NOAA Technical Report NMFS 47

Reproduction, Maturation,and Seed Productionof Cultured Species
Proceedings of the Twelfth
U.S.-Japan Meeting on Aquaculture,
Baton Rouge, Louisiana,
October 25-29, 1983
Carl J. Sindermann (Editor)

U.S. DEPARTMENT OF COMMERCE
National Oceanic and Atmospheric Administration
National Marine Fisheries Service

February 1987

NOAA TECHNICAL REPORT NMFS
The major responsibilities of the National Marine Fisheries Service (NMFS) are to monitor and assess the abundance and geographic distribution of fishery resources, tounderstand and predict fluctuations in the quantity and distribution of these resources, and to establish levels for their optimum use. NMFS is also charged with the development
and implementation of policies for managing national fishing grounds, development and enforcement of domestic fisheries regulations, surveillance of foreign fishing off UnitedStates coastal waters, and the development and enforcement of international fishery agreements and policies. NMFS also assists the fishing industry through marketing service
and economic analysis programs, and mortgage insurance and vessel construction subsidies. It collects, analyzes, and publishes statistics on various phases of the industry.
The NOAA Technical Report NMFS series was established in 1983 to replace two subcategories of the Technical Reports series: "Special Scientific Report-Fisheries" and"Circular." The series contains the following types of reports: Scientific investigations that document long-term continuing programs of NMFS; intensive scientific reports on
studies of restricted scope; papers on applied fishery problems; technical reports of general interest intended to aid conservation and management; reports that review in considerable detail and at a high technical level certain broad areas of research; and technical papers originating in economics studies and from management investigations. Since
this is a formal series. all submitted papers receive peer review and those accepted receive professional editing before publication.
Copies of NOAA Technical Reports NMFS are available free in limited numbers to governmental agencies, both Federal and State. They are also available in exchange forother scientific and technical publications in the marine sciences. Individual copies may be obtained from: U.S. Department of Commerce, National Technical Information Service.
5285 Port Royal Road, Springfield, VA 22161. Although the contents have not been copyrighted and may be reprinted entirely, reference to source is appreciated.
13. Guidelines for reducing porpoise mortality in tuna purse seining. by James M.
Coe, David B. Holts, and Richard W. Butler. September 1984, 16 p.

31. Shark catches from selected fisheries off the U.S. east coast. by Emory D. Anderson. John G. Casey, John J. Hoey, and W. N. Witzel I. July 1985.22 p.

14. Synopsis of biological data on shortnose sturgeon. Acipenser breviroslrum
LeSueur 1818, by Michael J. Dadswell, Bruce D. Taubert. Thomas S. Squiers, Donald
Marchette, and Jack Buckley. October 1984. 45 p.

32. Nutrient Distributions for Georges Bank and adjacent waters in 1979, by A. F.
J. Draxler, A. Matte, R. Waldhauer, and J. E. O·Reilly. July 1985, 34 p.

15. Chaetognatha of the Caribbean sea and adjacent areas, by Harding B. Michel.
October 1984, 33 p.
16. Proceedings of the Ninth and Tenth U.S.-Japan Meetings on Aquaculture, byCarll. Sindermann (editor). November 1984, 92 p.
17. Identification and estimation of size from the beaks of 18 species of cephalopods
from the Pacific Ocean, by Gary A. Wolff. November 1984. 50 p.
18. A temporal and spatial study of invertebrate communities associated with hardbottom habitats in the South Atlantic Bight, by E. L. Wenner, P. Hinde, D. M. Knott,
and R. F. Van Dolah. November 1984, 104 p.
19. Synopsis of biological data on spottail pinfish, Diplodus holbrooki (Pisces:
Sparidae), by George H. Darcy. January 1985, II p.
20. lchthyoplankton of the Continental Shelf near Kodiak Island, Alaska, by Arthur
W. Kendall, Jr., and Jean R. Dunn. January 1985, 89 p.
21. Annotated bibliography on hypoxia and its effects on marine life. with emphasison the Gulf of Mexico, by Maurice L. Renaud. February 1985, 9 p.
22. Congrid eels of the eastern Pacific and key to their Leptocephali, by Solomon
N. Raju. February 1985, 19 p.
23. Synopsis of biological data on the pinfish. Lagodon rhomboides (Pisces:Sparidae),by George H. Darcy. February 1985, 32 p.

33. Marine flora and fauna of the Northeastern United States. Echinodermata:Echinoidea, by D. Keith Serafy and F. Julian Fell. September 1985, 27 p.
34. Additions to a revision of the shark genus Carcharhinus: Synonymy of Aprionodan and Hypoprion, and description of a new species of Carcharhinus (Carcharhinidae),by J. A. F. Garrick. November 1985,26 p.
35. Synoptic review of the literature on the Southern oyster drill Thais haemasloma
floridana. by Philip A. Butler. November 1985,9 p.
36. An egg production method for estimating spawning biomass of pelagic fish:
Application to the northern anchovy, Engraulis mordax, by Reuben Lasker (editor).
December 1985. 99 p.
37. A histopathologic evaluation of gross lesions excised from commercially important North Atlantic marine fishes, by Robert A. Murchelano, Linda Despres-Patanjo,
and John Ziskowski. March 1986, 14 p.
38. Fishery atlas of the northwestern Hawaiian Islands, by Richard N. Uchida andJames H. Uchiyama (editors). September 1986. 142 p.
39. Survey of fish protective facilities at water withdrawal sites on the Snake andColumbia Rivers, by George A. Swan. Tommy G. Withrow, and Donn L. Park. April
1986, 34 p.

24. Temperature conditions in the cold pool 1977-81: A comparison between southern
New England and New York transects, by Steven K. Cook. February 1985,22 p.

40. Potential impact of ocean thermal energy conversion (OTEC) on fisheries, by
Edward P. Myers, Donald E. Hoss, Walter M. Matsumoto. David S. Peters, Michael
P. Seki, Richard N. Uchida, John D. Ditmars. and Robert A. Paddock. June 1986,
33 p.

25. Parasitology and pathology of marine organisms of the world ocean, by William
J. Hargis, Jr. (editor). March 1985, 135 p.

41. A stationary visual census technique for quantitatively assessing community structure of coral reef fishes, by James A. Bohnsack and Scott P. Bannerol. July 1986, 15 p.

26. Synopsis of biological data on the sand perch, Dipleclrum formosum (Pisces:
Serranidae), by George H. Darcy. March 1985,21 p.

42. Effects of temperature on the biology of the northern shrimp, Pandalus borealis,in the Gulf of Maine, by Spencer Apollonio, David K. Stevenson, and Earl E. Dunton,
Jr. September 1986. 22 p.

27. Proceedings of the Eleventh U.S.-Japan Meeting on Aquaculture, Salmon
Enhancement, Tokyo, Japan. October 19-20. 1982. by Carl J. Sindermann (editor).
March 1985. 102 P
28. Review of geographical stocks of tropical dolphins (Slenella spp. and Delphinusdelphis) in the eastern Pacific. by William F. Perrin, Michael D. Scott. G. Jay Walker.
and Virginia L. Casso March 1985, 28 p.

43. Environment and resources of seamounts in the North Pacific, by Richard N.
Uchida, Sigeiti Hayasi, and George W. Boehlert (editors). September 1986, 105 p.
44. Synopsis of biological data on the porgies, Calamus arclifrons and C. proridens(Pisces: Sparidae), by George H. Darcy. September 1986, 19 p.
45. Meristic variation in Sebastes (Scorpaenidae), with an analysis of character associa-

Prevalence, intensity, longevity, and persistence of Anisakis sp. larvae and LacisIOrhynchus lenuis metacestodes in San Francisco striped bass, by Mike Moser, Judy
A. Sakanari, Carol A. Reilly. and Jeannette Whipple. April 1985.4 p.

tion and bilateral pattern and their significance in species separation. by La-chai Chen
September 1986, 17 p.

30. Synopsis of biological data on the pink shrimp, Pandalus borealis Kr6yer, 1838,
by Sandra E. Shumway. Herbert C. Perkins, Daniel F. Schick, and Alden P. Stickney.
May 1985. 57 p.

46. Distribution and relative abundance of pelagic nonsalmonid nekton off Oreg,'nand Washington 1979-84, by Richard D. Brodeur and William G. Pearcy. December
1986. 85 p.

19.

NOAA Technical Report NMFS 47

Reproduction, Maturation,and Seed Productionof Cultured Species
Proceedings of the Twelfth
U.S.-Japan Meeting on Aquaculture,
Baton Rouge, Louisiana,
October 25-29, 1983

Carl J. Sindermann (Editor)
Panel Chairmen:
Conrad Mahnken, United States
Takeshi Nose, Japan
Under the U.S.-Japan Cooperative Programin Natural Resources (UJNR)
February 1987

u.s. DEPARTMENT OF COMMERCE
Malcolm Baldrige, Secretary

National Oceanic and Atmospheric Administration
Anthony J. Calio, Administrator

National Marine Fisheries Service
William E. Evans, Assistant Administrator for Fisheries

PREFACE
The United States and Japanese counterpart panels on aquaculture were formed in 1969 under the United StatesJapan Cooperative Program in Natural Resources (UJNR). The panels currently include specialists drawn from thefederal departments most concerned with aquaculture. Charged with exploring and developing bilateral cooperation, the panels have focused their efforts on exchanging information related to aquaculture which could be of benefit
to both countries.
The UJNR was begun during the Third Cabinet-Level Meeting of the Joint United States-Japan Committee on
Trade and Economic Affairs in January 1964. In addition to aquaculture, current subjects in the program include
desalination of seawater, toxic microorganisms, air poUution, energy, forage crops, national park management, mycoplasmosis, wind and seismic effects, protein resources, forestry, and several joint panels and committees in marine
resources research, development, and utilization.
Accomplishments include: Increased communication and cooperation among technical specialists; exchanges of
information, data, and research findings; annual meetings of the panels, a policy-coordinative body; administrative
staff meetings; exchanges of equipment, materials, and samples; several major technical conferences; and beneficial
effects on international relations.

Conrad Mahnken - United States
Takeshi Nose - JapanThe National Marine Fisheries Service (NMFS) does not approve, recommend
or endorse any proprietary product or proprietary material mentioned in thispublication. No reference shall be made to NMFS, or to this publication furnished by NMFS, in any advertising or sales promotion which would indicate
or imply that NMFS approves, recommends or endorses any proprietaryproduct or proprietary material mentioned herein, or which has as its purpose
an intent to cause directly or indirectly the advertised product to be used orpurchased because of this NMFS publication.

11

CONTENTS
Fukuhara, Osamu
Recent trends in mariculture and seed productionof fish in southern Japan 1
Yano, Isao
Maturation of Kuruma prawns Penaeus japonicus
cultured in earthern ponds 3
Sanbonsuga, Yoshiaki
Cultivation and seedling production of brown algae in Japan 9
Fukuhara, Osamu
Seed production of red sea bream Pagrus major (Sparidae) in Japan 13
Melancon, Earl, Jr., Roanne Trapani, Greg Scott, and Len Bahr
Culture of the oyster Crassostrea virginica (Gmelin) from seed to market
on bedding reefs in Baritaria Bay, Louisiana, U.S.A. 21
Chatry, Mark
Seed oyster production in Louisiana and prospects for enhancement 27
Lawrence, Addison L., and Jay V. Huner
Penaeid shrimp culture in the United States: A brief overview stressing species,
seed production, and growout 31
Cange, Stephen W., David L. Pavel, Mark S. Lamon, and James W. Avault, Jr.
Development of larval rearing systems for the Malaysian prawn
Macrobrachium rosenbergii in southern Louisiana 43
Rhodes, Robert J., and James W. Avault, Jr.
Changing sexual-state relationships of a population of red swamp crawfish
Procambarus clarkii 51
Rhodes, Robert J., and James W. Avault, Jr.
A note on recruitment cycles for the red swamp crawfish
Procambarus clarkii 53
Wolters, William R.
Production of triploid channel catfish fry 55
Culley, Dudley D., Kirk A. Easley, Lynn Denson, Barbara A. Carr, and M. Arif Othman
An investigation of potential ovulatory stimuli for the bullfrog
Rana catesbeiana 59
Lawson, Thomas B.
Treatment of fish hatchery water for solids and turbidity removal 67iii

Recent Trends in
Mariculture and Seed
Production of Fish in
Southern JapanOSAMU FUKUHARA
Nansei Regional Fisheries Research Laboratory
Ohno-cho, Saeki-gun, Hiroshima 739-04, Japan

150
Yellow tail
Serlola qulnqueradlata
Seriola aureovlttata
Serlola purpurascens

c:
o

o
.;:
Il)

E

.

"0

c:

100

I I)

::>

o

;;
c:
c:
o

U
::>

"0

Sea Bream
Pagrus major
Acanthopagrus schlegell
Evynnls Japonlca

50

o

D:.

oL~=::::t:~c=::L::.:...i..._L---l--L__L-....I
71

72

73

74

75

76

77

78

79

80

81

Year

Figure I-Annual production of three species each of yellowtail and red sea bream
in mariculture, 1971-81. (Source: Statistics and Information Department, [Japanl
Ministry of Agriculture, Forestry and Fisheries)

Marine aquaculture recently yielded more than 8 % of the total
fisheries production in Japan, ranging from 8.3 to 8.9% between
1977 and 1981. Yellowtail and sea bream are the major species
in marine cage culture of fin-fish. Annual yield of yellowtail varied
from 1.1 to 1.5 % of the total fisheries production during the period
1977-81. However, yellowtail production from culture declined
gradually for the past two years. This trend is a result of a shift
in market preference for culture species, changing from yellowtail
to other species, especially sparid fish. This trend is mainly due
to the excessive production of yellowtail (Fig. 1), beyond the market
demand.
More than 80% of yellowtail and sea bream are cultured in thesouthern areas of Japan. This paper describes briefly the recent
trends in mariculture in southern Japan. Southern Japan is a prosperous area for the development of coastal aquaculture, with 18
prefectures facing the southwest waters of the Pacific Ocean (westfrom Wakayarna) , Seto Inland Sea, and East China Sea, where warm
water masses of the Kuroshio and the Tsushirna currents influence
the coastal water temperatures (except for the central part of the
Seto Inland Sea). The total production figures for the two species
are compared in Table 1 with those for the southern areas of Japan.
The total production of yellowtail increased continuously until 1979,
and tended to decrease slightly thereafter. Production in southern
Japan, however, increased steadily as did sea bream production,
in total and in southern Japan.
Mass production of red sea bream (Pagrus major) started about
a decade ago, which was the time of the beginning of farming
fisheries. After establishing the rearing techniques for red sea bream,
attempts were made to adapt the methodology to culture of othercommercially important marine species such as black sea bream
and Japanese parrot fish. Thereafter, marine fish rearing from eggs
for stocking purposes prevailed among fisheries farmers, mainly
those involved in net cage farming.
Presently about 20 species are reared in farming centers and in
private hatcheries, mainly for net cage culture and/or planting offish in coastal waters within the boundaries of the 18 prefecturesof southern Japan. The farming centers concentrated their research
efforts on improving the releasing techniques. Recently, plantingof widely migrating fishes such as red sea bream has been carriedon by the farming center of the central government, Japan Farming Fisheries Association. On the other hand, the farming centersof the prefectural governments, including the Fisheries Cooperative
Associations, direct their efforts primarily towards the stocking ofspecies that stay close to inshore areas of the prefectural coastal
zone, so that they may be harvested by local fisherman in domestic
waters.
The diversity of species produced through artificial propagation
results from two causes: One is marketability of the product andthe farmer's interest; the other is determined by the effective running of the rearing facilities. Basically the food habits of the Japanesepeople call for a variety of products, and preferences are not focusedon one particular fish. This phenomenon favors diversification assoon as production techniques become available. Seed production
is concentrated during spring and summer, and is rarely carriedout during the winter season (Table 2). During winter, seed production of ayu, Japanese flouder, and mud dab is continued mainly at the farming centers and private hatcheries.
All fry of ayu are planted in rivers every year, and Japaneseflounder are preferred for net cage culture among farmers due tofast growth and marketability.

Table I-Total annual production for 1977-81, and its percentage in southern Japan, of yellowtail and sea bream culture.
Yellowtail

Sea bream

Southern Japan

Year

Total
(m!)

1977
1978
1979
1980
1981

115,098
121,956
155,053
149,449
150,907

Southern Japan

(%)

Total
(ml)

77.7
80.7
83.1
84.9
88.0

8,245
11,315
12,492
14,973
18,243

76.9
80.0
80.2
81.7
82.6

(%)

Table 2-Species, period, and number of prefectures in which fish are reared for seed production of commercially important species.
No. ofpref.

Species
Red sea bream, Pagrus major
Black sea bream, Acanrhopagrus schlegeli
Japanese parrot fish, Oplegnalhus fascia/us
Ayu fish, Plecoglossus allivelis
Japanese flounder, Paralichlhys olivaceus
Mud dab, Umanda yokohamae
Puffer, Takifugu rubripes
Red grouper, Epinephelus akaara
Rockfish, Sebas/es sehlegeli
Marbled rockfish, Sebasliseus marmoralus
Japanese sea bass, Laleolabrax japonieus
Rabbit fish, Siganus fuseeseens
Yellowfin bream, Aeanlhopagrus la/us
Yellowtail, Seriola aureovillata
Yellowtail, Seriola quinqueradiata
Lumpfish, lnimieus japonieus
Japanese whiting, Sillago japonieus
Chicken grunt, Parapristipoma trilineatum
Green snapper, Lelhrinus nebulosus

Jan

Feb

Mar

Apr

15
10
7
7
13

Oct

Nov

Dec

*--_._---* -- *

5* -- *

2
2
I
I
I

--*

*-*- ** -- ** -- ** -- ** -- *

2
II
1*------

In southern Japan, almost 20 million young red sea bream arereared for releasing and for cage culture (Table 3). Red sea bream
culture seems to be gradually replacing yellowtail culture in cages,
at least during the past several years. This trend is closely related
to the successful artificial rearing of the red sea bream to stocking
size. Yellowtail culture still depends on catching wild young fish
for stocking.
Seed production is already progressing towards commercialization for black sea bream, Japanese parrot fish, and Japanese flounderamong fisheries farmers. Other species are still cultivated on an
experimental scale. It is hoped that rearing techniques will develop
which will result in improved survival of the juvenile stages. Successful rearing of yellowtail fry from eggs, which were spawned
spontaneously, provides some hope that the supply of seed fish fornet cage culture might be possible in the future, and would decrease
the fishing pressure on wild stocks from which young fish arepresently caught.

Size ranges (em)
4-6

6-8

8-10

Total

Red sea breamBlack sea bream
Japanese parrot fish
Ayu fishJapanese flounder
Mud dabPufferRed grouper
RockfishMarbled rockfish
Japanese sea bass
Rabbit fish
Yellow fin bream
Yellowtail
Yellowtail
LumpfishJapanese whiting
Chicken grunt
Green snapper

330
490
10

849
39
64

329

9
6
10

10
105

427
5
168

413
47
4
452
164
25

510

1,930
582
98
452
606
110
1,631

5

Sep

10
4

2-4

10

Aug

* - - - - - - - _...._ - - - - - - *

1-2

8
2

Jul

5

Species

4
10
12

JUIl

-----_._*

Table 3-Number of young fISh (x 104) by size produced in 18
prefectures of southern Japan, 1982.

2

May

*--

10
5

225

14
12
12
10
I
I
5

REFERENCES

Blanks indicate <10,000 fish. Refer to Table 2 for latin names offishes.

_STATISTICS AND INFORMATION DEPARTMENT, [JAPANI MINISTRY OF
AGRICULTURE, FORESTRY AND FISHERIES.
1983. Fisheries statistics of Japan, 1981.
1983. Annual report of statistics in fisheries and aquaculture, 1981.
[JAPANI MINISTRY OF AGRJCULTURE, FORESTRY AND F1SHERJES.
1<)83. Annual report on Japan's fisheries, fiscal year 1982, a summary.

2

INTRODUCTION

Maturation of
Kuruma Prawns
Penaeus japonicus
Cultured in Earthen Ponds_

In decapod crustaceans, control of the reproduction of animals ofcommercial interest is a major problem for programming theirculture. Reproductive maturation in captivity could provide a reliable
year-round supply ofjuveniles, serve in developing selective breeding programs, and be of general usefulness for obtaining diseasefree spawners. Several species of penaeid shrimp have been matured
and spawned in captivity using unilateral eyestalk ablation or temperature, photoperiod and diet controls (Johnson and Fielding 1956;
Idyll 1971; Caillouet 1973; Arnstein and Beard 1975; Duronslet
et al. 1975; Laubier-Bonichon and Laubier 1976; Santiago 1977;
Halder 1978; Laubier-Bonichon 1978; Primavera 1978; Brown etal. 1979; Lawrence et al. 1979; Lumare 1979; Kelemec and Smith
1980; Chamberlain and Lawrence 1981). Successful maturation of
Penaeus setiferus and P. merguinesis in ponds has been reported
by Conte et al. (1977) and Lichatowich et al. (1978), respectively.
In Japan, P. japonicus is cultured mainly in earthen ponds using
postlarvae which have been reared in indoor tanks. Since all prawns
are harvested within a I-year period, the maturation of P. japonicus
in ponds has not yet been established. The purpose of this research
was to determine whether P. japonicus could mature under pond
culture conditions.

ISAO YANONational Research Institute of Aquaculture, Fisheries Agency,
Nakatsuhama, Nansei-cho, Mie, Japan

MATERIALS AND METHODS_

A sample of 1,774 (795 females, 979 males) 5-month-old hatcheryreared Kuruma prawns (Penaeus japonicus) was obtained from the
Hakatajima Prawn Farm. The wet weight of the specimens was
13-22 g in September 1980. No ovarian development was observedin female prawns at this time.
In September 1980 all prawns were released into a 0.7 ha pond
located on the grounds of the Momoshima Center of the Japanese
Sea Farming Association (Hiroshima Prefecture) (Fig. I). Mean

Fukuyama

a

~

1?km

Figure I-Location of the experimental pond, Momoshima, Hiroshima Prefecture, Japan.

3

water depth in the pond was 1.5 m (0.5-2.0 m). A water gate installed in the pond was used to enhance tidal water exchange.
Predatory fish were kept out by means of a screened fence. Pelleted
diets were fed once daily at sunset.
Ovarian development was examined monthly from September
1980 to 1981 on females sampled randomly from the pond. Data
were collected on total body weight, body length, gonad weight,
and signs of mating (stopper in thelycum). Oonadosomatic indices(OSI) were determined from these data. For histological observation, small pieces from the middle area of the ovary were removed
and fixed in Bouin's solution. The fixed ovary pieces were dehydrated in ethanol, embedded in a paraffin-celloidin mixture, sectioned (61ffil), and stained in Delafield hematoxylin plus eosin. Fifty
to sixty oocytes per ovary were measured for diameter. The development of oocytes was classified into eleven stages according to morphological characters, namely 1) synapsis stage; 2) chromatin
nucleolus stage; 3) early perinucleolus stage; 4) late perinucleolus
stage; 5) oil globule stage I; 6) oil globule stage II; 7) yolkless stage;8) primary yolk granule stage; 9) secondary yolk granule stage;10) pre-maturation stage; and 11) maturation stage. Degree ofovarian maturation was recorded for each sample.
Spawning and hatching were carried out in 0.5 ton fiber reinforced plastic tanks filled with filtered sea water (50 lim). Water
temperature was maintained at 3-6°C above that of the pond in order
to induce spawning. Eighteen ripe females were selected and eachplaced in individual spawning tanks. Spawning and hatching rates
were determined by means of egg and nauplii density counts.

20

•

90

80

• • • • • • •

0;70

~60

•

II>

~50
>

]40

•

• • 1819
0

0

0
0

..r:;

0

15

II>

0

13~

0 30

20

g,

14~

0

0

al

E
16..':.

0

0

17_

0
0

•

12

•

11

0

10

10

S

0

N

0

J

M

F

A

M

J

J

A

S

Figure 2-Body weight and body length of P. japonicus cultured in an earthen
pond, September 1980-1981.

80
70

; 60

a:

RESULTS

.,,50

_

l:

!O40
:E
30

Growth of prawns

20
10

Rapid growth was observed between May and September whilewater temperatures remained above 18°C. Mean weight of female
prawns was 36.6 g during October 1980, with a final mean weightof 79.0 g recorded in September 1981. Body length increased from
113.9 to 191.0 mm between September 1980 and 1981 (Fig. 2).
Mating rate was 2.8 % in September, increasing to 100% in
December 1980 (Fig. 3). A high percentage (88-100%) of matedfemales continued to be observed through September 1981 with the
exception of May (20%).

o

SONDJ

FMAMJJAS

Figure 3-Mating rate of P. japonicus cultured in an earthen pond, September
1980-1981.

