with the naked eye when a broodstock individual is held in your

hand . They are usua lly crawling over the entire body but are most

easily seen on the head region . Low level infestations are quite

common on wild f ish and even on healthy brGodstock . However , the

populations of this parasite can explode into epidemic proportions

within two to three weeks and must be treated before they reach

this stage.

Materials Required :

1 . top loading balance

2 . 500 to 1000-liter tank

3 . seawater, fresh water and aeration systems

4 . 5-l iter bucket or pitcher

5 . Trichlorofon (Argent Chemical , Redmond Washington ,

USA)

Step 1 . Isolation : If the number of infected broodstock is

low they can be isolated into a treatment tank as described in

the section on first aid. If there are a large number of

infected individual s , the entire holding facility must be

treated .

step 2 . Reagent: Trichlorfon (also known as dipteryx or

masoten) has been found to be the most effective chemical

treatment for dealing with caligus sp . It is an organo

phosphate compound in whitish powder form ( 8 0-97% active

ingredient ) . It is also effective on other metazoan parasites

- ( e . g . , leeches , monogenetic trematodes , and other parasitic

protozoans ) .

It is extremely toxic to invertebrates

( especially shrimp , crabs and mollusks) and contact with the

skin and inhalation should be avoided . This reagent breaks

down rapidly when exposed to light , high temperature and high

pH . For best results , the treatments should be initiated

during the morning hours . NOTE : IT IS UNLAWFUL TO TREAT FISH

MEANT FOR HUMAN CONSUMPTION WITH THIS CHEMICAL.

Step 3 . Treatment : A one hour exposure to 0 . 5 ppm of

trichlorfon is the recommended treatment . Determine the

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amoµnt of reagent needed using the _ equation below. The

calculation for determining the amount of Trichlorfon ( 8 0%

active ingredient) needed for a 1000-liter treatment tank is :

Trichlorfon = ( 1 0 0 0 l ) x ( 0 . 0 0 1 g/ l ) x 0 . 5 ppm x ( 1 0 0 / 8 0 )

Based on the above calculation , 0 . 6 3 g of trichlorfon is

required. Weigh out the amount and dissolve in five liters of

water obtained from the treatment tank . Turn off the water

supply and pour the reagent into the treatment tank , making

sure that it is evenly distributed . Make sure that the

aeration is on and treat the fish for one hour . After one

hour , flush the tank . This treatment should be repeated as

needed depending upon the quantity of parasites rema ining.

The holding facility from which the fish came should also be

treated or cleaned and al lowed to sun dry . When large number

of broodstock are infected , the entire holding facility must

be treated .

LITERATURE CITED

Bardach , J . E . , J . H . , Ryther and w . o . McLarney . 1972 . Aquaculture:

the- farming and husbandry of freshwater and marine organisms .

Wiley Interscience , New York , 868 pp .

B i llard , R . , c . Bry and c . Billet . 19 8 1 . Stress , environment ,

and reproduction in teleost fish . In : A . P . Pickering ( ed . )

Stress and Fish , pp . 185-208 .

Dhert , P . P . Lavens , and P . Sorgeloos . 1992 . stress evaluation :

a tool for quality control of hatchery-produced shrimp and

fish fry . Aquaculture Europe , Vol . 17 ( 2 ) : 6-10 .

FitzGerald , William Jr . , M . Bauerlein and T . Behrenfeld . 19 9 1 .

Prel iminary spawning and culture of groupers in Palau and

Guam .

Final Report , Pacific Aquaculture Association .

- Honolulu, Hawaii USA, pp . 1-53 .

Fulks W . and K . L . Main. 19 9 1 . Rotifer and microalgae culture

systems . Proceedings of a u . s . ·-Asia Workshop . Honolulu,

Hawa i i , January 28-3 1 , 199 1 .

Published by The Oceanic

Institute , Honolulu , 364 pp .

Johannes , R . E . 198 1 . Words of the Lagoon : Fishing and Marine Lore

in the Palau District of Micronesia-. University of Cal ifornia

Press , Berkely CA. 2 4 5 pp .

Lubzens , E . 19 87 . Rais ing rotifers for use in aquaculture . Hydro

biologia, 147 : 245-255 .

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Lubzens , E . , A . Tandler and G . Minkoff . 19 8 9 . Rotifers as food

in aquaculture . Hydrobiologia. 186/18 7 : 387-4 0 0 .

Myers , R . F . 199 1 . Micronesian Reef Fishes . A practical guide to

the identification of the coral reef fishes of the tropical

central and Western Pacific. Coral Graphics , Barrigada ,

Territory of Guam . 298 pp .

Sorgeloo s , P . , P . Lavens , Ph . Leger , w . Tackert and D .

versichele . 198 6 . Manual for the cu�ture and use of brine

shrimp Artemia in aquaculture . Artemia Reference Center , State

University of Ghent , Belgium. 3 19 pp .

Sorgeloos , P . P . Lavens , Ph. Leger and w . Tackaert . 19 9 1 . The

use of Artemia in marine fish culture . Paper presented at :

Finfish in Asia 1 9 1 Conference , Tungkang , Taiwan . Dec . 17-19 ,

19 9 1 . 12 pp .

Sweetman , J . W . 1993 . Perspectives and critical success factors

in the present farming of fish . Paper presented at World

Aquaculture ' 9 3 , Torremolinos , Spain, May 2 6-28 , 19 9 3 .

European Aquaculture Society , Special Publication No . 19 .

Oostende , Belgium. pp . 288 .

Tackaert , w . , P . Abelin, Ph. Leger and P . Sorgeloos . 1989 .

Stress resistance as a criterium to evaluate quality of

postlarval shrimp reared under different feeding procedures :

In: Proceedings III S imposio Brasileiro sabre cultivo de

camarao . vol . 1 . MCR Aquacultura , Joao Pesoa , Bras i l . pp .

393-403 .

Tamaru , c . s . , c . -s . Lee and H . Ako . 199 1 .- Improving the larval

rearing of striped mullet , (Mugil cephalus) by manipulating

quantity and quality of the rotifer, Brachionus plicatilus.

In : Rotifer and microalgae culture systems . Proceedings of a

U . S . - Asia workshop . ( Fulks , w . and Main, K . Eds . ) . The

Oceanic Institute , Hawaii , USA, pp . 89-103 .

Tamaru, c . s . , F . Cholik, J . C . -M. Kuo and W. FitzGerald, Jr. 1993a.

status of the culture for striped mullet (Mugil cephalus)

milkfish ( Chanos chanos ) and Grouper (Epinephelus sp . ) . World

Aquaculture 1 93 , Torremolinos , Spain, May 2 6-28 . European

Aquaculture Society , Special Publication No . 19 . pp . 296-2 97 .

Tamaru , c . s . , w . J . FitzGerald, Jr . and v . s . Sato . 1993b . In :

Christine Carlstrom-Trick ( Ed . ) , Manual for the artificial

propagation of striped mullet ( Mugil cephalus) .

Guam

Aquaculture Development and Training Center . 165 pp . ( In

press ) .

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Watanabe , T . , c . Kitaj ima and s . Fuj ita . 19 8 3 . Nutritional

values of live food organisms used in Japan for mass

propagation of fish: A Review . Aquaculture , 3 4 : 115-143 .

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