11

10
9

Changes in gonodosomatic index
8

Oonadosomatic index (OSI) peaks occurred in late spring-early
autumn 1981. The OSI of females increased from 0.4% to 1.5%
between September 1980 and April 1981. Some females reacheda 10% OSI between May and September 1981 (Fig. 4).

~7

(ii6
Cl

5
4

Development of oocytes

3

2

In September 1980 the ovaries contained immature or immaturedeveloping oocytes such as synapsis stage, chromatin nucleolus
stage, early perinucleolus stage, and late perinucleolus stage.
Oogonia were found throughout the experiment. Oil globule stage
I or oil globule stage II oocytes, which are the early developing
stages, were observed in early April 1981. In mid to late April andearly May 1981, yolkless stage, primary yolk granule stage, and

SON

0

J

F

M A M

J

J

A

S

Figure 4-Gonadosomic index (GSI) of P. japonicus cultured in an earthen pond,
September 1980-1981.

4

Seasonality of reproductive maturation

secondary yolk granule stage oocytes were observed in the developing ovaries. From May to September some oocytes reached theprematuration and maturation stages. The ripe eggs at the maturation stage have many club-shaped cortical rods in the peripheral
cytoplasm and contain extensive accumulations of yolk granules
dispersed throughout the cytoplasm (Fig. 5). The cytoplasmic membrane and the cortical rods of the oocytes were found to be separated
from the external media by a thin egg membrane (0.1 JAm thick)that surrounds the egg. Meiotic reduction division of the oocytes
was observed to reach only the metaphase of the primary maturation division. Therefore, polar bodies were not observed in theovarian oocytes.

Four stages of reproductive maturation were determined as follows:Stage I: immature-The ovaries are extremely thin, translucent,and white. Synapsis, chromatin nucleolus and early perinucleolus
stage oocytes are found. Multiplication of oocytes is characteristicof this stage.
Stage II: immature-developing-Ovaries are slightly thicker thanthe previous stage. Fresh ovaries are translucent, light orange
colored and smoothly textured. Late perinucleolus stage oocytes
appear. This stage is characterized by the appearance of follicular
cells on the surface of the oocytes.
Stage III: developing-The ovaries are turgid opaque and lightgreen. Oil globule stage I and II, yolkless, primary and secondary
yolk granule stages are observed. This stage is characterized by
the synthesis of vitellogenin and accumulation of yolk (Yano,
unpubl.).
Stage IV: ripe-The ovaries are very turgid, broad, opaque anddark green. The outline of the ovary is distinct and the appearance
is granular. This stage is characterized by the migration of thenucleus, ovulation and maturation division of the oocytes.
Stages I and II were observed in all samples in autumn and winter1980. A small percentage of stage III females was first observedin early April 1981. Stage IV females first appeared in mid May
and continued through September 1981 (Fig. 6). Spawning wassuspected in the pond since stage II females were sampled in August
1981. However, no larvae were found in the pond after plankton
tows.

Figure 5-Ripe eggs (maturation stage oocytes) of P. japonicus showing theappearance of many club-shaped cortical rods in the peripheral cytoplasm.
H & E stain. X 200.

~~

'c 30

Temperature•
20

Variation of oocyte size

10

There was a rapid increase in the sizes of the oocytes from the oil
globule stage I to maturation stage, but immature oocytes (synapsis stage, chromatin nucleolus stage, early perinucleolus stage, andlate perinucleolus stage) exhibited only a gradual size increase (Table1). Ripe eggs at the maturation stage measured 250 JAm in diameteron the average.

• •• • • • •

• •

• • •

0

Photoperiod

14

~12 •

"f

•

10

• • •

• •

• •

• • •

A

J

•

s

100

%90
Table I-Changes in the sizes of Kuruma prawn
oocytes in different stages of development.
Developmental stage

Oocytediameter <!,m)Synapsis stage
Chromatin nucleolus stage
Early perinucleolus stage
Late perinucleolus stage
Oil globule stage 1
Oil globule stage II
YolkJess stage
Primary yolk granule stage
Secondary yolk granule stage
Prematuration stage
Maturation stage

10
16-34
30-80
68-88
80-16088-144110-146128-160
136-237200-264224-288

SON
E:3Stage I

0JQ]Stageli

F

M

M

rl!lJStagel

J

A

S

.StagelVFigure 6-Interaction of temperature, photoperiod, and reproductive maturation in P. japonicus cultured in an earthen pond, September IfJ80..1981.

5

Spawning and hatching rates

Table 2-Spawning record of Kuruma prawn (Penaeus japonicus) cultured
in an earthen pond in 1981.

Seven females of the group of eighteen spawned, resulting in aspawning rate of39%. Hatching rates of two females were 78.9%(380,000 eggs) and 81.6% (500,000 eggs). The hatching rates forthe other five females ranged between 0 and 80.9% (Table 2).

DISCUSSION_

It is clear from the results that Kuruma prawns can be successfully
matured under pond culture conditions. Increased ovarian development (spring 1981) and rapid maturation (summer and autumn 1981)
were observed as water temperatures increased above 18°C. Thechange from 12 to 13 hours of daylight in April was also observedto accelerate the ovarian maturation rate. These facts indicate that
reproductive maturation is triggered by rising water temperatureand increased photoperiod in the spring. Undeveloped ovaries inautumn 1980 (compared to the rapid somatic growth of this period)
suggest that sexual maturation may not come about by falling watertemperature and reduced photoperiod. It is thought, therefore, thatwinter temperature inhibits somatic growth and ovarian development, either directly or indirectly by limiting food intake andavailability. Optimal maturation temperature for Kuruma prawns
is about 25°C. This is indicated by the fact that at 32°C (August)and 18°C (May) rapid ovarian maturation was not observed.
The role of the male prawns in the pond is essential since female
prawns are stimulated to molt during periods of high water temperature. Male spermatophores are shed during molting, thus new
mating for fertilization is required.
Yatsuyanagi and Maekawa (1955) observed the ovarian maturation of wild Kuruma prawns sampled from the Inland Sea near thearea of this experiment. Results indicated that Kuruma prawns
cultured in ponds matured earlier (April) than those growing at sea.
Minimum size at maturity in the pond was also slightly larger (159mm in body length) than that of wild females (135-140 mm in bodylength) (Yatsuyanagi and Maekawa 1955; Kajiyama 1933).
Nothing has been known of the correlation between mating andovarian maturation in P. japonicus. Results suggest that mating does
not directly accelerate ovarian maturation of Kuruma prawns, sinceovarian maturation was observed in spring after mating had occurredin autumn of the previous year.
The period of time required for maturation following the appearance ofthe oil globule stage I oocytes may be as short as 2 months.
Oocytes at this stage may be used as a maturation indicator, since
vitellogenin (egg yolk precursor) can be first observed in the blood
serum at this time (Yano, unpubl.). Oocyte diameter (>100 IJm)
can also be used as an indicator of the first stages of vitellogenesis.
The potential of Kuruma prawns for multiple spawnings during asingle spawning season can be demonstrated by the presence ofoogonia in the ovary at all times.
Kuruma prawns were not observed to spawn in the pond. However, when ripe females were thermally stimulated under laboratory conditions, spawning was achieved. This indicates that thermal
stimulation is necessary to induce spawning.
The spawning (39%) and hatching (80%) rates observed during
the experiment compare favorably with those of wild females(Yamaguchi Prefecture 1961). Hatchery techniques may be improved upon in order to achieve better results. With successful
reproductive maturation and spawning of Kuruma prawns under
pond culture conditions, new perspectives for the aquaculture industry will surely arise.

Spawnerno.

Body length(em)Total no.
eggs

Total no.
nauplii

Hatching rate

Date

I
2
3
4
5
6
7

30 MayII June25 June26 June15 July
25 July
25 July

18.2
18.6
16.6
17.3
16.7
18.7
18.5

130.000
68,000380,000
410,000
405,000
480,000
500,000

11.00055,000300,000
60,000072,000408,000

8.5
80.9
78.9
14.6
0.0
15.0
8\.6

ACKNOWLEDGMENT(%)_

This work was supported in part by a research grant from theJapanese Sea Farming Association.

CITATIONS_ARNSTEIN, D. R., and T. W. BEARD.
1975. Induced maturation of the prawn Penaeus orienta/is Kishinouye in thelaboratory by means of eyestalk remova1. Aquaculture 5:411-412.
BROWN, A., 1. MCVEY, B. S. MIDDLEDITCH, and A. L. LAWRENCE.
1979. Maturation of white shrimp (Penaeus setiferus) in captivity. Proc. World
Maricult. Soc. 10:435-444.
CAILLOUET, C. W.
1973. Ovarian maturation by eyestalk ablation in pink shrimp Penaeus duorarwn
Burkenroad. Proc. World Maricult. Soc. 3:205-225.
CHAMBERLAIN, G. w., and A. L. LAWRENCE.
198\. Maturation, reproduction, and growth of Penaeus vannamei and P.
styLirostris fed natural diets. Proc. World Maricult. Soc. 12:209-224.
CONTE, F. S., M. S. DURONSLET, W. H. CLARK, and 1. C. PARKER.
1977. Maturation of Penaeus styLirostris (Stimpson) and P. setiferus (Linn.) in
hypersaline water near Corpus Christi, Texas. Proc. World Marieult. Soc.
8:327-334.
DURONSLET, M. 1., A. I. YUDIN, R. S. WHEELER, and W. H. CLARK.
1975. Light and fine structural studies of natural and artificially induced egg
growth of penaeid shrimp. Proc. World Maricult. Soc. 6: 105-122.
HALDER, D. D.
1978. Induced maturation and breeding of Penaeus monodon Fabricius underbrackish water pond conditions by eyestalk ablation. Aquaculture 15: 171-174.
IDYLL, C. P.
1971. Induced maturation of ovaries and ova in pink shrimp. Commer. Fish.
Rev. 33:20.
JOHNSON, M. C., and 1. R. FIELDING.
1956. Propagation of the white shrimp, Penaeus setiferus (Linn.), in captivity.
Tulane Stud. Zoo!. 4:175-190.
KAJIYAMA, E.
1933. Spawning and growth of P. japonicus. Annu. Rep. Hiroshima Prefect.
Fish. Exp. Sin. 12: 134-159.
KE\.EMEC, 1. A., and I. R. SMITH.
1980. Induced ovarian development and spawning of Petuleus plebejus in a recirculating laboratory tank after unilateral eyestalk enucleation. Aquaculture
21 :55-62.
LAUBIER-BONICHON, A., and L. LAUBIER.
1976. Reproduction contrOlee chez la crevette Penaeus japonicus. FAO Tech.
Conf. Aquacult., Kyoto, Japan, 26 May-2 June 1976.
LAUBIER-BONICHON, A.
1978. Ecophysiologie de la reproduction chez la crevette Petuleus japonicus.
Troris annees d'experience en milieu controle. Oceano!. Acta. 1:135-15').
LAWRENCE, A. L., D. WARD, S. MISSLER, A. BROWN, 1. MCVEY, and B. S.
MIDDLEDITCH.
1979. Organ indices and biochemical levels of ova from penaeid shrimp maintained in captivity versus those captured in the wild. Proc. World Maricult.
Soc. 10:453-463.
LICHATOWICH, T., T. SMALLEY, and F. D. MATE.
1978. The natural reproduction of Penaeus merguiensis (DE MAN, 1888) inan earthen pclld in Fiji. Aquaculture 15.377-378.

6

LUMARE, F.
1979. Reproduction of PeTUleus kerathurus using eyestalk ablation. Aquaculture
18:203-214.
PRIMAVERA, 1. H.
1978. Induced maturation and spawning in five-month-old PeTUleus monodon
Fabricius by eyestald ablation. Aquaculture 13:355-359.
SANTIAGO, A. C.
1977. Successful spawning of cultured PeTUleus monodon Fabricius after eyestalkablation. Aquaculture 11: 185-196.
YAMAGUCHI PREFECTURE.
1961. Seed production of PeTUleusjaponicus. Annu. Rep. Yamaguchi Prefect.
Inland Sea Fish. Exp. Sin. 11 :354-385.
YATSUYANAGI, K., and K. MAEKAWA.
1955. The ecology of PeTUleusjaponicus in Inland Sea. Annu. Rep. Yamaguchi
Prefect. Inland Sea Fish. Exp. Stn. 7: 1-15.

7

INTRODUCTION

Cultivation and Seedling
Production of Brown Algae
in Japan_

The Japanese archipelago extends over a long distance from northto south, and is influenced by both the warm Kuroshio Current andthe cold Oyashio Current. For these geographic and oceanographic
reasons, as many as 860 species of seaweeds grow in Japanesewaters (Okamura 1932), many having been harvested and used as
human food and fertilizer since ancient times (Miyashita 1974) andas industrial materials, providing agar, alginic acid, potassium andiodine (0 kazaki 1971).
Japanese fisheries statistics for 1980 report that about 183,000
tons (fresh weight) of wild seaweed were harvested, mainly forhuman food. Total value of the crop was estimated at 41 billion
yen ($170 million). Brown algae, mainly Laminaria and Undaria,
account for approximately 78 % of the total harvest. Cultivated algae
were not taken into account in these figures. Many fishermen havelong obtained their livelihood from the harvest of wild seaweeds
in Japan.
Large brown algae are also economically important because they
serve as food for useful shellfish such as the abalone and sea urchin,and they also provide spawning grounds and shelter for many
juvenile fish. Therefore, brown algae are both directly and indirectly
important for fishermen. For this reason, fishermen have attempted
to maintain and increase the brown algae resources, with the support of the Japanese government.

YOSHIAKI SANBONSUGA
Hokkaido Regional Fisheries Research Laboratory
Fisheries Agency, Kushiro,
Hokkaido 085, Japan

ABSTRACTThis report briefly outlines the current brown algal cultivation and seedling production method used in Japan. Since the 19th Century, many cultivation efforts
such as placing stones in the sea and weeding out competing seaweeds have been
made to maintain and increase the brown algal resources, with the support of
the National Government. Another method used for the cultivation of brown algae
is called "suspended cultivation" or "raft cultivation". Raft cultivation of Undaria started in the early 1960's, and is the main method practiced at present.
The amount of Laminaria production through raft cultivation has also increased
markedly during the past ten years. The value of this crop is about 30% of thatof the wild harvest at present. In 1980, the total amount of seedling strings produced was 2,192 and 883 km for Undaria and Laminaria, respectively.

CULTIVATION OF BROWN ALGAE_

Laminaria
Among the brown algae, Laminaria is the favorite edible seaweed
in Japan. Much like the red alga Porphyra, it is used both as foodand for seasoning. Laminaria is called "Kombu" in Japanese, theword meaning "delight". It is served for happy events such as
betrothals, and used as a New Year's decoration. Laminaria is indispensable to the Japanese people. Its annual yield amounts to about
125,000 tons in fresh weight and, as indicated above, has a value
in excess of 30 billion yen ($100 million). The commercially important species of Laminaria are L. japonica Areschoug, L. japonica
var. ochotensis Okamura, L. diabolica Miyabe, L. angustata
Kjellman, and L. angustata var. longissima Okamura.
All of the species and varieties are harvested from Hokkaido andnorthern Honshu, where many fishermen depend on the harvest
for their livelihood. Thus efforts are made, such as placing stonesin the sea, to provide new substrates for the settlement of Laminaria
spores. Efforts are also made to remove competing seaweeds. This
work is supported with financial aid from the National Government.
Another method used for the artificial cultivation of Laminaria
in Japan is called "suspended cultivation" or "long-line cultivation." Since Laminaria plants are harvested when they reach anage of about two years, they have to be grown through two winters.
Thus the culture raft and other culturing equipment has to be solidand firmly anchored. This means that in Japan, the cultivation of
Laminaria is economically unrewarding. However, through development of the "forced cultivation" technique, one-year-old
Laminaria plants can be produced that have the characteristics andquality of two-year-old plants (Hasegawa 1971). Because of thisadvance, the amount of commercial Laminaria production throughraft cultivation has increased markedly for the past ten years in thesouthern part of Hokkaido and northern Honshu (Table I). At present, raft cultivation is also practiced in the middle and southern9Table I-Quality and value or Laminaria production by long-line cultivation, 1970-80.
Year1970 1971

quality(metric tons) 284
value(million yen) 53

1972

1973

1974

1975

1977

1978

1979

1980

665 3,340 7,681 10,201 15,759 22.096 27,260 21,890 25,291 38.561
95

481 1,589

1,868

2,365

regions of Japan, where Laminaria have never grown naturally.
In 1980, the amount of cultivated Laminaria was about 39,000 tons
in fresh weight, and is valued at about 30% of that of the wild harvest
from natural reefs.
Brown algae are major constituents of the natural sublittoral flora
in Japan. The predominating genera are Alaria, Laminaria, andCostaria in the northern part of Japan, and Eisenia and Ecklonia
in the middle and southern parts of Japan. As mentioned earlier,these brown algae are important sources of food for useful sea
animals such as the abalone and sea urchins. For this reason, theJapanese government program of "coastal fishing ground improvement and development" involves the artificial propagation of large
brown algae. In order to increase food supply and favorable conditions for sea animals, the economic efficiency of suspended raftcultivation of Laminaria has been studied recently in Japan.

3,877

3,736

3,336

5,111

8,984

fruiting plants. Laminaria spores that settle on the strings are grownindoors for one or two months. Settled Undaria spores are directlytransferred to the sea. It is possible for an individual fisherman tocollect spores and grow Undaria seedlings in his own culture tanks.
However, for Laminaria seedlings it is less costly in most cases
to purchase them from a seedling producer, usually a fisheries
cooperative which produces large quantities of seedlings. Undaria
seedlings are produced all along the Japanese coast, but seedlingsof Laminaria are produced only in Hokkaido and on the north Pacificcoast of Honshu, and are supplied from these regions to the southernpart of Japan. Japanese fisheries statistics for 1980 report that the
total amount of seedling strings produced was 2.192 and 883 km
for Undaria and Laminaria, respectively. Most of the seedlings wereused for human food production.

PROSPEcrs FOR THE FUTURE CULTIVATION
OF BROWN ALGAE IN JAPAN_

Undaria
Undaria, which like Laminaria is one of the most important edible
seaweeds in Japan, is harvested all along the Japanese coast except
in the cold regions around Hokkaido and in warm regions such as
Okinawa. Raft cultivation of Undaria started in the early 1960's
in northern Honshu and spread to other suitable growing regionsin Japan over the past 20 years. At present, Undaria is produced
mainly by raft culture. In 1980 the production of cultivated Undaria was estimated to be approximately 114,000 tons fresh weight.
The value of this crop is more than seven times that of the harvestof wild plants from natural reefs.
The commercial cultivation of another edible brown alga, Cladosiphon, has recently started. This plant is grown in warm water
regions such as Okinawa. The 1980 production was about 3,000
tons fresh weight.

SEEDLING PRODUCTION OF
BROWN ALGAE

1976

I have presented here only a brief outline of the current brown algal
cultivation and seedling production methods used in Japan. Suspended cultivation of seaweeds on longline rafts is very efficientwhen compared with the propagation on, and harvest of, plants from
reefs. Therefore, it is likely that the future cultivation of seaweeds
will be mainly through suspended cultivation, even though
Laminaria is propagated and harvested on reefs at present. The consumption of brown algae such as Laminaria and Undaria as humanfoods is limited and will probably not increase markedly in Japan.
Thus the mass cultivation of these seaweeds will provide crops thatcan be used for other purposes. The artificial propagation of abalone
and sea urchins in Japan is now being studied, and the productionof seaweeds as food for these animals is being seriously considered.
This is important because the natural stands of seaweeds along thecoast are insufficient to support large numbers of grazing animals.
Thus it is likely that there will be a major effort made to produce
brown algae such as Laminaria, Costaria, Undaria, Eisenia, andEcklonia for sea animal food. If this is successful, it will make itpossible to increase the production of abalone and sea urchins in
the near future.
As noted earlier, the Japanese government has begun to encouragetechnical innovation in the mass cultivation and utilization of seaweeds. Similar programs are now in progress in the United States,
where the cultivation of large brown algae also holds the prospect
for producing resources from the sea. However, there are still many
unsolved problems. Consequently, I think that it is very desirable
for us to have opportunities to exchange opinions and knowledge
with research workers in other countries, especially the UnitedStates.

_Since all of the above mentioned large brown algae belong to theorder of Laminariales, their life history pattern is identical. The
life history of all laminarialian plants consists of a macroscopic
sporophytic stage and a microscopic gametophytic stage. The sporophyte produces spores which germinate and grow to form microscopic dioecious gametophytes. The female gametophyte produces
eggs, and the male produces antherizoids. After fertilization, theeggs form zygotes that develop into juvenile sporophytes, which
in tum grow into large plants (Fig. 1). In the cultivation oflaminarialian plants, "seedling production" refers to the collection of spore~and rearing them through the gametophytic phase to the point where
juvenile sporophytes are produced. Mass seedling production is initiated by collecting fruiting sporophytic plants. Special "Kuremona"
string is soaked in a dense suspension of spores obtained from the10

CITATIONS_HASEGAWA, Y.

1971.

Forced cultivation of Laminaria. Proc. 7th Seaweed Symp., p. 391-393.

MIYASHITA, A.

1974. Seaweeds. Hosei Univ. Press, Tokyo (In Jpn), 315 p.
OKAMURA, K.

1932. Thedistribulion of marine algae in Pacific waters. Rec. Oceanogr. Works
Jpn 4(1):31-150.
OKAZAKI, A.

1971. Seaweeds and their uses in Japan. Toka; Univ. Press, Tokyo, 165 p.

Zoospore

Oogonium

Juvenile sporophyte

Macroscopic stage~I

Microscopic stage

Figure I-Life history of LaminariD.

11

INTRODUCTION

Seed Production of
Red Sea Bream
Pagrus major (Sparidae)
in Japan_

During the past decade extended research efforts have been directed
towards an improvement in rearing techniques for early life history
stages of marine fishes. The development of mass rearing methods
has already played an active role in the history of marine aquaculture
in Japan. The drastically increasing demand for aquaculture products
has recently been accelerated by the establishment of the 200-mile
economic zone. As a consequence, governmental programs are concentrating on the improvement and development of coastal fishinggrounds, induding mass releasing programs for important local
species. Under these circumstances, it was decided to continue theconstruction of Fish Farming Centers at strategically important
points along the coast line of Japan (Fig. 1).
Each of the Farming Centers produces large quantities of small
fish, prawns, and molluscs in order to release them into the naturalenvironment, thereby supporting the recruitment of exploited stocks.
Furthermore, experiments are under way to explore the most effective methods to utilize the adult fish. Among the major species
produced at the Farming Centers, red sea bream (Pagrus major)
is one of the most important and popular species, both in the northern and southern areas.
This paper describes the present status of artificial propagationof Pagrus major in Japan and outlines specifically the rearing techniques employed in governmental Farming Centers and in private
hatcheries.
Egg collection in the past depended largely on weather conditions and availability of parental fish from wild stocks, proper sex
ratio in captivity, maturity stage, and egg numbers spawned per
female. Therefore, treatment procedures using hormone injection
have been employed to obtain viable eggs. However, egg production could not be stabilized in the initial phase of the work. After
natural spawning succeeded in captivity (Noguchi 1968), viable eggs
were collected mainly and more regularly from brood stocks maintained in the tanks. Artificial breeding has advanced since then by

OSAMU FUKUHARA
Nansei Regional Fisheries Research Laboratory
Ohno-cho, Saeki-gun, Hiroshima 739-04, Japan

.0
• 0

•

•

Run by the central government
Run by prefectural governments
Construction completed

o 0 Under construction•

Figure I-Location of governmental and prefectural Fish Farming Centers(modified from Annual Report on Japanese Fisheries, Ministry of Agriculture,
Forestry and Fisheries).

13

CULTURE OF BROOD STOCK

establishing techniques that allow natural spawning and mass production of suitable organisms at the same time (Fukuhara 1975).
Seasonal activities for artificial propagation are outlined in Figure
2. All procedures indicated vary slightly in technical detail, depending on the facilities and strategies employed at each location.

Feb.

Apr.

Mar.

May

:

July

June
:

. . Transfer from cage to

Brood stocks are usually maintained outside during the spawningseason in facilities installed in the sea (mainly floating net cages)(Fig. 3). This is done because the maintenance of adult fish is easierand cheaper in the sea than on land. The transport of spawners is
generally done about 4 to 6 weeks before the spawning season, fromfloating net cages to specially prepared spawning tanks of an appropriate size.
Tank volumes of approximately 50 to 100 m 3 water are quite
commonly used for spawning tanks, and a I: I sex ratio of males
and females is used. The age of the spawners varies between 4 and7 years. They are maintained at a density between 0.7 and 1.5 individuals i m 3 . High stocking densities and uneven sex-ratio of
spawners in the spawning tanks cause negative effects on successful spawning (Fushimi 1972). Fresh small fishes (anchovy, sandlance, and mackerel) are the preferred diet prior to and during the
spawning season.
The spawning period extends for 50 to 70 days from March toJune. The onset of the spawning season occurs earlier in the Pacificregion than in the Seto Inland Sea and the Japan Sea (Fig. 4). In
some districts the energy from power stations is utilized to induce
an early spawning through elevated temperature. Eggs spawned during the initial phase of the spawning season are unsuitable for rearing because of their low quality, resulting in low hatching rates.
Eggs spawned during the peak spawning season (late April to lateMay) are suitable for rearing. Ambient temperatures during the
spawning season range from 14° to 23°C. During one spawningseason about 100 to 400 x l<r eggs are collected from one 3-to-6
year-old female within 50-70 days in captivity.

Aug.

sp~Wning ta~kspawrii~gc:;~~~;. . . . . .
.

Main hatching period
Rearing in tank

.

i

1

.
,
==='"""'i"'"~:::Transfer to outdoor: facility_:

IltiflCulture in net cage

Figure 2-Distribution of seasonal activities related to the artificial propagationof red sea bream, Pagrus major, in Japanese hatcheries. Time periods for eachactivity vary according to the spawning.

Figure 3-FIoating net cages for cultivation of brood stocks of red sea bream. Cages are usually installedin front of the farming facilities. Parental fish are transferred to spawning tanks on land prior to thespawning season. Cages are covered with fishing net to prevent predation by birds.

14

130"

SEA

JAPAN

34 Kumamoto~Ukuoka

Nagasaki

o

~-fJ

toV
'?

~March~AprllKagoshima~ May

L-L-.J
100KmoFigure 4-Major spawning season for Pagrus major in various prefectures of Japan.

INCUBATION_

Pelagic eggs of Pagrus major are collected by mtering the overflow
water from the spawning tank with a fine-meshed net. Eggs aretransfered daily to an incubation net. There they are kept in a flowthrough system until hatching (Fig. 5). Sometimes eggs are introduced directly to rearing tanks at a density of 3000 to 9000
eggs/m 3 , where they hatch without extensive control of theirmortality.

LARVAL REARING_

Tank capacity used for larval rearing ranges between 10 and 200
m3 ; the usual average size is 20 to 100 m3 (Figs. 6, 7). Hatchedlarvae are maintained at densities of 5,000 to 50,000 indivlduals/m 3 . Optimum stocking density has been determined experimentally to be 400-5000 individuals/500-L tank volume (Hirata<:t al. 1975).
The feeding regime for larval rearing is shown in Fig. 8. Unlike
the present practice, oyster larvae (Crassostrea gigas) were formerly
employed as food organisms. The main reason why oyster larvae
have been omitted from the list of feeding items is the difficulty
in preparing large quantities. Cultured rotifers (Brachionus plicatilis)
are now fed initially at a density ranging between 5 and 10 individuals/mL in the rearing tank. Chiorella species are also introduced to the rearing tanks at concentrations between 30 and 100
cells/mL. This is done during the period of rotifer feeding. The
inoculation of Chiorella is intended to avoid starvation of rotifers
offered as food, thereby maintaining their nutritional quality. Furthermore, they help to reduce the disintegration of faeces. Rotifers
are the principal plankton food for the larval stages; subsequently

Figure S-Pelagic eggs of Pagrus major are coUected from the spawning tankthrough fine-meshed nets, which are filtering the overflow water. Nets areexchanged every day and eggs transferred to incubation facilities.

15

Artemia nauplii, frozen rotifers, copepods (Tigriopus japonicus) ,and other organisms are fed in addition to rotifers. The larvae arereared in a standing water tank up to an initial period of 2 to 3 weeks.
Thereafter water exchange and regular removal of settled solids
from the bottom is undertaken periodically by a siphon. SubsequentIy, water exchange frequencies are increased with larval growth,

gradually integrating a "flow-through" system. With regard to aeration, a close relationship was found between the volume of air andthe aeration intensity and abnormalities in air-bladder and vertebral development (Utsunorniya and Suizu 1980; Kitajima et aI. 1981;
Iseda 1982). Gentle aeration ranging between 10 and 100 mLl(min'm 3) is used in the initial rearing.

Figure 6-View of indoor tanks employed to rear larval marine fISh as well as kuruma prawn; tank volume
200 m J •Figure 7-View of outdoor tanks used for initial rearing of laval Pagrus major. The facility is suitable
for mass production.

16

ROfIFER CULTURE

Days after hatching

o

10

20

_

40

30

r------,-------.,..--------,-'-----------,

RotiferDuring the past decade, culture techniques for rotifers have been
improved remarkably; much of the progress can be attributed tothe development of adequate feeds, resulting in improved nutrition.
Recently, the quality of yeast has been improved by the additionof polyunsaturated fatty acids, especially of the w-3 type (Kitajima
et al. 1980; Watanabe et al. 1979). These feeds are useful when
cultured ChIarella is unavailable, and to save tank capacity. Standard amount of yeast is about I g per one million rotifers in culture.
ChIarella as well as concentrated ChIarella are used successfully
among culturists and farmers in large-scale culture units. Alcohol
fermentation by products have been tried experimentally to culture
rotifers (Fukuhara et al. 1982; Higashihara et al. 1983).
When using processed materials as feeds to culture rotifers,
natural food in form of ChIarella is still provided to the rotifers
just before feeding them to the fish larvae (Fig. 11). This shorttime exposure of rotifers to algae will enrich and improve the nutritional value of the rotifers (Kitajima et al. 1979). A tank capacityof <100 m 3 is mainly used to culture rotifers (Fig. 12). The density of rotifers at the start of culture varies between 30 and 60
animals/mL. They are harvested for feeding when they exceed adensity of 70 to 100 animals/mL.

Arte'~m~ia~Zlll!itarJ2[MillFrozen rot::.ife:r~s~mr;iElli£
Copepo..:.d,;;,ez:q:llli±2llil

Minced meat and/or chopped' meat~=illFigure 8-VariODS food organisms and feeding periods in rearing larval Pagrus
major in tank or cages. Bars represent earliest (lower line) and latest (upper line)
point in time at which food is employed.

CULTURE IN NET CAGES_PROBLEMS AND PERSPECTIVESFOR FARMING FISHERIESThe transport of the metamorphosed young fish is carried out after
acclimatizing them step-by-step from the rearing facility on land
to the conditions in the sea. Usually the rearing period on land iscalled "initial breeding. " The raising of fry in the sea, where they
are then used for augmenting natural stocks is called' 'intermediate
culture. " The sizes of net cages used during the intermediate culture
range from 3 x 3 x 3 to 6 x 6 x 6 m. Stocking densities of fry range
from 100 to 600 fish/m 3 , depending on mesh-size of the net andthe tidal conditions on the farming grounds (Fig. 9). Nets of thecage are changed depending on fish growth and accumulation offouling organisms such as ascidians, barnacles, mussels, and seaweed and debris on the net surface (Fig. 10).

CHLORELLA CULTURE_

At the present time, the number of fingerlings of Pagrus majar produced for releasing or for cage culture may exceed one million
annually in one Farming Center or hatchery (Fig. 13). Every procedure, however, in artificial propagation still depends largely on
weather conditions and/or biological aspects (availability of eggs
and food organisms). Therefore, yield in rearing seedlings fluctuates widely with each trial. To obtain stable production, more
multifaceted research is required to provide useful scientific andtechnical data to optimize rearing practices. Furthermore, the biological quality of produced seeds and its effect on subsequent postrelease survival is nearly unknown.
In the past decade most efforts concentrated on increasing thenumber of fish raised per production unit. It is a prerequisite fora more effective use of the young fish produced, especially forreleasing programs, that more information on the differences in the
biological characteristics of natural and reared animals should becollected (Fujiya 1974). Some acclimation and familiarization innatural surroundings, or training of the animals produced shouldbe tried to obtain best results in the farming fisheries.

_

The tank volume used for culturing ChIarella varies between 10
and 800 m 3 • Tank sizes required for culturing ChIarella are 1.5
to 3 times greater than those needed for rotifer cultivation. Initially,
agricultural fertilizers are added to the tanks which are filled withsea water of ambient salinity. Fertilization amounts to 70-100 gammonium sulfate/m3 , 10-50 g calcium superphosphate/m 3 , and5-50 g urea/m3 . Some other ingredients are also added for plankton culture if necessary. Then the ChIarella inoculation in introduced
into the tank at a concentration of about 300 to 600 x 1()4 cells/mL.
Tank water is aerated heavily through perforated air pipes and/or
air stones. At peak culture bloom of the phytoplankton, cell numbers
reach a density of up to 2500-4000 x 1()4 individuais/mL. The maximum temperature for adequate growth of ChIarella is 18°C. During the summer season, reproduction of ChIarella is often decreased
due to high temperatures. In such cases the salinity of the culture
medium is reduced by dilution down to values of 10-20%. Thisis achieved by addition of fresh water. Growth of ChIarella canbe reactivated by this method at high temperatures. Sodium hypochloride is often used to treat bacteria and Protozoa in the tank during
culture. Both batch and continuous cultures are tried in hatcheries.
In the case of batch culture, the period is mostly more than 3 weeks.

CITATIONS_FUJIYA, M.
1974 Fish farming and the constraints in Japan. In Proceedings of the First U.S.hp.n ",cering on aquaculture, Tokyo, Japan, October 18-19, 1971. NOAA
Tech. Rep. NMFS CIRC-388, p. 27-32.
FUKUHARA, O.
1975. The present status and some unsolved problems on artificial propagationsof marine fishes. Spec. pub!., of Aquaculture Division, Nansei Reg. Fish.
Res. Lab., Hiroshima, Japan. (in Jpn.)
FUKUHARA, 0., H. YUKAWA, and T. NISHIMURA.
1982. Culture of rotifer, Brachionus plica/iUs (Muller), in an air lift aquarium
using alcohol fermentation slops. Bull. Nansei Reg. Fish. Res. Lab. (14):1-11.
FUSHlMI, T.
1972. Studies on egg-taking from cultured red Sea bream, Pagrus major (Temminck et Schlegel)-III. Tech. Rep. Farm. Fish. 1(1): 15-20 (in Jpn.).

17

HIGASHIHARA, T., S. FUKUOKA, T. ABE, I. MIZUHARA, 0. IMADA, and
R. HIRANO.
1983. Culture of the rotifer Brachionus plica/ilis using a microbial flock produced from alcohol fermentation slop. Bull. Jpn. Soc. Sci. Fish. 49(7): 10011Ol3.
HIRATA, M., T. FUJITA, S. HAMADA, and F. NAGATA.
1975. Studies on mass production of red sea bream. Tech. Rep. Farm. Fish.
4(2):61-68 (in Jpn.).
ISEDA, H.
1982. Prevention of demonstration in the juveniles of red sea bream, Pagrus
mnjor reared in ponds - III. Bull. Kumamoto Prefect. Fish. Exp. Stn.
(2):25-45.
KITAJIMA, c., S. FUITA, F. OHWA, Y. YONE, and T. WATANABE.
1979. Improvement of dietary value for red sea bream larvae of rotifers
Brachionus plica/ilis cultured with baker's yeast Saccharomyces cerevisia<.
Bull. Jpn. Soc. Sci. Fish. 45(4):469-471.
KJTAGIMA, c., T. ARAKAWA, F. OOWA, S. FUJITA, 0. IMADA, T. WATANABE,
and Y. YONE.
1980. Dietary value for red sea bream larvae of rotifer Brachionus plica/ilis
cultured with a new type of yeast. Bull. Jpn. Soc. Sci. Fish. 46(1):43-46.
KITAJIMA, c., Y. TSUKASHIMA, S. FUJITA, T. WATANABE, and Y. YONE.
1981. Relationship between uninflated swimbladders and lordotic deformity inhatchery-reared red sea bream Pagrus major. Bull. Jpn. Soc. Sci. Fish.
47(10): 1289-1294.
NOGUCHI, T.
1968. Natural spawning of red sea bream in an aquarium. Fish Culture 5(3):81-85 (in Jpn.).
UTSUNOMIYA, T., and H. SUIZU.
1980. Relationship between aeration volume and inflating of the air bladder oflarval red sea bream in initial rearing. Rep. Yamaguchi Outsea Farm. Fish.
Center (4):9-17 (in Jpn.).
WATANABE, T., F. OOWA, C. KJTAJIMA, S. FUJITA, and Y. YONE.
1979. Relationship between the dietary value of rotifers Brachionus plica/ilis
and their content of w2 highly unsaturated fatty acids. Bull. Jpn. Soc. Sci
Fish. 45(7):883-890.
Figure 9-Raft facilities holding separate floating net cages are used forintermediate culture. The facilities anchor from each corner to bottom of
about 8-15 m depth.

18~r ....._ _- -Figure to-Bamboos are usuaUy used to make culture rafts. Arrangement of the raft facilities is dependent
on various conditions, such as current, ship way, and depth, in farming ground.

~I Chlorella~otife)-I~I'=---------..

Chlorella + Yea.'

~

I

·

B

I/)Chlorella~
r

Yea.t

Figure ll-ConceptuaI diagram of rotifer culture for larval feeding of fish.
Chlorello<ultured rotifers are fed directly to larva; rotifers fed a combinationof Chlorello and yeast, or yeast alone, are activated by Chlorello prior to larval
feeding.

19

Figure 12-Indoor tank is convenient in culturing rotifers.

Figure 13-Raised seeds of Pagrus major. More than 5 million fish are released annually along the coastal
areas.

20

INTRODUCTION

Culture of the Oyster
Crassostrea virginica (Gmelin)from Seed to Market
on Bedding Reefs inBarataria Bay, Louisiana,
USA_Louisiana consistently ranks among the top states nationally in oyster
production and has led the Gulf states during the past thirty years,
averaging 4 million kg (9 million pounds) of oyster meat annually.
During recent years the Louisiana oyster fisheries have supported
an annual $11 million industry, with the bedding of oyster seed
and their subsequent harvest contributing significantly to thismonetary figure.
Commercial oyster production in Louisiana has undergone a transition from harvesting only on natural reefs to the culture of oysters
on planted reefs leased from the state. In the mid-19th century, Louisiana oyster fishermen realized that private ownership (today actually leased from the state for $2 per acre) of a percentage of thestate's natural oyster reefs and bedding grounds would help insure
the longevity and productivity ofthe industry; subsequently, legislation was passed allowing public and private ownership. Louisiana's
oyster grounds have, from that time to the present, been divided
into two regions: state controlled "red-line" areas, and water bottoms set aside and leased to private individuals (Perret et al. 1971).
As of May 1980, approximately 92,737 hectares (229,060 acres)were privately leased, while approximately 323,887 hectares(800,000 acres) were within the red-line areas. Of the state's redline areas, 6,661 hectares (16,453 acres) are specifically reserved
within five oyster seed reservations located across the Louisiana
coast. Oyster populations on the seed reservations are fished when
open to the public, and approximately 80,972 hectares (200,000
acres) of the remaining red-line areas are fished annually between
September and March either for seed or direct-market oysters(Dugas 1977). Also, privately leased water bottoms are fished by
the owners, especially if there is a limited availability of oysters
on the state's reefs. This does not mean that all 92,737 hectaresof privately leased water bottoms are fished annually, but it is an
integral part of the industry, especially on bedding leases where
oyster seed are planted for later harvest. Privately held leases canbe legally fished year-round.
Perhaps there is no better area in Louisiana for discussion of oyster
culture on bedding reefs than in Barataria Bay, part of one of themost productive estuarine systems in North America-Barataria
Basin (Day et al. 1972). This is an area where the relatively highsalinity of the Gulf of Mexico influences the development and survival of seed oysters (Fig. I). Van Sickle et al. (1976) estimatedthat 3,329 hectares (8,223 acres) of water bottom were leased in
Barataria Bay and adjacent marshes by the private sector in 1975.
Although dock-side production figures are generally incomplete,
this basin is one of the major oyster producing areas in the state.
However, only a percentage of bedding leases in the lower bay areplanted and fished annually, leaving the remaining fallow beds alone
for one or two years to recover from dredging and hopefully to
reduce the incidence and concentration of predators, especially theoyster drill Thais haemostoma.

EARL MELANCON, JR.
ROANNE TRAPANIIGREG SCOTP
Department of Biological Sciences
Nicholls State University
Thibodaux, LA 70310

LEN BAHR3
Coastal Ecology Section
Center for Wetland Resources
Louisiana State University
Baton Rouge, LA 70803

ABSTRACTLouisiana leads the Gulf states in oyster production. A significant part of theindustry is the extensive bottom culture using oyster seed. This paper discusses
commercial bedding procedures and culture techniques and gives specific examplesof the problems inherent in this type of culture.
Oysters (optimum size range 25-75 mm in length) are harvested by commercial fishermen in the fan from state-managed reefs and transported and plantedon private reefs located in the more saline waters near the coast where they exhibit rapid growth and good meat yield. Once bedded in the fall, the oysters areusually not harvested again until the next spring and summer when they are
greater than 75 mm in length and ready for market.
In the fan of 1982, large seed oysters were bedded (44%>75 mm) on the Independence Island reef. Through the spring and summer, predation by thegastropod Thais haemastoma and the protozoan parasite Perkinsus marinus waslight, probably due in part to the low salinities in Barataria Bay during the springand summer of 1983. It is probable that had the bay's salinity remained high,oyster mortalities would have increased and reduced the harvest and financial
gains of the commercial fishermen.

CULTURE TECHNIQUES_In contrast to the intensive off-bottom culture practiced in Japanand other countries, Louisiana's oyster industry is an extensive
bottom-type semiculture. Oyster fishermen, i.e., culturists, must
rely on nature to supply them with oyster seed. Once the seed istransplanted to the private bedding reefs, it becomes the fisherman's
responsibility to oversee the survival, growth, and eventual harvestof the oysters for market.

'Present address: Virginia Institute of Marine Science, College of William and Mary,
Gloucester Point, Virginia 23062.
'Presenl address: Fisheries Department, Louisiana State University, Balon Rouge,
LA 70803.
'Present address: 141 Washington Street, Baton Rouge, LA 70803.

21

50

-r---------------------,PERCENT

40

30

20

10

1-25

26-50

51-75

76-100SIZE IN MILLIMETERS (MM)Figure 2-Size frequency of normally bedded oysters in Barataria Bay, planted
in November (from St. Amant 1957).

Figure I-Barataria Bay, Louisiana.

In the early years of this century, the Louisiana oyster industry
experienced a transition from sail-powered to motorized vessels andfrom hand tongs to mechanical dredges to harvest oysters. These
two advances allowed fishermen the opportunity to expand theirrange and to increase their productivity. The term' 'lugger" is used
to denote the traditional oyster vessel. Oyster vessels used in
Barataria Bay typify the traditional large lugger and range from15.5 to 18.2 m (50-60 ft) in length and 5.4 to 6.7 m (18-22 ft) in
width. The hull is constructed of either cypress or steel and is
powered by a diesel engine. The two dredges, each 1.4 m (56 in.)wide, fish simultaneously on both sides of the vessel and are alternately winched aboard to dump the oysters on the deck; by thismethod one dredge is always fishing. A crew consists of a captainand two or three mates.
Seed oysters for the bedding reefs are obtained from statemanaged oyster reefs on which hydraulically dredged clam shells(Rangia sp.) or naturally occurring oyster shells are used as cultch
for the oyster spat to set. The seed oysters are harvested one year
after setting and transplanted to private bedding reefs from September through November when they are 25 to 75 mm (1-3 in.) in
length. Figure 2, taken from St. Amant (1957), shows the preferred
size range of bedded seed oysters. In lower Barataria Bay, the commercial fishermen prefer 50 to 60 mm length seed oysters because
they believe the larger size will insure a higher survival rate during transporting and bedding, and from predators.
With the relatively high salinities and temperatures that usually
prevail in the lower bay, predation and disease are quite prevalentand the naturally occurring subtidal oyster populations will succumb quic!dY. For these reasons the bay's bedding reefs are often
devoid of any natural oyster populations and are used only for shortperiods of time in the cultivation of transplanted seed stock. Throughthe fall and winter months, after the fishermen have bedded theirseed, they return either to the state's public reefs or to other privately
leased natural reefs to fish for market-size oysters for direct saleto shucking houses or canning factories. Not until the following

spring and summer do most fishermen return to the bedding reefs
to harvest the now 75 to 125 mm length oysters for market. If thebedded oysters are large, some fishermen may begin harvesting by
November or December instead of waiting until spring. Seed oysters
remain on the bedding reefs no longer than six to ten months becauseof predation and disease.

INDEPENDENCE ISLAND REEF_

One example of a bedding reef is Independence Island, located at
the southern end of the bay behind the barrier island Grande Terre.
The name implies the presence of a land mass, but the island longago eroded away leaving only a cluster of wood pilings visible above
the water. These pilings are all that remain of an old oyster campthat once housed as many as 30 to 40 oyster fishermen, i.e.,laborers, during the first quarter of this century and before the advent of refrigeration, motorized vessels, and mechanical dredges.
The massive, subtidal, hard oystershell reef is still present, however, and is used extensively by commercial fishermen to plant theirseed oysters. In the fall of 1982, six oyster vessels bedded oysters
on the massive reef. Each lease, except numbers 4 and 5, was approximately 0.4 hectares (I acre) in area; numbers 4 and 5 wereapproximately half that size.

SIZE, DENSITY, AND
GROWTH OF BEDDED OYSTERS_

As stated earlier, seed stock in Louisiana is dependent primarilyon the natural spat set that occurs on the state-managed oyster
grounds. In September of 1982 when the state reefs were opened
to commercial fishermen, they quickly discovered that the majority of oysters available as seed stock were large. Such oysters could
hardly be called true seed, but the fishermen had no choice but to
22

50

50

PERCENTPERCENT
40

LIVE

40

LIVE

30

30
N = 722
SEPTEMBER-OCTOBER
20

20

10

10

1-25

26-50

51-75

76-100101-125

1-25

26-50

51-75

76-100101-125

126-150

SIZE IN MILLIMETERS (MM)SIZE IN MILLIMETERS (MM)Figure 3-Size frequency of bedded oysters on Independence Island reef, planted
fall 1982.

Figure 4-Size frequency of bedded oysters on Independence Island reef, spring
1983, after fall-winter growth.

bed the large oysters or not bed at all. A survey revealed that approximately 44 % were greater than 75 mm in length, the minimum
legally harvestable size for direct sale (Fig. 3). It is not unusual,
however, to have market-size oysters mixed with the seed since
the majority of the stock comes from the natural reefs located on
the public grounds. The sizes of oysters available for seed fromthese natural reefs are dependent on many environmental and market
factors over which the Louisiana Department of Wildlife and Fisheries (LDWF) has little or no control. Often, only when the LDWF
plants cuItch material, i.e., clam shell, and regulates when the area
can be harvested, can a crop of true seed (25-75 mm) be produced
for the industry.
Day et al. (1972) estimated the density of an average plantingof 37 to 50 mm seed oysters in Barataria Bay at 110 oysters per
square meter. The mean density of oysters on lease 1 was 42 ±25.4/m2 , while on lease 2 it was 103 ± 46.0/m2 . There is often
a large variation in density from one location to the next withina lease due largely to the method of bedding the seed; water pressure
from a deck hose is used to distribute the seed across the lease whilethe vessel is circling, often resulting in patchiness and uneven
distribution.
The distance oyster vessels must travel from the public grounds
to their bedding reefs often compounds a fisherman's problem offinding adequate seed. It is not uncommon for fishermen beddingin Barataria Bay to travel over 129 km (80 miles) one-way, requiring as much as 11 hours travel time. Due to the distance, Barataria
Bay is considered the maximum range of bedding from the public
reefs located east of the Mississippi River. Once bedded, the vessels
return to the public reefs for another load. A fisherman may bed
8 to 20 loads, depending on the availability of oysters and market
conditions, with each load averaging 200 to 250 barrels of seed.
On the public reefs, every fisherman has the legal right and opportunity to harvest oysters. Competition, therefore, is keen and afisherman cannot afford the luxury of wasted time, especially withsuch a finite resource. Except to get fuel or supplies, the vessels
seldom stop until all the desired seed has been bedded.

Fishermen determine the quantity of seed oysters being transported by observing the area occupied by the compactly piled unsacked load on the foredeck. Most fishermen, through experience,know the carrying capacity of their vessel, thus enabling them todetermine the number of barrels planted by keeping a record of
the number of barrels hauled per trip. By volume, one barrel ofoysters is equivalent to three Louisiana bushels, 0.1 cubic meters,
which also equals two oyster sacks. The larger the oyster seed, thefewer the number of oysters in a barrel and therefore the fewer
the number of oysters planted per trip. However, dredged material
is not totally comprised of oysters; on the contrary, old dead reefshell and clam shell are also hauled on deck. The fishermen, again
through the experience gained by operating their dredges, try tokeep the quantity of reefshell to a minimum by scraping off only
the top layers of reef where the live oysters rest. At the present
time, Louisiana does not require the return of discarded oyster shells
from the shucking houses to the state's reefs.
A resurvey in March 1983 revealed a shift in the oyster population's size-distribution indicating that significant growth had occurred (Fig. 4). The size-distribution figures were somewhat
clouded, however, by the presence of a relatively large number of
1-25 mm oysters which indicated that a natural spat set had occurredon the reef since planting. It is not unusual to have a fall-winterspat set occur, but these oysters were too small to be harvested forspring and summer sale and either died from dredging and predators or were later harvested and transported to less saline waters
for future sale.

23

PREDATION AND DISEASE

_

.

30

('-

I

, • "I

Many marine organisms prey upon the immobile oyster, but thethree principal organisms causing extensive oyster mortalities on
the bedding reefs are the predaceous conch Thais haemastoma; thedisease producing protozoan Perkinsus marinus, colloquially known
as dermo; and the black drum fish Pogonias cromis. The black drum
fish is dominant during the fall and winter months, while the conch
and protozoan become increasingly active during the spring andsummer with a rise in water temperature above 20°C (68°F) andsalinity above 15 ppt. It is because of T. haemastoma and P. marinusthat bedded bay oysters are usually harvested by early summer.

"

"1/

25
20

40

\

\v".o;oo\, ,•,"
'---

30

\

20

15
10

10

A

SON

D

F

M

A

M

MONTH

Oyster drill

Figure 5-Temperature and salinity on Independence Island reef, 1982-83 season.

The oyster drill, T. haemastoma, is very common in the saline
waters of Barataria Bay and can cause extensive mortalities tobedded oyster populations. The combination of drilling with its
radula and the secretion of a paralytic substance is currently recognized as the gastropod's method of attack (Breithaupt and Dugas
1979). Interviews with the Independence Island fishermen andmonthly reef surveys taken through the spring and summer revealed
that few drills were evident on the leases. The drill infestation wasconsidered very light by the fishermen, and few of the gastropod's
eggs were evident on the harvested oysters and reefshells. Conversations with commercial fishermen and biologists at the LDWF
Lyle S. St. Amant Marine Laboratory on Grande Terre Island indicated that the salinity in the bay fluctuated dramatically and suddenly throughout most of the spring and early summer. The bay's
salinity is greatly influenced by precipitation in the northern areasof the basin and by the Mississippi River's discharge in the south;both set high records in the spring and summer of 1983. This perhaps
created an environmental barrier that prevented the oyster drill frombeing abundantly distributed across the reef. A survey of soft mud
areas adjacent to the reef revealed the presence of drills, indicatingthat they did inhabit the area but were relatively inactive. St. Amant(1957) discusses how the drill burrows into the soft mud andbecomes relatively inactive to protect itself from adverse environmental conditions such as fluctuating temperatures and salinities;
this may have been the case here. Figure 5 shows weekly temperature and salinity measurements. 4

do not develop dermo as readily or as severely as older oysters,
and it is not until the second or third summer of growth that many
succumb to the parasite (Mackin 1961). For these reasons, the oyster
population bedded on Independence Island in the fall of 1982 could
experience heavy mortalities the following summer since the majority would be in their second and third summer of growth.
A monthly survey of dermo was conducted from early springthrough summer. The spring (l March-29 April) and summer (8June-25 July) exhibited much similarity in the occurrence of dermo. Contrary to expectation, neither the frequency nor the degreeof infection increased from spring to summer. Dermo was abundant within the Independence Island population with 57 % harboring the parasite during the spring and summer. The weighted
incidence (WI) remained light, however, averaging 1.06 and 1.18
during the spring and summer, respectively (using the WI scale of
Quick and Mackin 1971). Similarities between spring and summer
probably were partially due to the relatively low salinities thatprevailed during 1983. Dermo is known to become increasingly
active with the synergistic effects of rising temperatures and salinities (Soniat 1985). In this instance, the two environmental parameters did not coincide. During the spring the water temperatures
were still relatively low while salinities remained high. In contrast,
during the summer the opposite prevailed with temperatures highand salinities low. If a hot dry spring and summer had occurred,higher salinities would have prevailed and higher oyster mortalities
probably would have occurred resulting in considerable financial
loss to the fishermen.

Dermo
Dermo is a parasite that has plagued the Louisiana oyster fishermen for decades and it is the primary cause for what they referto as the "summer mortalities". Infection by dermo has significant economic implications. Fishermen know that leaving bedded
oysters on the reefs through late summer may mean financial
disaster. Although the parasite is found in other organisms, dermo
is usually considered specific for the American oyster because of
the large-scale mortalities associated with its presence in theirtissues. Most oyster tissue will harbor the parasite, but the gills,
rectum, mantle, and adductor muscle appear to be the most infectedareas (Overstreet 1978). It is not until the oyster has a moderateto-heavy infection that it begins showing any signs of weight lossor other visible characteristics (Ray et al. 1953). Young oysters

Black drum
A third predator that can cause extensive damage to a bedded oyster
popuiation is the fish P. cromis. During the fall and winter months,
this fish can often be found in large schools of 100 or more andcan literally devour a bedded oyster population within a few days.
One has only to look at the jaws and teeth of these large fish tosee how they can easily crush an oyster's shell. Usually after beddingin the fall, the fishermen will not disturb their lease again until they
are ready to harvest for sale. Fishermen believe that disturbing theoysters after bedding will increase the chance of attracting the drum.
Whether fact or superstition, the fishermen of Barataria Bay aregoverned by this belief. This had an effect on our sampling schedule.

'Temperature and salinity data for Independence Island, 1982-83, courtesy of personnel from the LDWF"s Lyle S. SI. Amant Marine Laboratory, Grande Terre Island,Louisiana.

24

OVERVIEW

When the fishermen began harvesting their leases in the spring,
there were no apparent signs of widespread mortalities due to drum
predation.

HARVEST STATISTICS

The oyster bedding industry is dependent on a successful seed cropfrom the state reefs. Many believe that the present economic conditions in Louisiana do not warrant the development of commercial seed hatcheries even though reliance on natural seed brings fluctuations of scarcity and abundance. To insure the longevity anddevelopment of the industry to its maximum potential, a consistentand large supply of 25 to 75 mm seed oysters is mandatory. Possible solutions, other than hatcheries, to the problem of scarce seedstock are (1) supervised shell plants for cultch material (a practicealready implemented by the LDWF) , and (2) controlled andregulated freshwater diversion onto the state's prime oyster seedgrounds to slow or reverse the trend of encroaching high-salinity
waters.

_At no time during this study was an unusually high degree of oyster
mortality observed; mortality was estimated by counting the numberof empty hinged boxes. The observed monthly mortality was neverin excess of 5-10% of the population during any given spring andsummer month. Other scientists have found mortality ranges of30-100% (Mackin and Hopkins 1961), 15-77% (Owen 1953), and
16-44% (St. Amant 1957) during a bedding season.
It has been estimated that a boatload of seed oysters planted in
the fall of one year will yield one to three boatloads of marketableoysters by the following summer (Dugas 1982). Many seed oysters
may die either from the transplanting trip or from predators anddisease while on the bedding reefs. To insure a profit, the fishermen must compensate by bedding many seed oysters, although thisis often difficult for the bay fishermen because of the distance theymust travel and the limited availability of public seed. Fishermen
who bed in the basin to the east of the bay are closer to the public
reefs and can often make two trips to every one made by the bay
fishermen. Owen (1953) suggests that a fisherman can make a profit
if, for every sack of seed oysters planted, one sack of market oysters
is harvested. This would produce a one-to-one yield compensating
for oyster growth, mortality, and the bedding of accidentally collected old reef shells. Table 1 shows the number of barrels planted
and the number of sacks harvested by each vessel.
A thorough discussion of economic factors involved in the bedding
and harvesting operations of a commercial oyster business is beyond
the scope of this study. Most expenses during this study were incurred during transplanting of the seed from the public reef, expenses incurred with no monetary return until the following springand summer. All but one fisherman produced a yield ratio greaterthan one-to-one. Interviews with the bay fishermen indicated thatthey all made a profit on the 1982 seed crop.
The bay fishermen worked and harvested the bedding reefs fromearly spring through early summer, and in two cases through Julyto mid-August, thus barely giving them time to rest or to repair
their vessels before the new September season on the public reefs.
Oysters were harvested and sold to the shucking houses by the sack,usually containing 168 to 180 oysters each. At the shucking house,
the sacks were opened and oysters processed as a raw-shop product.

ACKNOWLEDGMENTS

Lease

No. sacks
beddedNo. sacks
harvested

Sack ratio
Bedded: Harvested

A-B
C
D
D
E
F

1-2
3
4
5
6
7

6,780
4,400
2,000
2.000
3,720
4,750

8,333
5,138
819
2,505
4,168
5,777

I: 1.23
1:1.17
1:0.41 'I: 1.25
1: 1.12
1: 1.22

Average_

Without the support and cooperation of the Barataria Bay oyster
fishermen, this project could not have been conducted. This study
was funded partially by the NSU Research Council and the Coastal
Fisheries Institute, Center for Wetland Resources, LSU. Logisticalsupport was furnished by Louisiana Universities Marine Consortium (LUMCON) and the LDWF's Lyle S. St. Amant Laboratory.
Part of this work is being included in the senior author's doctoral
thesis at the Coastal Fisheries Institute, LSU.

CITATIONS_BREITHAUPT, R. L., and R. J. DUGAS.
1979. A study of the southern oyster drill (Thais haemastoma) distribution anddensity on the oyster seed grounds. La. Dep. Wildl. Fish., Tech. Bull. 30, 20 p.
DAY, J. w., w. G. SMITH, P. R. WAGNER, and W. C. STOW.
1972. Community structure and carbon budget of a salt marsh and shallow bay
estuarine system in Louisiana. Sea Grant Publ. LSU-SG-72-04, Louisiana State
Univ., Baton Rouge, 80 p.
DUGAS, R. J.
1982. The Louisiana oyster, La. Dep. Wild!. Fish., Educ. Bull. 32, 33 p.
1977. Oyster distribution and density on the productive portion of state seedgrounds in southeastern Louisiana. La. Dep. Wildl. Fish., Tech. Bull. 23, 27 p.
MACKJN, J. G.
1961 . Oyster disease caused by Dennocystidium morinum and other microorganisms in Louisiana. Publ. Inst. Mar. Sci. Univ. Tex. 7:132-229.
MACKIN, J. G., and S. H. HOPKINS.
1961. Studies on oyster mortality in relations to natural environments and tooil fields in Louisiana. Publ. Inst. Mar. Sci. Univ. Tex. 7:1-131.
OVERSTREET, R. M.
1978. Marine maladies? Worms, germs, and other symbionts from the northern
Gulf of Mexico. Gulf Coast Res. Lab., Ocean Springs, Miss., 140 p.
OWEN, H. M.
1953. Growth and mortality of oysters in Louisiana. Bull. Mar. Sci. Gulf Caribb.
3(1):44-54.
PERRET, W. S., B. B. BARRETT, w. R. LATAPIE, J. F. POLLARD, W. R.
MOCK, B. G. ADKINS, W. J. GAIDRY, and C. J. WHITE.
1971. Cooperative Gulf of Mexico estuarine inventory and study: Phase I, Area
description, and Phase IV, Biology. La. Dep. Wildl. Fish., 59 p.
QUICK, J. A., Jr., and J. G. MACKIN.
1971. Oyster parasitism by lAbyrinthomyxa marina in Florida. Fla. Dep. Nat.
Resour. Mar. Res. Lab., Prof. Pap. Ser. 13, 55 p.
RAY, S. M., J. G. MACKJN, and J. L. BOSWELL.
1953. Quantitative measurement of the effect on oysters of disease caused by
Dennocystidium morinum. Bull. Mar. Sci. Gulf Caribb. 3(1):6-33.

Table I-Commercial oyster statistics for Independence Island Reef, 1982-83
season.

Vessel

_I: 1.13'Low ratio may be due to fall oyster drill predation; lease 4 not actually locatedon Independence 1., but near.

25

SONlAT, T. M.
1985. Changes in levels of infection of oysters by Perkinsus marinus, with specialreference to the interaction of temperature and salinity upon parasitism. Nonheast Gulf Sci. 7(2):171-174.
ST. AMANT, L. S.
1957. Growth and mortality rates of Louisiana oysters. La. Dep. Wild!. Fish.,Seventh Bienn. Rep., p. 71-95.
VAN SICKLE, V. P., B. B. BARRETT, T. B. FORD, and L. S. GULICK.
1976. Barataria Basin: salinity changes and oyster distribution. Sea Grant Publ.
LSU-T-76-Q02, Louisiana State Univ., Baton Rouge, 35 p.

26

INTRODUCTION

Seed Oyster Production in
Louisiana and Prospects
for Enhancement_

Despite the many and diverse methods of oyster culture practiced
throughout the world, the success of all methods is dependentprimarily upon consistent availability of low cost, high quality seed.
The Louisiana oyster Crassostrea virginica (Gmelin) fishery has
evolved over the past 150 years from the indiscrirninant harvestingof natural reefs to a regulated, semiculture operation which encourages judicious use of the State's seed resources. This paper
examines the development of the fishery and its sources of seedalong with efforts of the Louisiana Department of Wildlife andFisheries and other government agencies to ensure continued
availability of seed oysters.

MARK CHATRY
Lyle S. St. Amant Laboratory
Louisiana Department of Wildlife and Fisheries
P.O. Box 37
Grand Isle, LA 70358

DEVEWPMENT OF THE FISHERYAND SEED SOURCES_

Coastal Louisiana is characterized by extensive estuarine areas
created over the past 5,000 years by the deltaic processes of theMississippi River. Oyster populations have flourished and declinedin the vicinity of each of the emerging and retreating delta lobes.
While oysters have undoubtedly been exploited in this region sinceprehistoric times, the first commercial operations took place around1800 in the estuaries near the present Mississippi River Delta. In
those early days, oysters were harvested directly from the natural
reefs, usually by the hands of fishermen wading in waist-deep water.
Oyster culture as practiced in Louisiana today had its beginningin the mid-1800's. Immigrant fishermen from Dalmatia, who settled
in fairly large numbers in the Mississippi River Delta, realized thathigh quality oysters could be produced by transferring seed fromthe natural reefs to bedding grounds closer to the Gulf of Mexico.
These higher salinity areas did not support substantial natural populations, due to the inability of recently set oysters to survive the heavy
predation, but were excellent for fattening and growth of transplanted seed. Increased demand in the late 1800's for these superiortransplanted oysters, along with improved harvesting techniques,
namely oyster tongs and the dredge, prompted the fishermen to
recommend State Legislation which was both supportive of their
method of cultivation and protective of the natural reefs as a sourceof seed.
By the turn ofthe century the State's oyster producing areas had
been divided into public seed grounds and private bedding grounds.
The public grounds included the most productive natural reef areas
east of the Mississippi River (Fig. I). The private grounds were
designated as all oyster producing areas in the State, excluding the
public grounds, and were made available for leasing. Leasing procedures were placed under the jurisdiction of the Oyster Commission of Louisiana. Additionally, the Commission was made responsible for supplying a source of seed oysters on the public grounds.
Thus, the basic organization of the fishery had been established.
The State supplies the seed, and the private leaseholders grow theseed to market size. This system has endured over the years withthe fishermen dredging the public seed grounds each fall, loading
their boats with year-old 25-76 cm seed, and transporting and bedding the seed on their leases. An individual fisherman may make
20-25 trips per year from the seed grounds to his leases which may
be over 80 km away. The seed usually remains on the leases for
six to nine months before harvesting, with each boatload of seed
returning one to two boatloads of market oysters. If, in a given
year, there is an overabundance of seed on the public grounds,
market oysters (>76 cm) are dredged on the grounds the following
27

Lomoe u ••_ Leased~ -Public
PSG- Primary Seed Grounds
S R- Seed Reservations
T R- Tonging Reef

Figure t-Oyster growing areas of Louisiana.

year, and are either sold directly or used as seed.
In the mid-1900's the State established five seed oyster reservations, one east and four west of the Mississippi River, to supplement seed production on the public oyster seed grounds (Fig. 1).
The reservations are usually opened on alternative years, as opposed to the public seed grounds which are opened every year.

SEED PRODUCTION ENHANCEMENT

Table I-Oyster harvest in Louisiana, 1940-82 1•_

The Louisiana Oyster Commission, along with its antecessors theConservation Commission and the Seafood Division of the Department of Wildlife and Fisheries, has generally met its responsibility
to produce seed, as evidenced by the consistency of Louisiana oyster
landings over the years (Table 1). This has been accomplishedthrough identification of the factors limiting seed production andimplementation of several combative strategies.
Seed production is limited by extreme high and low salinities
throughout the peak spawning season (June-September) and by lackof suitable substrate for larval settlement. A narrow range of mean
spawning season salinities, 12-17 parts per thousand (ppt), is
associated with successful production of seed (Chatry et al. 1983).
Mean spawning season salinities <12 ppt inhibit gametogenesis andreduce larval survival, resulting in insufficient numbers of matureoyster larvae. At salinities>17 ppt, mature larvae are abundant
but setting and survival of recently set oysters is poor due to increased numbers of fouling organisms and predators.
Salinities on the public seed grounds have been increasing steadily
for the past 50-60 years due to improvements in the Mississippi
River levee system and from channelization, subsidence, and erosion of the coastal marshes. As saltwater intrusion progressed, thenarrow range of salinities associated with successful production ofseed has moved away from historically productive reefs and into
areas lacking suitable substrate for larval settlement. In order tomaintain or increase levels of seed production, the State has created

Year

Harvest (kg] meat)

1940
1950
1960
1961
1962
1963
1964
1965
1966
1967
1968
1969
1970
1971
1972
1973
1974
1975
1976
1977
1978
1979
1980
1981
1982

5,623
3,948
3,765
4,593
4,603
5,238
5,165
3,779
2,158
3,507
5,944
4,158
3,913
4,769
3,989
4,056
4,517
6,196
5,587
4,559
4,377
3,494
3,147
4,119
5,454

-Historical Catch Statistics: u.s.
Fish Wild!. Serv. Bur. of Commer.
Fish., and Nat!. Mar. Fish. Serv.,
NOAA.

new reefs through cultch plants, reestablished old reefs with thecontrolled diversion of freshwater from the Mississippi River, andis currently working with the U.S. Army Corps of Engineers tobuild additional, more efficient, diversions.
28

Cultch Plants

Freshwater diversion

Since 1926, the State has planted over 1 million m3 of cultch
material on public seed grounds and reservations for the purposeof creating new reef areas or increasing production on existing reefs.
Steam plant oyster shell, returned to Louisiana by Mississippi canneries, was the primary material used prior to 1956. In the late1950's and early 60's, reef oyster shell dredged from relict reefs
along the central Louisiana coast was used to supplement steam
plant shell (St. Amant 1959). Clamshell (Rangia cuneata) has been
the preferred cultch material since the mid-1960's due to its availability and because it generally produces well-shaped oysters that
require minimal culling.
Clamshell is dredged hydraulically from vast deposits in Lake
Pontchartrain, loaded onto flush deck barges, and transported tothe seed grounds. The shell is planted using a specially designed"spray barge" with a high-pressure water pump and four to six
nozzles (Fig. 2). Streams of water are directed at the loaded clamshell barge, and the shell is washed overboard in a thin, even layer
for a distance of 9-12 m from the barge. The speed at which thetwo barges are maneuvered determines the planting rate (usually
94-188 m3/ha).
The selection of a cultch plant site depends upon the suitabilityof the bottom and the anticipated salinity conditions. The bottom
must be of sufficient fIrmness and stability to prevent the cultch
material from sinking or being buried, and salinities should be within
the range previously described as optimal for seed production. IdealIy, the shell is planted immediately preceeding a spatfall. If no set
occurs within several weeks of planting, the shell becomes fouled
and therefore less attractive to setting larvae. Due to the protracted
spawning and setting season, however, successful shell plants havebeen made from April to September. Most shell plant failures are
due to inability to predict salinity conditions consistently and hence
predict the production of mature larvae or survival of recently set
oysters.
Successful shell plants in Louisiana have had cost:benefIt ratios
as high as 20: 1 (Schafer 1972). Unfortunately, the cost of clamshell is escalating rapidly. Research is underway to fInd suitable
substitutes for clamshell and to more precisely identify the environmental conditions associated with successful plants.

Despite the State's ambitious cultch planting program, there havebeen partial or total failures of oyster set on the public grounds foreight of the past ten years (Table 2). Most of the failures have been
related to the occurrence of high salinities over the entire seedgrounds. The two years in which the most successful oyster sets
were recorded were marked by openings of the Bonnet Carre
Spillway, a flood control structure of the Mississippi River (Fig.
1), which reduced salinities throughout the seed grounds. In bothcases, the large number of oysters resulting from the successful
set sustained the industry for several years. The Spillway, however,is used only when floodwaters threaten the City of New Orleans
and therefore cannot be relied upon to consistently reduce salinities for the production of seed oysters.

PLANT

Table 2-Seed abundance (25-76 cm
oysters) on Louisiana's primary seedgrounds, 1974-83.
Year

Thousands of Barrels 1

1974
1975
1976
1977
1978
1979
1980
1981
1982
1983

2,436
700
503
341
206
69
2,376
908
360
76

I 1 Barrel = 106 L or approximately 720 seed oysters.

The State has long realized the disastrous effects of saltwater intrusion on the productivity of the seed grounds and also the potential value of freshwater diversion. In 1958 a freshwater diversion
structure was built at Bayou Lamoque to supply Mississippi River
water to a portion of the seed grounds (Fig. 1). The structure has
been highly successful in increasing seed production, but its areaof influence is relatively small due to the limited capacity of thestructure and the circulation pattern of the region which tends tocarry the freshwater away from the seed grounds.
The U. S. Army Corps of Engineers has proposed a comprehensive plan for large-scale controlled freshwater diversion from theMississippi River to the estuarine areas of southeast Louisiana.
Tentative sites for the diversion structures are located near the topof their respective estuarine basins so that the greatest possible area
will be impacted. BenefIts to be realized from diversions include
reduced coastal erosion, preservation of fresh and brackish water
marshes, and general enhancement of fIsh and wildlife resources.
Seed oyster production will increase dramatically due to the establishment of favorable salinities over vast acreages of formerly productive reefs. The magnitude and timing of the diversions will becontrolled to create salinity conditions similar to those which
occurred during years of highly successful oyster set. As a result,seed abundance on the public grounds should consistently equal or
surpass the quantities recorded in 1974 and 1980. This increased
availability of seed should allow for considerable expansion of theoyster industry in Louisiana.

AREA----+

Sh.1l
Bar,.

High P'enur.
Spray
Ba'g_

Figure 2-Arrangement of tugboat, shell barge, and spray barge for the plantingof cultch material.

29

CITATIONSBoth cuItch plants and freshwater diversion, if properly executed,
enhance the production of seed oysters, Cultch plants, however,are merely stopgap measures which will lose their effectiveness as
salinity intrusion progresses. For this reason, freshwater diversion
offers greater hope for the future of the Louisiana oyster industry.

_

CHATRY, M., R. J. DUGAS, and K. A. EASLEY.
1983. Optimum salinity regime for oyster production on Louisiana's state seedgrounds. Contrib. Mar. Sci. 26:81-94.
SCHAFER, H. E.
1972. Louisiana oyster fishery. In Proc., Public Symposium on the Oyster
Fisheries of the Gulf States, October 6, 1972. Gulf Coast Res. Lab., Ocean
Springs, Miss.
ST. AMANT, L. S.
1959. Successful use of reef oyster shells as oyster cultch in Louisiana. Proc.
Natl. Shellfish. Assoc. 49:76-76.

30

INTRODUCTION

Penaeid Shrimp Culture in
the United States:
A Brief Overview Stressing
Species, Seed Production,
and Growout_

Americans consume huge quantities of shrimps annually, especiallythose species belonging to the family Penaeidae. Annual landings
appear to have stabilized around 90 million kg with a dockside valueof $500-600 million. Yet despite this substantial domestic production, an additional $1 billion plus worth of shrimp, mostly penaeids,
is imported each year. Thus, there is great motivation to develop
additional domestic sources of shrimps (Lawrence et al. 1981a, b;
1983a, b). Mariculture is the obvious avenue to pursue, and shrimp
mariculture research can be traced back to the 1950's when Lunz
initiated culture studies at the coastal Bears Bluff Laboratory in South
Carolina (Lunz 1951, 1956, 1958, 1967). Additional penaeid shrimp
growout studies in ponds have been conducted in states bordering
the Gulf of Mexico, including Florida (Caillouet et al. 1976),
Alabama (Tatum and Trimble 1978; Trimble 1980), Louisiana(Broom 1968; de la Bretonne and Avault 1970; Latapie et al. 1972;
Neal and Latapie 1972; Rose et al. 1975), and Texas (Parker 1972;
Parker and Holcomb 1973; Parker et al. 1974; Elam and Green
1974; Chamberlain et al. 1980, 1981; Lawrence et al. 1983c; Davisand Johns 1984; Johns and Davis 1984; Smith 1984). This area
produces the bulk of the nation's penaeid shrimp harvest, andcultural efforts are certainly a natural outgrowth of the region's
commitment to a shrimp fishery and its suitable, although not ideal,
climate for shrimp culture.
While early penaeid growout studies showed much promise, they
were continually plagued by problems associated with obtaining
hatchery-reared rather than wild-caught shrimp seed, with thegrowth responses and survival of native species, and with theabsence of well defined pond feeds. Although the natural life cycleof penaeid shrimps was well understood, it was difficult to produce shrimp seed with consistency when relatively scarce, matedfemale shrimp had to be caught in open waters by trawlers. Further, hatchery procedures were not well established, diseases werea problem, and female shrimp could not be matured and mated in
captivity at that time. A good review of the early problems encountered by shrimp culturists is provided by Hanson and Goodwin (1977).
The U.S. Government initiated studies in the 1960's to solve these
problems. These studies coincided with a significant amount ofresearch and development by a number of major U.S. corporations.
While many problems were overcome, commercial support for adeveloping U. S. shrimp mariculture industry either evaporated or
was transferred to more favorable regions in South and Central
America, where problems such as a seasonal growing period, private
and government resistance to development of wetlands, and highlabor costs could be more readily overcome. There, too, spawning
female shrimp were more readily available and wild seed shrimp
were abundant and could be taken from coastal waters without extensive objections from environmental groups and the existing
shrimp fishing industry. A summary on shrimp culture activities
in Equador, which is producing more shrimp in ponds than any
other country in the world, has recently been reported (Hirono
1983).
Significant strides have been made in overcoming impediments
to shrimp mariculture in the United States, including Hawaii. The
major remaining obstacle to extensive commercial penaeid shrimp
mariculture today is obtaining dependable supplies of viable fertilized eggs from captive shrimp broodstock (Lawrence et al. 1983a,b). However, one private firm is raising shrimp semi-intensively
in 55 hectares of open ponds in south Texas, and private firms aredeveloping 25 hectares of ponds in Hawaii (Anonymous 1983).

ADDISON L. LAWRENCE
Texas A&M University
P. O. Drawer Q
Port Aransas, Texas 78373
JAY V. HUNER
Center for Small Farm ResearchCollege of Agriculture
Southern University
Baton Rouge, Louisiana 70813

31Texas ponds have the capacity for growing 950-2,350 kg of
shrimp/ha in a 6.5-7.5 month growing period during the warm
months, and this shows an acceptable profit. The University of
Arizona is currently developing, with industrial support, intensive
raceway culture systems in Hawaii that are said to be capable ofyielding 5 kg of shrimp/m2 2 1/ 2 times per year. Although expensive, the economics of the system are such that commercial development is now expected in the next year (D. V. Lightner, Environ.
Res. Lab., Univ. Arizona, Tucson, AZ 85706, pers. commun.
1983). A project costing close to $50 million which utilizes thisintensive culture system is being planned by W. R. Grace and F.
H. Prince Companies in Singapore (Anonymous 1984). Finally,
abandoned coastal rice fields are being developed for low-input extensive shrimp mariculture in South Carolina on the southeastern
U.S. Atlantic Coast (T. I. J. Smith, Mar. Resour. Res. Inst.,
Charleston, SC, pers. commun. 1983).
The following sections outline current methodologies involved
in shrimp mariculture in the United States as of late 1983. Topics
include species of importance and life cycles, maturationlreproduction methodology, hatchery/nursery systems, and growout systems.

SPECIES OF IMPORTANCE
AND LIFE CYCLES

aztecus favors mud and is more abundant in the northern Gulf of
Mexico where mud substrates predominate. Life history studies ofthe various species include: P. setiferus (Lindner and Anderson
1956; Lindner and Cook 1970; Chamberlain and Lawrence 1983;
Renfro and Brusher 1982); P. duorarum (Costello and Allen 1970;
Renfro and Brusher 1982); and P. aztecus (Cook and Lindner 1970;
Renfro and Brusher 1982; Chamberlain and Lawrence 1983).
None of the three native penaeids has shown great promise asa culture species in North America; of the three, however, P.
setiferus is generally considered the best performer (Broom 1968;
Latapie et al. 1972; Neal and Latapie 1972; Parker 1972; Parker
and Holcomb 1973; Hysmith and Colura 1976). Because culturists
have obtained less production in ponds with the native species, they
have experimented with a number of exotic species. Two nongrooved species, Penaeus stylirostris and Penaeus vannamei, areespecially favored at this time. Both perform better than indigenous
species (Parker et al. 1974; Conte 1978; Trimble 1980; Ojeda etal. 1980). P. vannamei performs best in pond situations, but seedcannot yet be produced on a completely reliable basis (Chamberlain et al. 1980; Lawrence et al. 1983a). The one commercial operation in Texas had good results with P. vannamei in 1983, while
the Texas A&M University research station is studying both species.
Texas A&M is also evaluating P. monodon, the most utilized shrimp
in pond production in southeast Asia, and two native species, P.
setiferus and P. aztecus. The University of Arizona's Hawaiian work
is emphasizing P. stylirostris.

_

There are three commercially important penaeids in U. S. coastal
waters from the southeastern Atlantic states bordering the Atlantic
Ocean around the Florida peninsula into and throughout the Gulf
of Mexico. These are Penaeus aztecus, or brown shrimp; Penaeus
durorarum, or pink shrimp; and Penaeus setiferus, or white shrimp(Lawrence et al. 1983a). P. aztecus and P. durorarum are bothgrooved shrimps with closed thelyca, i.e., they have a pair of middorsal, lateral grooves extending from the rostrum to the rear of
the carapace. P. setiferus does not have these grooves and is referred to as a non-grooved shrimp with open thelyca.
Spawning takes place in open waters 30-200 m deep. Nauplius
larvae hatch within 12-24 hours. There are five or six nauplius stages
during which no food is consumed. The last nauplius molts into
a protozoea larva after 2-3 days. Protozoea feed primarily on algae(diatoms and phytoflagellates) and secondarily on small zooplankters
during a 3.5-5.0 day period in which they undergo three molts,
the last giving rise to the mysis stage. Mysis larvae feed primarily
on zooplankters, but do consume algae. There are three mysis stages
with the final molt giving rise to the postlarval stage. Postlarvae
feed on animal material and detritus. They are demersal, in contrast to earlier stages that are planktonic, and are identified by the
number of days elapsed since they became postlarvae: PL I , PLs ,PL4Q, etc.
During larval development, the planktonic shrimp are carried into
coastal estuaries by tides and other currents and arrive as postlarvae
or small juveniles. After a period of about 60-100 days, juveniles
reach a size of 5-15 g and begin to migrate from estuaries to open
waters. They are demersal during the day but rise to the surface
at night and move outward on the outgoing tide.
The grooved shrimps typically spawn in late winter and early
spring, with juveniles returning to open water in late spring andinto the summer. The non-grooved shrimps primarily spawn in lateto early summer, and postlarvae enter estuaries as the juvenile
grooved shrimps leave them. Non-grooved shrimps return to open
water during late summer and into the fall. There is always some
gegree of niche overlap, but not enough to prevent coexistence ofall species. P. duorarum tends to favor sandy substrates, while P.

MATURATION/REPRODUCTIONMETHODOWGY_

An abundant, reliable, and inexpensive souce of seedstock is anabsolute necessity for shrimp mariculture. This realization has
historically led farmers toward reduced dependence on wild populations. Originally seedstock (postlarvae and juveniles) were obtained
directly from the wild (Lunz 1951, 1956; Broom 1968; Parker andHolcomb 1973). Subsequently, mature, mated females were captured from the wild and spawned in captivity (Wheeler 1967; Gould
et al. 1973; Hysmith and Colura 1976). At present, shrimp arereared to adult size, then matured and spawned in captivity, completing the life cycle independent of wild populations. This experimental maturation and reproduction technology is now being
evaluated to develop a commercial level of frequency and predictability .
The ovaries of all penaeid shrimps extend dorsally far into theabdomen from the cephalothorax (Heinen 1976; Chamberlain and
Lawrence 1983). Although ovarian maturation is a continual, gradual process, mariculturists have developed a series of maturation
stages to assist in selecting ripe spawners. Female grooved shrimps
have closed thelyca and mate shortly after the molt preceding spawning. They store the sperm, and culturists depend on the ovarianstage to determine if a female will spawn during a particular evening(all shrimp invariably spawn at night). Female non-grooved shrimps
have open thelyca and spawn shortly after mating. Males deposit
spermatophores, and a sperm mass remains in the thelycum after
the spermatophore becomes detached if mating was successful. Normally, only open-thelycum females with advanced ovarian development mate. Culturists examine such females carefully after the early
evening mating period to determine if spermatophores or sperm
masses are present. Development of artificial insemination
technology (see below) is helping greatly to eliminate much of theuncertainty about mating status of female non-grooved shrimps.
32

1977; Santiago 1977; Brown et al. 1979; Kelemec and Smith 1980;
Beard and Wickins 1980; Chamberlain and Lawrence et al. 1981a,b). Chamberlain and Lawrence (1981a) concluded that a combination of animal sources (squid, shrimp, polychaetes, and clams) is
better than a single-food diet. However, some researchers have obtained maturation and reproduction of penaeid shrimp in captivity
using single-food diets, such as fish (Lichatowich et al. 1978),mussels (Laubier-Bonichon and Laubier 1979; Primavera et al.
1978; Lumare 1979), mysid shrimp (Alikuhni et al. 1975; Nurjana and Yang 1976), and squid (Chamberlain and Lawrence 1981a).
Other researchers have fed a combination of prepared dried feeds
and frozen or fresh animals (Aquacop 1975, 1979; Primavera etal. 1978; Emmerson 1980a; Lawrence et al. 1980). We are notaware of any published information indicating that maturation/
reproduction of penaeid shrimp in captivity can be obtained using
only prepared dried feed.
The ability to artificially inseminate shrimp was reported by Persyn (1977), who patented a method for artificial insemination of
open-thelycum Penaeus species. This technique involves mechanically removing the spermatophore, isolating the sperm mass, andplacing the sperm mass in the thelycum area of a gravid female.
Intraspecific-but not interspecific-crosses, using a similar technique, have been successful for several species of Macrobrachium(Sandifer and Smith 1979). Sandifer and Lynn (1981), using similar
techniques, successfully obtained interspecific crosses of two
Penaeus species. These crosses were obtained with male P.
stylirostris and female P. setiferus and with male P. setiferus andmale P. stylirostris. P. setiferus is native to the Gulf of Mexico,and P. stylirostris is native to the Pacific Ocean from Peru to Mexico. Sandifer et al. (1984) obtained successful intraspecific crosses
with P. vannamei and P. stylirostris by artificial insemination. Brayet al. (1982, 1983) not only successfully obtained viable offspringof P. setiferus using artificial insemination, but also increased the
production of nauplii from sourcing cruises by 300 %. This removes
some of the unpredictability of obtaining nauplii of open-thelycum
penaeids from sourcing cruises because only 11 % to 50 % of mature
P. setiferus females are mated (Burkenroad 1939; Eldred 1958).
Using artificial insemination, sourcing cruises could be an economically feasible means of supplementing the production of nauplii
from maturation and reproduction in captivity for supplying seedstock for commercial shrimp mariculture. Also, the successful interspecific crosses provide the basis for initiation of genetic selection
studies. Finally, artificial insemination will help solve the problemsof lack of mating and spermatophore loss in captivity, and provide
the basis for studies concerned with the quality of sperm and ova.
Some initial success also has been obtained on cryopreserving
shrimp nauplii, but not zygotes, protozoea, or mysis (Lawrence
and Baust 1980). Successful cryopreservation of shrimp gametes,
zygotes, or larvae would potentially decrease the cost of seedstock
produced in captivity and their transportation.

Maturation with spawning of marine shrimp in captivity was firstreported by Hudinaga (1942) using the temperate closed-thelycum
shrimp Penaeus japonicus. Moore et al. (1974) reported limited
maturation/reproduction in raceways using the closed-thelycum
shrimp P. californiensis. Duronslet et al. (1975) reported induced
egg growth in the closed-thelycum shrimp Penaeus aztecus, inraceways for both unablated and unlaterally eyestalk-ablated
animals. Maturation without spawning, and spawning without viablezygotes, were reported for P. setiferus and P. stylirostris (openthelycum shrimp), respectively, in O.I-ha hypersaline ponds (Conte
1975; Conte et al. 1977). The first real success in maturation andreproduction in captivity was reported by Aquacop (1975) for P.
merguiensis, P. japonicus, P. aztecus, and Metapenaeus ensis intanks. Several reports indicate that the following species require
unilateral or bilateral eyestalk ablation for ovarian maturation: P.
aztecus (Aquacop 1975), P. duorarum (Idyll 1971; Caillouet 1972),
P. kerathurus (Lumare 1979), P. monodon (Aquacop 1975, 1977a,b; Santiago 1977; Primavera 1978; Primavera and Borlongan 1978),and P. orientalis (Arnstein and Beard 1975). Maturation with spawning of viable zygotes has been achieved with unblated P. japonicus
in tanks using particular light intensities, photoperiods, and nutritional supplementation (Laubier-Bonichon and Laubier 1979;
Laubier-Bonichon 1978; Caubere et al. 1979). Aquacop (1975)
reported less ovarian maturation of P. merguiensis in a tank receiving 40% incident light vs. one receiving 10%. In addition,
Chamberlain and Lawrence (1981 b) reported that optimum lightintensity for P. stylirostris appears to be lower than for P. vannamei, with natural light supplementation beneficially affecting
reproduction of both species. Less stringl'nt photoperiods and lightintensities with diets containing at least some frozen natural foods
have been used to obtain spawning with viable nauplii in tanks forunablated P. japonicus, P. merguiensis, P. monodon, P. setiferus,
P. stylirostris, and P. vannamei (Aquacop 1975; Beard et al. 1977;
Primavera et al. 1978; Brown et al. 1980; Lawrence et al. 1980;
Chamberlain and Lawrence 1981a, b). However, with all of these
species except P. japonicus, unilateral eyestalk ablation enhanced
maturation and spawning with viable nauplii (Aquacop 1975, Beardet al. 1977; Primavera et al. 1978; Brown et al. 1980; Lawrence
et al. 1980; Chamberlain and Lawrence 1981a, b). Furthermore,
species such as P. indicus and P. stylirostris (Mexican strain), whichhave been matured in captivity without ablation (Aquacop 1979;
Emmerson 1980a), are annually ablated to increase spawning rates.
Recently, Chamberlain and Lawrence (1981 b) suggested that malegonadal development may be a limiting factor in reproduction ofmarine shrimp in captivity, as they obtained increased reproduction of P. vannamei by unilateral eyestalk ablation of males. Anotherfactor is deterioration of sperm quality in shrimp held in captivity(Trujillo and Lawrence in press).
Unfortunately, several investigators have obtained data indicatingthat unilaterally eyestalk-ablated, captive-reared shrimp have lower
fecundity and hatching rates than unablated wild shrimp (Nurjana
and Yang 1976; Lumare 1979; Emmerson 1980a; Primavera andPosadas 1980, 1981). Furthermore, ablated penaeids often arelimited to short periods of viable spawning before they must be
replaced with new brookstock (Aquacop 1979; Beard and Wickins
1980).
The nutritional requirements for penaeid shrimp maturation andreproduction are poorly understood, even though nutrition is oneof the most important factors in maturation. Several investigators
use only combinations of animal food sources, such as bivalves,
cephalopods, crustaceans, fish, and polychaetes, for penaeid shrimp
reproduction in captivity (Arnstein and Beard 1975; Beard et al.

HATCHERY/NURSERY SYSTEMS_

There are basically two hatchery systems: the extensive, or Japanese,
method using green water and large tanks; and the intensive, orGalveston, method using clear water and small tanks (Mock and
Neal 1974; Heinen 1976). In the Japanese method, gravid females
are stocked in large tanks where dense algal populations have been
established. These algae provide feed for the protozoeal stage.
Postlarvae can be held in tanks for 10 days or so postmetamorphosis.
Only newly hatched nauplii are stocked in the Galveston method.
33

Gravid females are allowed to spawn individually in relatively small
containers. Protozoea are fed living or frozen algal suspensions produced separately from the hatchery system itself. Postlarvae arenormally transferred at an earlier age, 5-7 days postrnetamorphosis,
than they would be in the Japanese system. Merits and demeritsof these two methods are discussed later; however, the intensive(Galveston) method, in some form, is preferred to the extensive(Japanese) method by shrimp culturists in the United States.
Since the pioneering work of Hudinaga in 1942, considerable progress has been made in the field of larval rearing (Hudinaga andKittaka 1967; Cook 1967; Cook and Murphy 1966, 1969; Mock
1971; Shigueno 1972, 1975; Tabb et al. 1972; Yang 1975; Heinen
1976; Mock et al. 1980a, b). A disturbing feature of current larvalrearing data is the tremendous variability in performance of the larvae, as expressed in terms of survival from the nauplius to the postlarval stage. For example, in some recent reports of rearing attempts
with several penaeid species of commercial interest, survival range
was 5-44 % for Penaeus japonicus (Hudinaga and Miyamura 1962;
Hudinaga and Kittaka 1967; Shigueno 1975), 1-100% for Penaeus
monodon (Liao and Huang 1972; Platon 1978; Beard and Wickins
1980), and 0-77% for Penaeus stylirostris (Mock et al. 1980a, b;
J. S. Wilkenfeld, Texas A&M Univ., Port Aransas, TX, unpubl.
data 1983). The potential problem this variability presents for commercial operations is accentuated by the inability of various researchers to duplicate their own results consistently (Cook andMurphy 1969; Shigueno 1969; Brown 1972; Liao and Huang 1972;
Mock et al. 1980b). The major factors contributing to inconsistent
postlarval production are nauplii quality, water quality, experimental
conditions (physical and environmental), and feeding regimes.
The effect of differences in quality of larvae batches on the resultsof larval rearing efforts has been noted by a number of researchers(Hudinaga 1942; Brown 1972; Wickins 1972; Mock and Neal 1974;
Beard et al. 1977; Cognie and Hirata 1978; Aquacop 1980;
Wilkenfeld et al. 1984; Kuban et al. 1984). These variations may
be due to individual differences between spawning females, whether
the females were obtained from the wild or matured in captivity,
how long the captive female had been in a "production" mode.
the diet fed to maturation stock, and the physical and hormonal
manipulations performed on broodstock animals to bring them toa reproductive state.
A number of methods are suggested in the literature for identifying "good" batches of eggs (Hudinaga and Miyamura 1962; Mockand Neal 1974; Yang 1975; Aquacop 1977a; Platon 1978; Primavera and Posadas 1980) and nauplii (Hudinaga 1942; Yang 1975;
Aquacop 1977a; Cognie and Hirata 1978; Emmerson 1980a; Mocket al. 1980a). However, all these methods are subjective and, thus,
dependent on the experience and interpretaion of the hatchery technician, and less desirable than a concise test which would givehatchery personnel the ability to consistently select nauplii batchesof good, or at least uniform, quality.
Sufficient information is currently available to establish desirable
water quality parameters for penaeid larval culture (Hudinaga 1942;
Cook and Murphy 1966, 1969; Cook 1967; Mock et al. 1974;
Shigueno 1975; Wickins 1976; Lawrence et al. 1981c; Castille andLawrence 1981). Fluctuations of incoming seawater quality can,however, have significant effects on the results of rearing efforts(Cook and Murphy 1966; Shigueno 1969; Mock 1971). This suggests that the selection of a synthetic media mix, at least for use
in small-scale bioassay systems, would significantly enhance thereproducibility of results. A small-scale system (one L volume) using
a synthetic sea salt media has recently been reported (Wilkenfeld
et al. 1983).

Interpretation and comparison of available shrimp research data
are difficult, and sometimes impossible, because of major differences in experimental techniques and systems used by various
research groups (Mock 1971; Zein-Eldin and Meyers 1973; Yang
1975; New 1976; Emmerson and Andrews 1981). Experimental
systems for larval culture recorded in the literature include assorted
glass beakers from 250 to 4000 mL in volume (Hudinaga 1942;
Cook and Murphy 1966; Brown 1972; Gopalakrishnan 1976; Beard
and Wickins 1980), inverted glass carboys of 19 L volume (Cook
1967; Mock and Murphy 1970; Wilkenfeld et al. 1981), medium
size tanks of 50-500 L volume (Cook and Murphy 1966; Beard etal. 1977; Beard and Wickins 1980; Emmerson and Andrews 1981),
and large-scale tanks ranging from 1,000 to 200,000 L (Hudinaga
and Kittaka 1967; Cook and Murphy 1969; Salser and Mock 1974;
Brown 1972; Tabb et al. 1972; Shigueno 1975; Heinen 1976; Mocket al. 1980a, b).
Various feeding regimes are mentioned in many of the papers
dealing with rearing of penaeid shrimp larvae. For the most part,however, the pattern established by Hudinaga (1942) of feeding
protozoea with phytoplankton, and mysis with phytoplankton supplemented with zooplankton, has persisted.
A number of different genera of diatoms and phytoflagellates havebeen used repeatedly with some measure of success, among them
Chaetoceros (Hirata et al. 1975; Yang 1975; Millamena andAujero 1978; Platon 1978; Simon 1978; Jones et al. 1979a),
isochrysis (Millamena and Aujero 1978; Samocha 1980; Wilkenfeld
et al. 1981), Nitzschia (Hudinaga and Miyamura 1962; Liao and
Huang 1972; Wickens 1976), Phaeodactylum (Tabb et al. 1972;
Wickins 1976; Samocha 1980), Skeletonema (Hudinaga 1942;
Hudinaga and Miyamura 1962; Hudinaga and Kittaka 1966; Cookand Murphy 1969; Brown 1972; Liao and Huang 1972; Mockand Neal 1974; Wickins 1976; Mock et al. 1980a), Tetraselmis(Mock and Neal 1974; Beard et al. 1977; Millamena and Aujero
1978; Platon 1978; Wickins and Beard 1978; Mock et al. 1980a;
Samocha 1980; Wilkenfeld et al. 1981) and Thalassiosira (Cook
1967; Cook and Murphy 1969; Brown 1972; Wickins 1976;
Emmerson 1980b).
Despite the large number of studies concerned with algal feeding
regimes, there are few data concerning identification of a single
algal species or combination of species which would be significantly
better than any other for rearing larval penaeid shrimp. Data
presented by Kuban et al. (1984) and Wilkenfeld et al. (1984) suggest that the diatom combination of Skeletonema costatum and
Chaetocerus gracilis is superior to the phytoflagellate combinationof isochrysis sp. and Tetraselmis chuii as a nutritional source forpenaeid larvae (P. stylirostris, P. vannamei, P. setiferus, and P.
aztecus). In another study, Thomas et al. (1976) found that Metapeneaus affinis larvae metomorphosed to the postlarval stage quicker
when fed Thalassiosira sp. rather than Tetraselmis gracilis. Continued studies to identify optimum algal species of species combination and optimum feeding densities will, no doubt, lead to
improved production capacity in hatcheries.
The need for an animal protein source as a component of the mysis
stage diet was first recognized by Hudinaga (1942). Since that time,
a number of live and frozen animal food sources (bivalve eggs andveliger larvae, small zooplankton, penaeid nauplii, ground fish,
rotifers, and brine shrimp nauplii) have been used successfully during the mysis substages (Hudinaga 1942; Cook and Murphy 1969;
Liao and Huang 1972; Kittaka 1975; Yang 1975; Heinen 1976;
Mock et al. 1980a; Wilkenfeld et al. 1981). However, the brine
shrimp Artemia has consistently been the food of choice as an animal
protein source because of its ease of use and ready acceptability
34

taken (Mock and Neal 1974; Heinen 1976). In the Japanese (or extensive) system of larval rearing, mixed phytoplankton and zooplankton blooms are developed in the hatchery tank by fertilization of the seawater prior to metamorphosis of the nauplii to theprotozoea stage (Shigueno 1975; Yang 1975). In the Galveston (orintensive) system, shrimp and feed organisms are cultured separateIy, and feeding is carried out only as needed, depending on residual
food remaining in the tank and the developmental stage of the larvae (Mock and Murphy 1970; Salser and Mock 1974; Mock and
Neal 1974; Wilkenfeld et al. 1984).
Heinen (1976) succinctly described the advantages and disadvantages of each system as follows: "Current indoor (intensive)
methods ... use greater larval densities, require less space and water,and offer more reliability; outdoor (extensive) methods are simpler,offer lower labor costs, require no separate algal culture, and aremuch less dependent on use of Artemia . .. ". Both methods are
dependent on critically timed blooms or hatches, and suffer fromthe potentially catastrophic hazard posed by failed or collapsed foodorganisms cultures (Shigueno 1975; Simon 1978). In addition, theGalveston method of algae culture would not be practical in terms
ofthe manpower and space that would be required to produce sufficient quantities to be fed live in a large-scale commercial facility(Hidu and Ukeles 1962).
Feeding frozen larval foods is an alternative that reduces theburden and risks of dependence on continuous culture of live larval food organisms (Brown 1972). The earliest attempt at feedingfrozen foods was reported by Hudinaga and Kittaka (1966) using
oyster eggs. Results were generally poor, and this was attributed
to either deteriorating water quality as a result of food breakdown
in the tank, or breakdown of the frozen food in storage.
More recent fmdings on the use of frozen foods for larval shrimp
rearing show greater promise (Brown 1972; Mock and Neal 1974;
Salser and Mock 1974; Millamena et al. 1977; Lawrence et al. 1978;
Millamena and Aujero 1978; Baust and Lawrence 1979; 1980a;
Mock et al. 1980a, b; Wilkenfeld et al. 1981). Frozen foods, whenused, have improved the reliability of supply, but in general are
not considered to be as good as the living organisms (Hudinaga
and Kittaka 1966; Kittaka 1975; Heinen 1976; Sorgeloos 1980).
Better cryopreservation techniques for larval foods must be developed in order to overcome the problems of reduced nutritional value
and deteriorating water quality, which can deleteriously affect the
production capabilities of larval rearing facilities (Hudinaga andKittaka 1966; Kittaka 1975; Fluchter 1980; Mock et aI. 1980b; Baustand Lawrence 1980b, c; Wilkenfeld et al. 1981).

by many larval aquatic organisms including shrimp (Reeve 1969;
Tabb et al. 1972; Wickins 1972; Salser and Mock 1974; Kittaka
1975; Yang 1975; Sorgeloos 1979, 1980; Johns et al. 1980; Mocket al. 1980b).
The major drawbacks to the use of Artemia are the high priceand occasional unavailability of the cysts (Hudinaga and Miyamura
1962; Benijuts et al. 1975; Sorgeloos 1979, 1980). There is a needfor less expensive, more readily available, animal protein sourcethat can be used as a partial or complete substitute for Artemia inthe mysis and early postlarval diets.
A number of experiments have already been performed in which
the rotifer Brachionus plicatilis was fed to either the protozoea or
mysis stage of penaeid shrimp with varying degrees of success(Hudinaga and Kittaka 1966; Kittaka 1975; Yang 1975; Platon 1978;
AI-Attar and Ikenoue 1979; Emmerson 1980b; Fontaine and Revera
1980; Mock et al. 1980a). Despite the recognized potential advantage of rotifers as a food organism for larval shrimp (small size,
slow moving, easily digested, potential replacement for Artemia)(AI-Attar and Ikenoue 1979; Fontaine and Revera 1980; Mock et
al. 1980a), there is not yet sufficient information available todemonstrate at what larval substages and at what densities rotifers
can be advantageously fed, and to what extent they can be successfully substituted for Artemia.
The nematode Panagrellus redivivus is another animal protein
source which has recently shown some promise as a potential substitute for Artemia (Fontaine et al. 1981; Wilkenfeld et al. 1984).
In the above reports, P. redivivus were consumed by late protozoeal and mysis larvae, though sufficient survival and growth data
are not yet available to draw firm conclusions about their nutritional value. Preliminary indications are th:lt the lipid content of
P. redivivus compares favorably with that of freshly hatched Artemia
nauplii (about 24% on a dry weight basis) (Benijuts et al. 1975).
Penaeids and other crustacean larvae are known to require lipid
content in their diet (Jones et al. 1979a, b; Emmerson 1980b), andthus research into the potential use of P. redivivus as an animal
protein source is of particular importance.
Recently, several studies have shown that the food value of rotifers
and Artemia can be affected by the nutrient source used to rear thelarval food organism itself (Wickins 1972; Scott and Middleton
1979; Kitajima et al. 1979; Watanabe et al. 1979; AI-Khars et al.
1980). The concept of improving the nutrient value of live larval
foods through media manipulation is of great importance, androtifers in particular, because of their size and method of feeding,are an excellent model organism for testing this approach. A few
experiments have already been performed in which the food valueof Brachonus plicatilis was modified in order to improve performance of fish larvae fed upon them (Kitajima et al. 1979, 1980).
Fontaine and Revera (1980) suggested a similar approach to be
used with rotifers fed to penaeid larvae. They described a simple
technique for causing B. plicatilis to gorge themselves on the desired
food organism, appropriately terming the process "biological microencapsulation". Subsequent biochemical analysis indicated verysignificant nutritional changes (e.g., lipid) in the fed rotifers(Lawrence and Middleton, unpubI.). Further research using rotifers
as carriers for selected food particles should contribute significantly
to our knowledge of the nutritional requirements of larval penaeid
shrimp.
It is clear from the literature cited thus far that current larval
feeding regimes are almost totally dependent on the use of living
foods. These living foods, particularly algae, require highly technical culture procedures.
Two basic approaches for culturing live larval foods have been

GROWOUTSYSTEMS-PONDS_

Many studies have been conducted on pond culture of shrimp. In
the United States, shrimp mariculture research in ponds was initiated by Lunz using Penaeus aztecus, P. duorarum, and P. setiferus(Lunz 1951, 1956, 1958, 1967; Lunz and Bearden 1963). He
demonstrated that ponds in which flooding was accomplished bytidal flow, or which were built on higher land with water suppliedby irrigation pumps, were suitable for growing shrimp for both bait
and food. Using Purina Catfish Chow, Broom (1968) obtained 530
to 909 kg/ha (472 and 809lbs/acre) and food conversion ratios of
2.3:1 and 3.0:1 in ponds for P. aztecus and P. setiferus, respectively. Latapie et al. (1972) and Neal and Latapie (1972), also conducting research in ponds at Grand Terre, Louisiana, obtained 933
kg/ha (831 Ibs/acre) and food conversion ratios as low as 1.1: 1.
Each of these last three papers concluded that P. setiferus grew35

faster and had higher survival than P. aztecus. This was also theconclusion of Parker (1972), Parker and Holcomb (1973), andHysmith and Colura (1976) for P. setiferus and P. aztecus in pondson the upper Texas Gulf Coast. Wheeler (1967, 1968) demonstrated
that the addition of inorganic fertilizer to ponds at Galveston enhanced the growth of P. aztecus by increasing natural productivity.
Quick and Morris (1976) postulated that the increased growth ofbrown shrimp (P. aztecus) in ponds containing dredged materials
was due to greater natural productivity as compared to ponds without
dredged material. More recently P. stylirostris have been grownin ponds with natural productivity enhanced by the mixing of secondarily treated sewage with seawater (Landau et al. 1982).
Lunz and Bearden (1963), Broom (1970), Parker et al. (1972),
Conte (1975), and Rose et al. (1975) concluded that certain fish
were a source of predation and competition for shrimp and should
be removed from shrimp ponds. Gould et al. (1973) obtained production as great as 596 kg/ha using the brown shrimp P. aztecus,
and concluded that failure to control predation and competition
would markedly reduce yields in ponds. In 1974, Parker et al. produced 1,234 kg/ha of 61 count (heads-Qn) P. vannamei using a 3-unit(nursery, intermediate, and growout) pond system. These shrimp
had a value of more than $5,500/ha. They also reported that P.
vannamei was better in terms of pond production than P. setiferus,
which in turn was better than P. aztecus and P. occidentalis. Elamand Green (1974) produced 224 and 667.5 kg/ha with food conversion rates of 1.8 and 2.3, respectively, using P. setiferus in ponds
located in the middle Texas coast. More recently, more than 2,200
kg/ha of 41 count (heads-on) of P. vannamei were obtained in single
pond units in four months (Chamberlain et al. 1981). These shrimp
had a value of over $4,500/ha using ex-vessel prices. Investigators
also have reported that polyculture of such diverse species as fishand shrimp increases the productivity of ponds (Gundermann andPopper 1977; Liao and Huang 1972; Rossberg and Strawn 1980;
Tatum and Trimble 1978; Trimble 1980). Furthermore, George(1975) observed that Metapenaeus dobsoni and P. indicus performed
better than M. monoceros and M. affinis in controlled polyculture
experiments. The polyculture of two very closely related penaeid
species, P. vannamei and P. stylirostris, in ponds resulted in an
increase in gross revenue over that of either species when monocultured (Chamberlain et al. 1981). The staggered stocking concept was tested by Ojeda et al. (1980), and their results indicatedthat ponds stocked in three increments over time had better survival and gave higher yields than those expected from single-stocked
ponds at the same density.
Food is a major cost item in the operation of a shrimp farm (Parker
and Hayenga 1979; Adams et al. 1980; Farmer 1981; Pardy et al.
1983). Although "suitable feeds" are available for pond culture,
there is considerable room for improvement. Marine shrimp are
omnivores and detrital feeders (Williams 1955; Dall1968; Caillouet
et al. 1972, 1976; Cuzon et al. 1974; Venkatararniah et al. 1975,
1978; Moriarty 1976, 1977; Jacob et al. 1980) and with the helpof bacteria, even utilize dissolved organic matter (Lawrence et al.
1975; Castille and Lawrence 1979). In contrast, Marter (1980) concluded that P. monodon is more of a predator for slow-moving benthic macroinvertebrates than a scavenger of detritivore. However,there is no doubt that natural productivity in ponds is of considerable
nutritional importance to shrimp. In fact, after analysis of stomach
contents of Metapenaeus macleayi, Maguire and Bell (1981) concluded that 42 % of the shrimp diet came from natural productivity
even though the shrimp were being fed 5 % of their biomass eachday with a formulated feed. Also indicating the importance of natural
productivity to shrimp in ponds, Latapie et al. (1972) and Rubrightet al. (1981) obtained 47 to 300 kg/ha and 245 kg/ha, respectively,in ponds not receiving any fertilizer or feed. By adding organic
fertilizer to ponds Caillouet et al. (1972, 1976) reported 180 to 401
kg of shrimp/ha. Quick and Morris (1976) and Rubright et al. (1981)
obtained 250 kg/ha and 375 to 590 kg/ha of shrimp, respectively,by adding inorganic fertilizer to ponds. The addition of fertilizer
increases natural productivity (Stahl 1979; Boyd et al. 1981; Geiger
1981; Rubright et al. 1981) resulting in increased production of
the cultured animal (e.g., shrimp).
Preliminary economic analysis of pond production for shrimp
where the growing season is less than one year (e.g., Texas) has
indicated that greater than 600 kg/ha per crop are needed to support a commercial operation (Parker and Hayenga 1979; Adams
et al. 1980; Griffin et al. 1981; Pardy et al. 1983). Natural productivity is not adequate to support the stocking densities necessary
to consistently achieve production rates greater than 600 kg/ha.
Broom (1970) stated that shrimp production in ponds not fertilized
and fed seldom exceeds 224 kg/ha and that yields are much higher
in ponds receiving feed. Therefore, in most studies of shrimp pond
production, fertilizer and formulated feed are added to the ponds(Neal and Latapie 1972; Gould et al. 1973; Parker et al. 1974;
Hysmith and Colura 1976; Chamberlain et al. 1981;~aguire andBell 1981; Rubright et al. 1981; Maguire and Hume 1982).
Surprisingly, very few studies are concerned with developing anadequate feed for pond production of shrimp. Broom (1972) concluded that shrimp fed 26-30%, as compared with 60%, dietary
protein grew well in ponds. Parker and Holcomb (1973) evaluatedshrimp production in ponds for two feeds, each containing 50%
protein; in one feed, 60% of the protein was derived from fish meal;in the other, only 20 % was from fish meal. They also tested a 35 %protein diet prepared by Ralston Purina. No differences in production were reported for these different diets. Aquacop (1977b) foundthat a diet of approximately 40 % protein gave excellent growth forP. monodon (3-25 g in 133 days). SEAFDEC tested this same dietand obtained 25 g P. monodon from 3 g animals in 7 months (Cookand Rabinal 1978). Excellent results have also been obtained witha 36.58 % protein feed made in Taiwan by the President Company(Cook and Rabinal1978).1n the study by Chamberlain et al. (1981),
more than 2,200 kg/ha were produced using a Ralston Purina feed
which was analyzed to contain approximately 20% protein. A 25%
protein commercial feed from Texas Farm Products tested with P.
vannamei and P. setiferus produced growth of 1-2 g per animal
per week in ponds (Lawrence and Johns, unpubl.). Maguire andHume (1982) obtained data suggesting that a 27% protein feed issatisfactory for shrimp in ponds.

INTENSIVE GROWOUT SYSTEMS_Intensive growout systems concentrate shrimp in a small area. Circulation and aeration are increased to remove wastes and oxygenate
the water. Both open (once-through) and closed (recirculation)systems have been developed. While these systems have been
biologically successful, none has been commercially successful todate. However, the open system developed originally by the Universities of Arizona and Sonora (Mexico), with industrial sponsorship,
is said to be perfected to the point that commercial development
will soon proceed at a Hawaiian site (D. V. Lightner, Environ.
Res. Lab., Univ. Arizona, Tucson, AZ 85706, pers. commun.
1983).
A brief description of the Arizona/Sonora system is in order.
Growout units are called" aquacells," with representative dimen36

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sions of 3.35 x 61 x 0.3 m. Each unit typically contains two longraceways covered with air-inflated plastic domes. Sea water fromwells is delivered through side manifolds and is turned over seventimes a day. Water remains clear and shrimp remain visible.
The hatchery phase of this intensive system takes roughly 20 days
(Galveston method), the post-hatchery nursery phase some 18
weeks, and the final growout phase 21 weeks. After this 220-230
day period, shrimp are harvested at a large size of21 g. Final carrying capacity is 5.6 kg/m2 • Based on 1980 data, 18 growout units
could produce 3,632 kg of shrimp per month. Pertinent references
include Mahler et al. (1974) and Salser et al. (1978).

CONCLUSIONS_

Penaeid shrimps are high value, glamorous crustaceans. They arebeing cultured commercially in Central and South America, Southeast Asia, and Japan, where climatic, biologic, and economic conditions, and availability of excellent native shrimp for stocking
ponds, favored development of shrimp culture before the necessary
technology was perfected in the United States. However, it appears
that where suitable sites are available, penaeid shrimps can becultured profitably in extensive semi-intensive and intensive pond
systems, and intensive raceway systems. We are optimistic that continued demand and high prices for penaeid shrimps, with continued
development of technology, should lead to the development of acommercial shrimp mariculture industry in the United States.

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1981. Studies on the egg quality of PelUleus monodon Fabricius, based on morphology and hatching rates. Aquaculture 22:269-277.
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1976. Experimental extensive culture of penaeid shrimp in a simulated dredg<XI
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REEVE, M. R.
1969. The laboratory culture of the prawn PaLaemon serratus. Fish. Invest.
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RENFRO, W. C., and H. A. BRUSHER.
1982. Seasonal abundance, size distribution, and spawning of three shrimps(Penaeus azlecus, P. setiferus, and P. duorarum in the northwestern Gulf of
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1975. Extensive culture of penaeid shrimp in Louisiana salt-marsh impoundments. Trans. Am. Fish. Soc. 104:246-307.
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1980. Comparative growth and survival of brown shrimp cultured with Florida
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41

INTRODUCTION

Development of Larval
Rearing Systems for
the Malaysian Prawn
Macrobrachium rosenbergii
in Southern Louisiana_

Experimental culture of the Malaysian prawn Macrobrachium rosenbergii has been conducted in Louisiana during the past 6 years by
the Louisiana Agricultural Experiment Station (LAES) and the Louisiana Department of Wildlife and Fisheries. At least 120 experimental ponds have been stocked with prawns since 1979 (Table I).
Pond culture experiments have involved studies with prawn feeds(Perry and Tarver 1981, in press), stocking sizes (Pavel et al. 1985),
stocking densities (Cange et a1. 1983), polyculture with finfish(Huner et al. 1980; Miltner et al. 1983), and rotation with crawfish, Procambarus clarkii and Procambarus acutus acutus (Cangeet al. in press). These studies have shown significant increases inyield with supplemental feeding and increased stocking size; an inverse linear relationship between stocking density and prawn sizeat harvest; compatible polyculture with catfish (letalurus punctatus) ,tilapia (Oreochromis aureas), chinese carps (Hypophthalmichthys
molitrix and Cetenopharyngodon idella), and golden shiners (Notemigonus crysoleucas); and successful rotation with crawfish. Results
indicate good pond culture potential for prawns in southern Louisiana. However, the availability and cost of seed stock for pond
stocking has greatly restricted development of pond culture efforts.
Postlarval prawns utilized for our initial growout studies werepurchased from commercial hatcheries in Hawaii and Florida.
Hatchery prices ranged from 2 to 3C/postlarva plus air freight toLouisiana (total cost 4-5C/postlarva). We had no control over thecondition or quality of purchased postlarvae. Typically, many postlarvae were dead, injured, or under stress after hours in air transit.
Moreover, postlarvae had to endure a lengthy automobile ride froman airport to the culture facilities. A closer and more economicalsource of prawn seed stock was needed to maximize research anddevelopment efforts. This paper reviews hatchery systems and techniques developed for production of postlarval prawns in Louisiana.

STEPHEN W. CANGEDAVID L. PAVELMARK S. LAMONJAMES W. AVAULT, JR.
School of Forestry, Wildlife, and Fisheries
Louisiana Agricultural Experiment Station
Louisiana State University Agricultural Center
Baton Rouge, Louisiana 70803

ABSTRACT
Prawn (MQ£TObrlU:hium rosenbergil) culture research has been conducted in Louisiana during the past six years by the Louisiana Agricultural Experiment Station(LAES) and the Louisiana Department of Wildlife and Fisheries. Most of theresearch thus far has been conducted in replicated earthen ponds, where investigators have compared prawn feeds, stocking sizes, stocking densities, polyculture strategies, and rotation schemes. However, high costs and difficulty inobtaining postlarval prawns for pond stocking have led to recent developmentof larval rearing systems at the LAES. Rearing system designs and initial results
are discussed in this paper.

PILOT SCALE HATCHERY_

A pilot-scale prawn hatchery was constructed in 1982 at LAES's
Ben Hur Farm, Baton Rouge, LA. This simple recirculating system
consisted of three 1,600-L cylindrical fiberglass rearing tanks, one
1,400-L biofilter trough, and an elevated 1,600-L fiberglass brackish
water mixing tank (Fig. 1). Brackish water (120f0o) was preparedin the mixing tank and flowed by gravity into the rearing system.
Water was recirculated continuously through the bioftlter by a l/8-hp
magnetic-drive pump at a rate of 70 Llminute (approximately one
complete rearing system turnover per hour). The filter trough contained a 30-cm layer of biodek' covered with a 15-cm layer of clamshell (Rangia sp.). A rotating biodisc filter mounted atop the filter
trough turned continuously at a rate of 6 rpm (Fig. 2). A 1.5-kw
cable heater (range 1-100°C) located in the bottom of the filter tankmaintained water temperature at 28-30°C. Each rearing tank wasalso equipped with two 200-watt submersible heaters.
A 212-llm screened PVC pipe (20-cm diameter X 9O-cm length)
was placed around the drain pipe in each rearing tank to retain larvae (Fig. 3). Air pumped through polyethylene air rings attachedto the base of each pipe reduced screen clogging. Each rearing tankalso contained four to six 7.5 X 3.75 X 3.75 cm glass-bonded air
diffusers which aerated the water continuously. A 1.5-hp Condecarbon-vane air compressor supplied continuous aeration. A stainless'Laminated sheets of corrugated black polyethylene utilized commercially for treatment of municipal and industrial wastewater.

43

Table I.-Summary of prawn research in freshwater ponds at Ben Hur Research Area (LAES) and in brackish
water ponds at Rockefeller Wildlife Refuge, 1979-84.

StudylocationYear

Stockedponds(n) (ha)Prawn
stocking
density(per halPrawn
stocking
size(g)Growout
period(d)Supplemental
feed

Averageharvestweight(g)

Survival(%)

Yield(kg/ha)

LAES

6

0.048

25,000

0.01

140

Yes

32

57

442

Rockefeller

3
3
3

0.04
0.04
0.04

12,50025,00037,500

0.01
0.01
0.01

163
163
163

No
No
No

15
11
12

82
77
61

159
191
218

LAES

6 0.048
3 0.048

25,00050,000

0.01
0.01

144
144

Yes
Yes

37
24

16
22

122
266

Rockefeller

2
3
2

0.04
0.04
0.04

25,00049.00074,000

0.01
0.01
0.01

140
140
140

Yes
Yes
Yes

21
17
12

80
72
56

408
619
510

1981

Rockefeller

4
3
4
4

0.04
0.04
0.04
0.04

12,50025,00050,00075,000

0.01
0.01
0.01
0.01

146
146
146
146

Yes
Yes
Yes
Yes

34
23
21
19

67
64
59
51

266
353
607
644

1982

LAES

18 O.04b

2,500

0.5-1

107

Yes

71

96

170

Rockefeller

6
6

0.04
0.04

44,00044,000

0.01
0.01

122
122

Yes
Yes

20
24

74
73

578
704

LAES

I
9
9
5

0.68
0.04 8
0.048
0.12'

6,875
5,000
5,000
5,000

0.25
0.01
0.2
0.01

122
106
106
92

No
Yes
Yes
Yes

41
31
39
25

92

256

89
82
86

119

Rockefeller

5
5

0.04
0.04

44,00044,000

0.01
0.01

141
141

Yes
Yes

17
20

76
84

588
733

LAES

4
4
4

0.04
0.04
0.04

4,942
9,884
14,826

0.08
0.08
0.08

106
106
106

Yes
Yes
Yes

54
46
34

60
75
46

115
234
256

1979

1980

1983

1984

stocked with catfish
bstocked with catfish and chinese carps'stocked with blue tilapia
8

top view

D

A

F

1600 L rearing tanks

Standpipe/drain

B Filter troughG

Screened PVC pipe

C Saltwater mixing tank

H

Gearmotor

D

PVC supply line

E

PVC return line

Bio-disc filterJ

1/8 hp pump

I
,.,

n

~

_~I·-.. -

T

I

•

•

•

•

rr\I

I

side view\lIUUL'\ilil!

Figure 1-Initial recirculating system constructed at Ben Hur Farm for rearing larval Malaysian
prawns (Maerobraehium rosenbergii) in South Louisiana, 1982.

44

Rotating Fiberglass DiscsI

Steel~~~
Shaft'd~in

Figure 2-Detail of filter trough used in larval prawn rearing system.

A"·--'1'~"·"·iiii~"·iZill!·""·="""~"""·~"- 8.";===:.~~~._.

''';!.

A Air inlet-=+---c

B Band clamp-,+--()<: 20-cm PVC pipeE*-+--F----G[)

5-cm PVC drainE212-~m screenF

Air supply line (PVC)
to bot tom rings(;

Inside PVC support ringH

5-cm PVC couple
Inner air ringH
T.:lL_-1

J

Outer air ringK

5-cm PVC male adapterL

Air ring connector

M

Support ring air holeJlK

Side View

J8
E

1flG9>~::-Yfr-

A..

cl

D
I
M
Top View

Figure 3-Detail of screened standpipe used to retain larval prawns in culture tanks.

45

steel "in-line" filter vessel containing a 5-llm polypropylene tilterbag removed carbon dust from the air lines.
Brine shrimp (Artemia salina) were hatched in six 58-L conebottom polyethylene tanks (Fig. 4). Three 75-watt lamps suspended
above the hatching tanks supplied constant lighting. Each tank wasaerated with an air diffuser and heated to 26-30°C by a 2oo-watt
submersible heater.
Brine shrimp hatching water was prepared by adding 10 giL NaCI
or synthetic sea salts to Baton Rouge city water. Cysts were notdecapsulated prior to hatching. Approximately 150 mL of cysts wereadded to 45-L of prepared water in each tank. Brine shrimp were
subsequently harvested 24 and 30 hours later. The harvest procedureentailed removal of the airstone and disconnection of the heater ineach tank. After 15 to 20 minutes, the contents of each tank separated
into distinct layers. Hatched cysts floated, while unhatched cysts
sank to the bottom. Newly hatched brine shrimp formed a dense
layer immediately above the unhatched cysts and were harvested
through the drain pipe.
Artificial lighting in the hatchery room was supplied by fluorescent lights and followed an approximate 12: 12 photoperiod. Threewindows on the north wall also supplied natural lighting.

LARVAL PRODUCTION

Larval production cycles were initiated by stocking 3-6 gravid
female prawns (30-50 g each) with gray-colored eggs into each1,600-L rearing tank. Hatching occurred 24-72 hours later, andall adult prawns were removed from tanks 72 hours after stocking.
Hatching resulted in an initial density of approximately 30-60 prawn
larvae/L.
Brine shrimp were fed to larval prawns (~ stage II) twice per
day in proportion to the volume of water in each tank. Brine shrimp
densities were gradually reduced from lQ-15/mL initially, to 1-2/mL
immediately prior to prawn metamorphosis. The amount of brine
shrimp utilized to maintain these concentrations was not quantified,but depended upon both prawn consumption and brine shrimp mortality in prawn culture tanks.
Initially (larval stages II-III), heavy feeding of brine shrimp(12-15/mL) caused a rapid formation of an "orange deposit" ontank bottoms. Consequently, siphoning of tanks was extremely important during this period to maintain water quality. As the rearing
cycle progressed, brine shrimp stocking density decreased, while
prawn feeding activity increased. Typically, brine shrimp stocked
at 5/mL in the evening were reduced to 1-2/mL by morning, withlittle evidence of an orange deposit on the tank bottoms.
Steamed chicken egg was fed to prawn larvae beginning at approximately stage N. Egg mix was prepared daily by mixing
powdered eggs, water, and powdered protein hydrolysate, thensteaming it until a firm consistency was obtained. The steamed mix
was washed through a sieve series to obtain the proper particle size
for larval consumption (Table 2). Egg particles were then collected
in a beaker, covered with water, and refrigerated until needed (maximum 3 days). The feeding frequency and amount of egg mix fed
to larvae were determined visually. Feeding frequency depended
upon larval consumption, and ranged from once/day initially (stageN) to four times/day prior to metamorphosis (stage XI). The amount
fed was proportional to the number of prawn larvae present in each_

The recirculating saltwater system was conditioned by stocking each
culture tank with a few tilapia or brood prawns. After about 30
days, the recirculation water pump was turned off and all animals
were removed. Water was drained completely from all culture tanks.
Each culture tank was thoroughly cleaned and refIlled with newly
mixed brackish water (120100). The water pump was then turnedon and the system allowed to recirculate 1-2 days. Water temperature was stabilized at 28°C.

~A

jliS;~~:C~~~C;:;;;::E=;:;;:~;;;;;=C;:;~-air,.,::, ..~
...
., ,
;: :
.

......

~

7j1l

"

.

"
"

;: t

'.' ,
,"

.. .

"

"(j
"
~~

.

~;

.

·~-.~":r'li·,<01>"'])'-' .~. :.•-: .• :~."'-~.
• • • ',

,

F'N::
.. S

E-7-: :...... :0.

4

..

.....

\0-"'

1~:.':'.~ •. ;~-",~.~_·';l. ~ ~.

GA

75-watt lamps

EStandpipe/drain

B

58 L polyethylene tanks

F

AirstoneC

Harvest valve

G

Wood support rack

D

Drain troughFigure 4-Tank system constructed to hatch brine shrimp (Artemia salina).

46

~;.

tion process is provided by two ultraviolet sterilizers, each containing two 6O-watt UV lights. Bacteria and other pathogenic organisms
are killed as the culture water flows through each unit. Filtered wateris pumped back to the culture tanks by a V2-HP magnetic drive pumpat a maximum rate of 175 LPM. Water flow into each culture tankis regulated by a 12-cm PVC ball valve and quantified by a variable
area flowmeter (range 1.8-18 LPM).
Each rearing tank is initially equipped with a 20-cm PVC pipe
frame covered with 21O-1lffi nitex screening to retain stage I-V prawn
larvae. However, as larval size increases (stages VI-XI) and brine
shrimp densities decrease (12/mL to lImL), a smaller lO-cm PVC
pipe frame covered with a larger 280-llm screening is used. One
7.5 x 3.75 x 3.75 cm glass-bonded air diffuser is placed undereach standpipe at the bottom of the tank cone to prevent settlingof animals and debris. Air flow into each culture tank is regulated
by a 12-cm PVC ball valve and quantified by a variable area
flowmeter (range 0-15 SCFM).

Table 2-Sieve series (USA standard) used to
produce food particle sizes for larval Malaysian prawns (Macrobrachium rosenbergil).
Larval Stage

Sieve
position

Ill-IV

V-VI

VII-IX

X-PL

Top
Intermediate
Intermediate
Bottom

1140
1120
1130
1160

1130
1120

1120
1116

1116
1114

1140

1130

1120

tank, and not to the water volume of each tank (as with brine
shrimp). Aeration was turned off prior to each feeding. Initially(stages IV to V), small amounts of steamed egg were spread byhand over the water surface in each tank to provide approximately
1 egg particle/I-2Iarvae. Larvae were relatively slow to accept egg
particles during this phase. As development advanced (stages V to
XI), egg was added until about 70-90% of the prawn larvae wereobserved holding particles within 1-2 minutes. This usually resulted
in a ratio of 1-2 egg particles/larvae. Extreme caution was taken
not to overfeed. Excess feed not only polluted the water, but also
clogged standpipe screens and caused tank overflows. Standpipe
screens were cleaned two to three times daily with a long-handled,soft-bristle brush, and bottoms were cleaned once daily with a siphon
hose.
Approximately 100,000 postlarval prawns were produced during
1983-84 utilizing these materials and methods. Final tank production rates ranged from 5 to 15 postlarvae/L. Complete mortality
was not experienced in any larval culture tank, and relatively low
production rates were attributed to low initial stocking densities.

NEW HATCHERY SYSTEMLARVAL PRODUCTION_

Stocking of larval culture tanks was initiated in January 1985. Twoto twelve gravid female prawns were placed directly into each
culture tank. All females were removed from the tanks 48-72 hours
later. Larvae were left in the tanks where they had hatched. Initial
larval densities were estimated in each tank by taking three to fourlOO-mL samples with a plastic tube while aerating the water
vigorously. The number of larvae counted in each sample was
extrapolated to a liter basis and then multiplied by the tank volume
to estimate total tank population. Larval densities in each tank were
estimated daily using the plastic tube sampler until about day 15,
and then by using a 500-mL beaker until tank harvest. At least fivelarvae from each tank were staged daily by microscopic examination. This provided a daily index for larval condition, growth, anddevelopment.
Brine shrimp were fed daily to prawn larvae until about day 25.
Brine shrimp densities in culture tanks were relatively low throughout the cycle due to limited hatching tank volume, and ranged from
2 to lO/mL. A mixture of chicken eggs, squid, and protein supplements was steamed, sieved, and fed daily to larvae beginning atstage III. The frequency and amount of egg mix fed to larvae were
determined visually, but generally increased with larval development. Feeding procedures closely followed those outlined earlier
for the previous hatchery system.
All tanks were harvested after 36-55 days by removing the standpipe in each tank and flushing all postlarvae into a screened bucket.
Harvest was delayed in several tanks to facilitate conditioning ofjuvenile nursery tanks. Tank populations at harvest were estimatedon a weight basis.
The production results from the first larval cycle are presented
in Table 3. After an average of 46 days, production averaged II
postlarvae/L. Postlarvae averaged 0.01 g each. The number of days
necessary for ~50% of the population in each tank to reach a particular larval stage are presented in Table 4. An average of 3 days
occurred between larval stages. Earlier stages were generally passed
through more quickly than later stages. The daily population density estimates from each tank are presented in Figure 7. Larval mortality rates were high through approximately day 15-17, but
decreased considerably thereafter.
We believe the low production average was due to the high initial mortalities experienced in all tanks. These high mortalities may
have been caused by low brine shrimp densities during the early_

Construction of a new 135-m2 hatchery research building wascompleted at the Ben Hur Research Area in the Fall of 1984. Thisfacility contains eight separate recirculating systems consisting ofover 96 culture tanks ranging in size from 55 to 450 L. Each researchsystem utilizes recirculating water pumped through a bioftlter andultraviolet sterilizer. The largest recirculating system is used forlarval prawn rearing and consists of twelve 400-L conical fiberglass culture tanks (Fig. 5). This system was designed to maximize
larval culture efficiency, while providing increased experimental
control and flexibility.
Brackish water is prepared in an elevated 1,600-L fiberglass tankby combining Baton Rouge city water with synthetic seasalts andtrace elements. The mixture is aerated vigorously for at least 24
hours, heated to 28°C, and then flows by gravity into the culture
system. Each 400-L culture tank has the capability of utilizing"flow-through" city or pond water independent of other tanks utilizing recirculating water.
Culture water is filtered by continuous recirculation throughbiological, mechanical, and ultraviolet mters (Fig. 6). Two 2,OOO-L
square polypropylene tanks serve as biomters. Surface area for
denitrifying bacteria is provided in the bottom of each ftlter tankby a 30-cm layer of biodek below a 30-cm layer of clam shell(Rangia sp.). In addition, each mter tank is equipped with a 1.5-kw
immersion tube heater to maintain water temperature at 28°C. Afterleaving the biofilters, rearing water is pumped through a particle
ftlter unit to remove suspended, colloidal, and dissolved solids. This
unit contains 9.3 m2 of 5-llm polypropylene ftlter mesh around a
3.6-kg canister of activated carbon. The final step in the water ftltra47

nm

a - 1900 liter salt-- Weighted-keel
air tubing(outer ring)water mixing tankb - 2000 liter bio-

- Weighted-keel
air tubing(inner ring)filter tanks

C - 450 liter conebottom culturetanks

k- Screened PVC

d - ~HP magnetic.-- Standpipe(drain)pipe (209 or
265 UIl1 mesh)drive water pumpnme - ~p magneticdrive water pumps

f - Particle filter
unit

g- I.S KW tube

m - filtered pond
water

n

UV - Ultraviolet
5 teril izers

heatermnh- Water flowmeters

Dechlorinatedcity water~- Three-way PVC

ball valve(]) - 1\.Io-way PVC

ball valve

Figure 5-Newly constructed recirculating system for rearing larval Malaysian prawns (Mtu:robnu:hiumrosenbergii) at Ben Hur Farm, Baton Rouge, Louisiana.

A

A Biological filter tanks
II Filter cartridge
C Ultraviolet sterilizer

D Clam shellin-_""'-_

E Biodek

F Filter media support
G Particle filter

H
out-

G

. . . . . ....,H Activated carbon
Ultraviolet lights

Figure 6-Detail of water filtration components of larval prawn rearing system.

48

Table 3-Production data from the initial larval prawn rearing cycle at the
new LAES hatchery in January 1985.
Days tofirst P.L.
(no.)

Days toharvest(no.)

P. L. density
at harvest(no.lL)

Avg. P.L.
wet weight
(g)

50
47
40
49
36
44
55
45
50
48
44
44

8.4
5.7
27.6
13.1
12.5
16.7
6.0
13.5
11.2
8.0
0.8
8.7

0.019
0.010
0.007
0.01 I
0.007
0.010
0.022
0.011
0.012
0.010

12

110
140
155

25
25
22
27
19
25
27
24
28
24
25
23

Average

141 ±98

24±2

46±5

11.0±6.7

0.010±0.005

Tank
no.
I
2
3
4
5
6
7
8
9
10
II

Initial larval
density'(no.lL)
37
290
127
50
98
66
333
2

350

300

250

:00

'50

0.009

(i± ISO)'00'Due to eggs hatching over a several day period, values represent highest density recorded from each tank.
2Values deleted because larvae from other tanks were added during the cycle.
50

Table 4-Number of days necessary
for ;'50% of prawn larvae to achievestages of development at the new
LAES hatchery in January, 1985.
Stage

10

15

20

25

30

35

40

45

50

55

DAYS POST-HATCH

Day(i ± ISO)

Figure 7-Daily estimates of larval prawn densities in ten culture tanks at Ben
Hur Farm in January 1985.

I

11
III
IV
V
VI

VII
Vlll
IX
X
XI
PL

3.1 ± 0.8
5.6 ± 1.3
8.3 ± 1.9
10.4 ± 1.9
13.1 ± 1.8
16.1 ± 2.3
21.1 ± 2.8
23.7 ± 3.5
25.2 ± 5.0
28.8 ± 4.0
33.7 ± 3.2

CITATIONS_CANGE, S. W., J. W. AVAULT, and W. G. PERRY.
1983. Macrobrachium rosenbergii culture in southwest Louisiana in brackish
water ponds. In Proc., 1st 1m. Conf. Warmwater Aquacult., p. 148-156.
Brigham Young Univ.
CANGE, S. W., O. PAVEL, and J. W. AVAULT.
In press. Pilot study on prawn/catfish polyculture with rice/crayfish rotation.
In Proc., 7th Int. Crayfish Symp., Lund, Sweden.
HUNER, J. V., W. G. PERRY, Jr., M. MILTNER, R. A. BEAN, and J. W.
AVAULT, Jr.
1980. A preliminary study into the polyculture of giant Malaysian prawns,
Macrobrachium rosenbergii, and channel catfish, Ietalurns punctatus, in Louisiana. Proc. La. Acad. Sci. 54:95-103.
MILTNER, M. R., A. GRANADOS, R. ROMAIRE, J. W. AVAULT, Jr., Z.
RA'ANAN, and O. COHEN.
1983. Polyculture of the prawn, Macrobrachium rosenbergii with fingerling andadult catfish. Ietalurns punctatus, and Chinese carps Hypopthalmichthys molitm,
and Ctenopharyngodon idella, in earthen ponds in South Louisiana. J. World
Maricult. Soc. 14: 127-134.
PAVEL, O. L., J. W. AVAULT, Jr., and S. W. CANGE.
1985. Polyculture of channel catfish, Ietalurns punetarus (Rafinesque), with postlarval and juvenile prawns, Macrobrachium rosenbergii (DeMan). In Proc.,
16th Annu. Meet. World Maricult. Soc., Jan. 13-17, 1985, Orlando, Fla.
PERRY, W. G., and J. TARVER.
1981. Malaysian prawn culture in brackish ponds in Louisiana. J. World
Maricult. Soc. 12(2):214-222.
In press. Production trials of prawn comparing a marine ralion, catfish diet andagricultural range pellet. In Proc., 38th Annu. Meet. S.E. Assoc. Fish Wildl.
Agencies, New Orleans, La.

stages of growth. In clear water systems, brine shrimp are usuallystocked initially at 10-15 brine shrimp/mL. However, we were only
able to supply approximately 2-10 brine shrimp/mL during the earlystages. Moreover, the delayed harvest of several tanks resulted inexcessive mortality of postlarvae. Water quality parameters were
within acceptable limits for larval prawn culture. Salinity ranged
from 12 to 13%0; temperature averaged 28.7 ± 1.3°C; pH
averaged 7.8; and ammonia concentration ranged from 0.001 to
0.002 mg/L.
Future research efforts will be directed toward reducing hatcherycosts for production of postlarval prawns. Experiments will be
undertaken to investigate the effect of brine shrimp density on larval growth and survival, and to assess several formulated diets which
could minimize brine shrimp usage.

49

INTRODUCTION

Changing Sexual-State
Relationships of a
Population of
Red Swamp Crawfish_

There is some general understanding of Procamharus clarkii population dynamics. Crawfish burrow underground in the summer, produce eggs with the fall or winter floods, and mature in the spring.
The relationship of sexual states has been treated only superficially with respect to crawfish population dynamics (Sheppard 1974;
Huner 1975; Huner and Avault 1976; Huner 1978).
Some crawfish population estimates of recruits and sex ratios havebeen calculated within a trapping season. Huner (1975) noted amale:female ratio of 1:1.09. Huner and Avault (1976) found themale:female ratio as I: I. Huner (1978) also described male:female
ratios within and between seasons as I: I for all study ponds. Sheppard (1974) in Alligator Bayou, Louisiana, consistently caught more
females than males from 1972 to 1974. Our objective in the present study was to assess changing sexual-state ratios of crawfish overtime, and the effect of nitrogen fertilization on these ratios.

Procambarus clarkii
ROBERT 1. RHODESJAMES W. AVAULT, JR.
School of Forestry, Wildlife, and Fisheries
Louisiana Agricultural Experiment Station
Louisiana State University Agricultural Center
Baton Rouge, Louisiana 70803

MATERIALS AND METHODS_

The field site was Louisiana State University'S Ben Hur Farm, 4.8
km south of the main campus at Baton Rouge, Louisiana. Sixteen
ponds, approximately 0.05 ha x 1 m deep, were planted with rice
forage and fertilized with 26 or 222 kg/ha of nitrogen (Rhodes1982).
Sex ratios were determined for crawfish caught by trapping andhand picking in drained ponds. Trapping continued from 2 November 1980 to 3 May 1981, with a total trapping effort of 2,840 trapsets/ha. 1 About 50% of the total crawfish catch was hand pickedat the time of pond draining (1-3 May 1981).
All captured animals were examined ventrally for sexual state(males, form-lor form 2; and females, virgin or inseminated).
Known inseminated and virgin states were determined by drawing
a No. 2 lead pencil across the annulus ventralus. If it was vertically out of round and/or striated, allowing the pencil to leave graphite
on it, the female was considered inseminated and/or sexually active. Otherwise, she was considered a virgin. Form-l males had
distinct hooks on the 3rd pair of walking legs; form-2 males had
less distinct hooks. Females were used as a general indicator for
separating parental stock from recruits, because once inseminatedtheir annulus ventralus remains striated. Males, however, canchange from form-l to form-2 and back again.

ABSTRACfGreater variation of individual sexual states of male crawfish (fonn-l and fonn-2)
and female crawfISh (virgin and inseminated) was found in samples taken toward
the end of the harvesting season. Mature males and females dominated at the
beginning and end of the harvest season. High-nitrogen fertilized ponds showed
different sexual-state ratios than low-nitrogen ponds. There was a significant (P< 0.05) difference between percentage ratios of adult females to subadult females.

RESULTS AND DISCUSSION_

Sexual states changed through time and with capture method. Individual ponds differed in the pulse of male and female populations and their sexual states. The ratio of all males (forms 1 and
2) to all females (inseminated and virgin) decreased from fall todraining in the spring (Table 1). For male crawfish, the ratios ofform-l to form-2 were nonsignificant (P > 0.05) in low-nitrogenand high-nitrogen ponds. The ratio of inseminated to virgin females
showed a very close and significant (P < 0.05) parallel (in highand low-nitrogen ponds) from the beginning of trapping to draining, separated by a 10-20% gap from December to April. The ratios
of form-2 males to virgins in both treated ponds was nonsignificant. The ratio of form-l males to inseminated females decreased'A "trap-set" is one trap. set and harvested in one day.

51

from November to January, increased until April, and decreased
upon draining. The high- and low-nitrogen fertilization treatments
were separated by a nonsignificant (P > 0.05) gap until the spring,when the high treatment was greater than the low treatment.
Sexual-states were similar for both the high- and low-nitrogen
fertilization treatments, giving the impression of stable, seasonal
trends. Previous researchers (Sheppard 1974; Huner 1975; Hunerand Avault 1976; Brown and Bowler 1977; Huner 1978) have statedthat sex ratios are very close to 1: 1 (ma1e:female) during and between harvests. Not only does a 1: 1 male:fema1e ratio indicate an
unrealistic state, but a deviation around the 1: 1 indicates a dynamicbalanced change to encourage compensatory growth in females whenmales are being harvested with traps (Brown and Bowler 1977;
Goldman and Rundquist 1977) or to inhibit female reproduction
when crawfish and/or male densities are high (Brown and Bowler
1977; Niemi 1977). In our study, the ratio of males to females was
greater than 1: 1 throughout the season. Under harvest, the crawfish population showed a decreasing ratio approaching 1: 1 throughtime. The natural ecosystem has shown a ratio less than 1: 1
throughout the year (Sheppard 1974).
Differential loss of a sex or age group in terms of a changing
ratio is a predictive tool for examining a population (Selleck andHart 1957; Paulik and Robson 1969). The high-nitrogen ponds produced 20-40% fewer form-1 crawfish than inseminated crawfish
until December. Indirect confirmation of the decrease in form-1
males is found in the appearance of virgin females in the fall, therapid increase ofform-2 (growth phase) males to the exclusion ofform-1 males until January, and the rapid increase in virgins ascompared with inseminated females and form-2 males. With adecrease in population at the beginning of the flooded season,
density-related growth would increase, explaining the increases inrelative appearances of individual sexual-state forms (Paulik andRobson 1969). The higher ratios of sexually mature combinationsin the high-nitrogen ponds at the end of the flooded season, while
lower for the sexually immature, are compensatory in adjusting thehigh-nitrogen population to the low-nitrogen population. Finally,
although virgins in the high-nitrogen populations lack the sharp
recruitment peak in the low-nitrogen populations, virgin recruitment takes place over a longer period. Again, this suggests increased
compensatory growth due to a decrease in parental stock.

CITATIONS

Table I-Monthly ratios of crawfISh group means from experimental crawfISh
ponds at Ben Hur Farm, La. State Univ., Baton Rouge, La.
Crawfish group Nitrogen
ratio
level
\1alc>: Females
lowcombined
highPaired , test I' = 1.260. P > 0.05.

Month
N

D

F

M

2.73 2.00 10.00 1.12 1.25
2.67 165 1.67 1.09 1.32
167 0.50 167 0.90 1.57

Pond
A draining
1.23
1.29
159

105
108
1.16

Adul!s:Sub-adul!slow
2195.67 2.54 0.88 0.72 066 9.05
combined 38.60 2.46 0.74 0.66 0.68 7.25
high
3.00 100 0.33 0.27 0.73 400
Paired' test
' = !.OO3, P > 0.U5.

2.89
3.25
4.37

Inseminated:Virgins
lowcombined
highPaired' test
, = 1.650, P < U.05.

5.00 105 1.14 11.42 33.68
16.00 4.00 0.96 107 7.77 20.10
3.00 2.00 0.43 0.84 2.92 10.06

Form-I :Form-2
lowcombined
high

45.33
27.80
150

1.36 0.33 0.50 0.41 7.70
1.33 0.25 0.45 0.46 6.89
100 0.00 0.13 0.67 5.05

I. \I
1.45
2.72

Paired' test
= 1.049, P > 0.05

I

Form-I: Inseminatedlow
combined
highPaired' test
' = 0.766, P> 0.05.

2.67 115 0.50 073 0.69
2.57 1.00 0.42 070 0.81
1.00 0.33 0.00 0.33 1.38

118
1.28
1.78

0.57
0.67
0.93

7.50 153 190
12.00 6.67 147 186
1.00 5.00 114 1.74

1.75
144
103

17.32
9.34
3.44

Form-2:Virginlowcombined
highPaired t test
t = 1.347, P> 0.05.
I_

Difference between low and high nitrogen levels.

HU:-IER, J. V., and J. W. AVAULT. Jr.
1<)76. Sequential pond flooding: A prospective management technique for extended production of bait-sized crawfish. Trans. Am. Fish. Soc. 105:637-642.
NIEMI, A.
1977. Population studies on the crawfish Asracus astacus L. in the River Pyhajki, Finland. In Lindquist, O. V. (ed.), 3rd lnt. Symp. Freshwater Crawfish,
Kuopio, Finland, p. 81-94.
PAULIK, G. J., and D. S. ROBSON.
1969. Statistical calculation for change-in-ratia estimates of population parameters. J. Wild!. Manage. 33: 1-27.
RHODES, R. J.
1982. Crawfish (Procambarus clarkii) production under two rice (Oryza sativa)
varieties, two levels of nitrogen fenilization and two crawfish stocking rates.
M.S. Thesis, La. State Univ., Baton Rouge, 86 p.
SELLECK, D. M., and C. M. HART.
1957. Calculating the percentage of kill from sex and age ratios. Calif. Fish
Game 43:309-316.
SHEPPARD, M. F.
1974. Growth patterns, sex ratio and relative abundance of crayfishes in Alligator
Bayou, Louisiana. M.S. Thesis, La. State Univ., Baton Rouge, 54 p.

BROWN, D. J., and K. BOWLER.
1977. A population study of the British freshwater crawfish Austropotamobius
pallips (Lereboullet). In Lindqvist, O. V. (ed.), 3rd Int. Symp. of Freshwater
Crawfish, Kuopio, Finland, p. 33-49.
GOLDMAN, C. R., and J. C. RUNDQUIST.
1977. Acomparative ecological study of the California crawfish, Paci!astacus
leniusculus (Dana), from two subalpine lakes (Lake Tahoe and Lake Donner)
In Lindqvist, O.V. (ed.), 3rd Int. Symp. of Freshwater Crawfish, Kuopio,
Finland, p. 51-80.
HUNER, J. V.
1975. Observations on the life histories of recreationally important crawfishes
in temporary habitats. Proc. La. Acad. Sci. 38:20-24.
1978. Crawfish population dynantics as they affect production in several small,
open commercial crawfish ponds in Louisiana. In Avault, J. W., Jr. (ed.). Proc.,
9th Annu. Meet., World Maricul!. Soc., p. 619-640.

52

A Note on Recruitment
Cycles for the
Red Swamp Crawfish
Procambarus clarkii

The red swamp crawfish, Procambarus clarkii, burrows into theground in the summer, bears young with fall or winter floods, andthe young mature in the spring. However, multiple recruitment canoccur without a definite explanation (Penn 1943; Huner 1975,
Romaire 1976; Witzig 1980). Knowledge of crawfish recruitment
cycles is limited to field observations. Crawfish ponds are typically flooded after September which results in young-of-the-year
recruitment (Huner and Avault 1976). A small, post-flooding
recruitment peak occurs in December and another smaller peak in
March (Romaire 1976; Huner and Avault 1976; Huner 1978).
In this study, recruitment was examined over 8 months inlaboratory aquaria. Eight 22-L and eight 2-L aquaria with undergravel filters were used. Four females and two males were randomly assigned to each 22-L aquaria, and one, two, or three females
and one male to each 2-L aquaria on 23 July 1980.
Crawfish were fed carrots and brown rice until December anda crustacean ration thereafter. They were checked daily for deaths
and for appearance of ovipositioned eggs. In mid-December, allsurviving females in oviposition were restocked into 22-L aquaria
to observe their life span and whether they could produce a second
clutch of eggs. One male was added to those aquaria lacking a surviving males. Again, deaths and appearance of ovipositioned eggs
were recorded, and the study terminated 1 October 1981.
In this study there was specific seasonal timing of egg releasesimilar to observed fall and spring recruitment in the field. Percentage of total ovipositioned females were:

ROBERT 1. RHODESJAMES W. AVAULT, JR.
School of Forestry, Wildlife, and Fisheries
Louisiana Agricultural Experiment Station
Louisiana State University Agricultural Center
Baton Rouge, Lousiana 70803

1980
Sept. Oct.
24
56

1981

Nov. Dec. Jan. Feb. March April
9
4
0
0
0
17

Of 95 females, 57 escaped, 38 were retained, and 34 ovipositioned.
Romaire (1976) and Witzig (1980) reported that from fall throughsummer in experimental crawfish ponds, there were one major andtwo minor peaks of recruitment. Recruitment was largest in October with smaller peaks in late December and early March. Similarseasonal peaks of recruitment (up to five) have been reported byother researchers in natural areas and commercial crawfish ponds(Penn 1943; Suko 1956; de la Bretonne and Avault 1976; Huner
1978; Huner and Avault 1976; Witzig 1980).
After females in aquaria went into oviposition, mortalities anddeformities greatly increased following molting. Females before
ovipositioning, and males throughout the study, molted with little
or no deformities. Major mortalities have occurred in the field withmolting of Orconectes virilus (Momot 1967). It appears that P.
clurkii can deliver one clutch successfully, but a second clutch isdifficult.
Parents grew before the second oviposition. Penn (1943) and Suko
(1958) noted growth and development of P. clarkii after egg release.
Suko (1958) had two females carry a second clutch 3 months after
the first. However, they did not grow between clutches and utilized underdeveloped oocysts from the first clutch for an oviposition of a minor amount, from 518 to 37 and from 420 to 171 ovipositioned eggs for the two females. Following is our data for females
having a second clutch of eggs in the laboratory:53

First clutch

Individual

Date

Femalelength
(mm)

A
B
C2

10/09/80
10/12/80
10/14/80

90
88
97

CITATIONS

Second clutch
Eggs
(no.)

Molt
(s)

161
309

I
I'
I

Date

Femalelength
(mm)

Eggs
(no.)

4/02/81
4/15/81
4/15/81

93
100
98

427
517
347

_

CLARKE, K. U.
1973. The biology of the arthropoda. American Elsevier Pub\. Co., Inc., New
York. 270 p.
de la BRETONNE, L., and J. W. AVAULT, Jr.
1976. Egg development and management of Procambarus clark;; (Girard) ina south Louisiana commercial crawfish pond. In Proc., Int. Symp. Freshwater
Crawfish 3: 133-140.
GREEN, J.
1961. A Biology of Crustacea. Quadrangle Books, Chicago, 180 p.
HUNER, J. V.
1975. Observations on the life histories of recreationally important crawfishes
in temporary habitats. Proc. La. Acad. Sci. 38:20-24.
1978. Crawfish population dynamics as they affect production in several small,
open commercial crawfish ponds in Louisiana. Proc., Annu. Meet., World
Maricult. Soc. 9:619-640.
Hl'NER. J. V., and J. W. AVAULT, Jr.
1976. Sequential pond flooding: a prospective management technique for extended production of bait-sized crawfish. Trans. Am. Fish. Soc. 105:637-642.
MERMILLIOD, w. J.
1976. Life history and ecology of the large river shrimp Macrobrachium ohione
Smith. M.S. thesis, La. State Univ., Baton Rouge, 87 p.
MILTNER, M. R.
1980. Evaluation of domestic rice, Oryza saliva, and Japanese rniIlet, Echinocloa!rumemaeea, as fodder for red swamp crawfish, Procambarus clarkii. M.S.
thesis, La. State Univ., 106 p.
MOMOT, W. T.
1967. Population dynamics and productivity Jf the crawfish, Oreonecles viritis,
in a marl lake. Am. MidI. Nat. 78:55-~1.
PENN, G. H., Jr.
1943. A study of the life history of the Louisiana red-crawfish, Cambarus clarkii,
Girard. Ecology 24:1-18.
ROMAIRE, R. P.
1976. Population dynamics of red swamp crawfish, Proeambarus clarkii(Girard), in ponds receiving fertilization, and two agricultural forages as supplemental feed. M.S. thesis, La. State Univ., Baton Rouge, 92 p.
SUKO, T.
1958. Studies on the development of the crawfish; VI. The reproductive cycle.
Sci. Rep. Saitama Univ., Servo Bull. 3:79-91.
WITZIG, J. F.
1980. Spatial and temporal panerns of macroinvertebrate communities in a small
crawfish pond. M.S. thesis, La. State Univ., Baton Rouge, 113 p.

'Probably more than one occurred, although only one was observed.
2The first clutch occurred in 4 ml of water in a bucket. Eggs and glair covered
her body, instead of the pleopods. An egg count was not made.

The females that go into oviposition (first cohort) in the fall may
also go into oviposition in late December if they develop the few
residual oocysts (Suko 1958), Females going into second ovipositionin late December normally do not go into oviposition in early Marchbecause of reduced water temperature and slower development(Mermilliod 1976), unless they molt and develop more oocysts during the remainder of the fall before cold winter temperatures reduce
growth.
It is improbable that sexual development and oogenesis could occur before winter for fall-spawned hatches (MermiJliod 1976; Witzig1980). A second cohort in December is thought to come fromjuvenile crawfish which previously burrowed underground whenponds were drained the previous spring/summer.
Recruitment rapidly diminishes in the spring (Huner 1978; Penn
1943; Romaire 1976; Witzig 1980), Inseminated females and maleswith sperm in sperm-ducts at the end of the harvesting season(May/June) produce the first recruitment of young when ponds areflooded in the fall (Green 1961; Clarke 1973), When fall flooding
occurs, surviving juveniles grow, resume oogenesis, and spawn 4-5
months later in December (de la Bretonne and Avault 1976) as thesecond cohort. These survivors probably comprise the bulk of theearly crop in the commercial crawfish ponds in Louisiana (de la
Bretonne and Avault 1976), An example of this was described by
Miltner (1980), Most of his total catch in older ponds was bimodal,
with fall and spring peaks, whereas in new ponds it was a unimodal,
spring catch.
In summary, recruits occur in three distinct peaks, fall, early
winter, and early spring, by two separate crawfish populations. The
fall and early spring recruits come from the same parental stockthat survive harvesting to have two ovipositions per female. The
early witner recruits are produced from the previous season'&crawfish which were juveniles at pond draining in spring/summer.

54

INTRODUCTION

Production of Triploid
Channel Catfish· Fry_

Channel catfish are widely cultured because they are easily managed
and have good food quality. Although much research has been performed on rearing, stocking, and nutrition of channel catfish, lessresearch has been directed toward genetic improvements that would
increase its value as a cultured species.
One genetic improvement that may provide benefits to fish cultureis the production of triploid individuals. Natural spontaneous
triploidy in fish is rare (Cuellar and Uyeno 1972; Gold and Avise
1976; Allen and Stanley 1978; Thorgaard and Gall 1979), but has
been successfully induced by cold-shocking fertilized eggs in plaice,
Pleuronectes platessa; its hybrid with the flounder, Platichthys flesus(Purdom 1972); three spine stickleback, Gasterosteus aculeatus(Swamp 1959a); blue tilapia, Tilapia aurea (Valenti 1975); carp,
Cyprinus carpio (Gervai et al. 1980); channel catfish, lctaluruspunctatus (Wolters et aI. 1981b); and rainbow trout, Salmo gairdneri (Lincoln 1982). Triploid individuals might be expected to belarger than their diploid counterparts, in accordance with the generalhypothesis that nuclear size increases in proportion to chromosome
number while the nuclear-cytoplasmic volume ratio is maintained(Fankhauser 1945; Swamp 1959b). Swamp (1959b), however,found triploid threespine sticklebacks to be no larger than diploids;
cellular size increased, but the number of cells per organ decreased.
Purdom (1976), Valenti (1975), and Wolters et al. (1982a) foundinduced triploid plaice-flounder hybrids, blue tilapia, and channel
catfish to be significantly larger than normal diploids. These triploids
may have differed from triploid threespine sticklebacks in their indeterminate growth pattern. The regulatory mechanism proposed
by Swamp (1959b) may have been absent, causing these triploidfish to be larger.
Channel catfish are often cultured for food consumption to sizes
near and above 0.5 kg. They begin primary oocyte production as
juveniles, and can be sexually mature as small as 0.33 kg (Bardach
et al. 1972; Meyer et aI. 1973; Grizzle and Rogers 1976). Polyploid fish have been found to have reduced gonadal development
and grow faster than diploids during periods of gonad development(Purdom 1976; Allen and Stanley 1978; Thorgaard and Gall 1979;
Gervai et al. 1980). Triploid channel catfish in commercial culture
may also avoid depressed growth and poorer feed conversion
characteristic of sexual maturation, and therefore be more profitableto raise than diploids. This paper describes methods to produce
triploid channel catfish fry and fingerlings, and possible benefits
to commercial producers.

WILLIAM R. WOLTERS
School of Forestry. Wildlife. and Fisheries
Louisiana Agricultural Experiment Station
Louisiana State Univesity Agricultural Center
Baton Rouge. Louisiana 70803

METHODS_

Spawning and triploid induction
Sexually mature channel catfish can be obtained by seining broodponds during the spawning season. Males are selected on the basisof prominent tertiary sex characters such as large, dark colored,
and muscular heads. Females near spawning are selected for full,well-rounded abdomens past the pelvic fins, and the female genitalarea becomes red, swollen, and often pulsates as spawning time
approaches (Meyer et aI. 1973; Smitherman et aI. 1978).
In order to induce triploidy, complete control over the spawning
process by the culturist is required. Such intensive methods arenecessary in order to obtain and properly manipulate the eggs andsperm within a short period of time.

55

Male and female catfish can be paired for spawning in suitable
sized aquaria, tanks, or concrete troughs utilizing dividers to form
pens. Care should be taken to place males and females of similar
size in the spawning tanks. Tanks can vary in size from 40 to several
thousand liters. Spawning shelters are generally required only inlarge tanks allowing more fish movement. Water supply into thetanks should be with 24-28°C well-aerated water at a rate necessary
to maintain adequate water quality. Female catfish can be induced
to ovulate with daily injections (4.4 mg/kg) of carp pituitary extract or human chorionic gonadotropin (11 00-1800 IU/kg). Twoto three injections may be needed depending on the condition of
the female.
When female fish are observed to be ovulating freely, the eggs
can be handstripped and fertilized with sperm from mascerated testesof donor males (Dupree et al. 1969). Beginning 5 minutes after
fertilization, eggs are cold-shocked at 5°C for 1 hour (Wolters etal. 1981b). After treatment, the eggs are transferred without acclimation to a standard hatching tray. Disease treatment of eggs
may be needed to control fungus from small pieces of testes imbedded in the egg mass during fertilization. After hatching, fry canbe reared to fingerling size by commonly used methods (Meyer
et al. 1973; Jensen et al. 1983).

Table I-Mean weights, feed consumption, and feed efficiency of diploid andtriploid channel catfish from 2 to 16 months of age (Wolters et al. 1982a).
Asterisks (0) denote significant difference in weight (P < 0.05) at age between
diploids and triploids (Wilcoxon rank sum).
VariableAge (months)
2
4
8
13
16

Triploids

4.41'
(72)
40.6 ± 2.39 (70)87.8 ± 1.09 (70)304.9 ± 17.4 (40)401.7 ± 39.9 (IS)

4.43'
(72)
40.7 ± 2.11 (70)92.9 ± 0.98 (70)*357.8 ± 25.9 (40)*466.0 ± 63.0 (15)*Feed conversion
Total fish weight gained (14 mo)b
Total feed given (14 mol
Feed conversion

2008 kg
26.10 kg
1.30

22.20 kg
26.40 kg
1.19

a<\11 fish were weighted as a group.
"Total gain includes fish that died or were removed for chromosome counts.

1.30 (Table 1). Further studies are needed to assess comparative
growth rates in ponds.
Mature diploid male channel catfish have the tertiary sexual
characteristics of broad muscular heads and dark coloration. The
sex of both male and female diploid catfish can be discerned rather
easily. Tertiary sex characteristics are not as evident in triploidmales, and it is difficult to sex triploid males and females externally. Experimental data has shown no significant difference from a
1: I sex ratio (P > 0.99) (Wolters et al. 1982a).
The testis of diploid channel catfish has anterior and posterior
regions based on the appearance of fingerlike projections of the surface. The anterior region (three-fourths of the testis) has large, thick,white projections. This region contains coiled seminiferous tubules
where spermatogenesis occurs. Projections of the posterior region
are pink and generally fused into a single mass (Sneed and Clemens
1963; Grizzle and Rogers 1976). The testes of triploid catfish at
8 months of age have been found to be smaller than those of diploid
catfish, and macroscopic differences between anterior and posterior
regions are less distinct. In histological sections, the seminiferous
tubules of diploid testes have spermatogenic epithelium composedof2-3 cell layers. Most cells had interphase nuclei, but only a few
cells appeared to be in active division. The large lumina of thetubules are filled with many spermatozoa. The spermatogenie epithelium of triploid testes was found to be much thicker with avariable number of cell layers. Many cells were undergoing active
division. The lumina of the tubules were smaller and contained no
spermatozoa.
Diploid ovaries in catfish at 8 months of age were found to be
3-4 times larger than triploid ovaries. Numerous developing oocytes
were macroscopically visible in the ovaries of diploid catfish.
whereas in triploids only an occasional oocyte could be seen. Diploidovaries examined in histological sections contained many primaryoocytes having strongly basophilic cytoplasm and lightly staining
nuclei. Many large, maturing oocytes were also present and could
be identified by their large, irregular nuclei and vacuolated cytoplasm associated with yolk formation. Sections of triploid ovaries
contained small cells that were all of similar size. Many of the cells
were undergoing active division. Occasionally a larger, apparently
developing, oocyte could be found (Wolters et al. 1982a).

Determination of ploidy levels
A determination of the degree of success in triploid induction should
be made. Triploidy (3n = 87) can be confirmed from chromosome
counts of cultured lymphocytes, mascerated kidney tissue, ordeveloping embryos (Kligerman and Bloom 1977; Hollenbeck andChrisman 1981; Wolters et al. 1981a). These methods are accurate
and direct, but relatively time-consuming, expensive, and require
skilled personnel. Triploidy in catfish can also be estimated frommeasurement of erythrocyte nuclei (Wolters et al. 1982b). Approximately 92 % accuracy can be obtained from this method; however,
it is relatively easy, rapid, and requires little specialized equipment.

RESULTS AND DISCUSSION

Diploids

Growth: mean weight (g) ± SE (N)_

Success of triploid induction
Females in good spawning condition should ovulate after 1 to 3
injections. With practice, 100% fertilization is possible, and theincidence of triploidy can be 100%. Cold shocks longer than I hourare not necessary and have caused 100% mortality in developing
embryos (Wolters et al. 1981b).
Although it has not been proven, triploidy probably results fromnonextrusion of the second polar body (Purdom 1969). Triploid
catfish are as viable as their diploid full sibs and tolerate the polyploid condition well. Triploid catfish do not appear to be different
morphologically from diploids; however, it has been noted that
triploid catfish appear to have a darker coloration than diploids.

Growth rates and gonad development
Experimental studies conducted in 870-L fiberglass tanks equippedwith biofiltration have shown that from 2 until 16 months of age,
diploid and triploid fish consumed equal amounts of food. However, triploid fish were significantly heavier than full-sibling diploids
after the eighth month because of better feed conversion: 1. 19 vs.
56

SMITHERMAN, R. 0., H. M. EL-IBIARY, and R. E. REAGAN, (editors).
1978. Genetics and breeding of channel catfish. South. Coop. Ser. 223, AJabama
Agric. Exp. Sin., Auburn Univ., Auburn.
SNEED, K. E., and H. P. CLEMENS.
1963. The morphology of the testes and accessory reproductive glands of thecatfishes (Ictaluridae). Copeia 1963:606-611.
SWARUP, H.
1959a. Production of triploidy in Gasterosteus aculeatus (L.). J. Genet. 56: 129J42.
1959b. Effect of triploidy on the body size, general organization and cellular
structure in Gasterosteus aculeatus (L.). J. Genet. 56:143-155.
THORGAARD, G. H., and G. A. E. GALL.
1979. Adult triploids in a rainbow trout family. Genetics 93:961-973.
VALENTI, R. J.
1975. Induced polyploidy in Tilapia aurea (Steindachner) by means of temperature shock treatment. J. Fish. BioI. 7:519-528.
WALDROP, J. E., and R. D. SMITH.
1980. An economic analysis of producing pond-raised catfish for food in
Mississippi: A January 1980 update. Res. Rep. 103, Miss. Agric. For. Exp.
Stn., Miss. State, MS.
WOLTERS, W. R., C. L. CHRISMAN, and G. S. LIBEY.
1981a. Lymphocytes culture for chromosomaJ analyses of channel catfish, lctalurus punctatus. Copeia 1981:503-504.
1982b. Erythrocyte nuclear measurements of diploid and triploid channel catfish, lctalurus punctatus. J. Fish. BioI. 20:253-258.
WOLTERS, W. R., G. S. LIBEY, and C. L. CHRISMAN.
1981b. Induction of triploidY in channel catfish. Trans. Am. Fish. Soc.
110:312-314.
1982a. Effect of triploidy on growth and gonad development of channel catfish. Trans. Am. Fish. Soc. 111:102-105.

Potential benefits to commercial culture
The faster growth rate demonstrated by triploid catfish in tankculture would be very beneficial in commercial pond culture. Fastergrowing fish obviously reach market size faster, resulting in asavings in labor and greater production per unit of time. Better feed
conversion in triploids possibly resulting from the sterile condition
yields savings in feed costs. Feed costs can be 51-57% of the annual operating costs for catfish production (Waldrop and Smith
1980). Triploid catfish with 8% better feed conversion than diploids
would significantly lower feed costs (Wolters et al. 1982a).
The most significant benefit to commercial production from
triploid catfish may occur during processing. Reduced gonad
development results in less wastage in processing. Preliminary datafrom processed triploid catfish have shown an average of 6 % higher
dress-out percentage over diploids (Wolters et al. unpubl.). Average prices in Jul y 1983 were $1 .57/1b. for frozen fish. An increase
in dress-out percentage of only I % would have increased processedweight by 300,000 kg (700,000 lb.) for the first 6 months of 1983,
with a value of $1.1 million to the industry.

CITATIONS_ALLEN, S. K., Jr., and J. G. STANLEY.
1978. Reproductive sterility in polyploid brook trout, Salvelinusfontinalis. Trans.
Am. Fish. Soc. 107:473-478.
BARDACH, J. E., J. H. RYTHER, and W. 0 McLARNEY.
1972. Aquaculture; th~ farming and husbandry of freshwater and marineorganisms. John Wiley and Sons, New York.
CUELLAR, 0., and T. UYENO.
1972. Triploidy in rainbow trout. Cytogenetics (Base!) 11:508-515.
DUPREE, H. K., O. L. GREEN, and K. E. SNEED.
1969. Techniques for the hybridization of catfishes. Publ. 221, U.S. Fish Wildl.
Serv., Southeast. Fish Cult. Lab., Marion, AJabama.
FANKHAUSER, G.
1945. The effects of changes in chromosome number on amphibian development. Q. Rev. BioI. 20:20-78.
GERVAI, J., S. PETER, A. NAGY, L. HORVATH, and V. CSANYI.
1980. Induced trip10idy in carp, Cuprinus carpio L. J. Fish BioI. 17:667-671.
GOLD, J. R., and J. C. AVISE.
1976. Spontaneous triploidy in the CaJifornia roach Hesperoleucus symmetricus(Pisces:Cyprinidae). Cytogenet. Cell Genet. 17: 144-149.
GRIZZLE, J. M., and W. A. ROGERS.
1976. Anatomy and histology of the channel catfish. Auburn Univ. Agric. Exp.
Stn., Auburn, Alabama.
HOLLENBECK, P. J., and C. L. CHRISMAN.
1981. Kidney preparations for chromosomal analyses of Ictaluridae. Copeia
1981:216-217.
JENSEN, J., R. DUNHAM, and J. FLYNN.
1983. Producing channel catfish fingerlings. Circ. ANR-327, Coop. Ext. Serv.,
Auburn Univ., Auburn, Alabama.
KLIGERMAN, A. D., and S. E. BLOOM.
1977. Rapid chromosome preparations from the solid tissue of fishes. J. Fish.
Res. Board Can. 34:266-269.
MEYER, F. P., K. E. SNEED, and P. T. ESCHMEYER, (editors).
1973. Second report to the fish farmers; the status of warmwater fish farming
and progress in fish farming research. U.S. Bur. Sport Fish. Wildl. Rcs. Publ.
113.
PURDOM, C. E.
1969. Radiation-induced gynogenesis and androgenesis in fish. Heredity 24:
431-444.
1972. Induced polyploidy in plaice (Pleuronectes platessa) and its hybrid withthe flounder (Platichthys jIesus). Heredity 29: 11-24.
1976. Genetic techniques in flatfish culture. J. Fish. Res. Board Can.
33: 1088-1093.

57

INTRODUCTION

An Investigation of_

Although the bullfrog Rana catesbeiana is widely used as a laboratory animal, culture techniques have not been developed andimplemented, with the exception of efforts by our laboratory. Lackof reproductive control, the inability of most laboratories to provide the required food to the postmetamorphic stages, inadequate
housing, improper environmental conditions, poor sanitation, andlack of disease control have been major obstacles to developing
colonies of bullfrogs for teaching and research, or mass culture foreducation and human consumption.
In recent years considerable progress at the Louisiana State
University Amphibian Laboratory has been achieved in developing techniques for continuous culture of this species (Culley et al.
1978). Most noteworthy of this research effort was the recent
development of standard techniques to induce spermiation fromlaboratory-reared or wild-collected bullfrogs (Easley et al. 1979).
However, control of ovarian recrudescence and ovulation has remained problematic.
Basic procedures for induction of ovulation in anurans with homoplastic pituitary injections and subsequent artificial fertilization of
extruded eggs have been outlined by Rugh (1965), di Berardino(1967), and the National Academy of Sciences (NAS 1974) withthe methodology resulting from studies by Rugh (1935), Wright(1961), and Wright and Flathers (1961). Although these techniquesof artifically ovulating wild-caught anurans are frequently used on
the bullfrog, ovulation is seldom achieved. Rugh (1935) treated two
female bullfrogs obtained during the breeding season with high levelsof pituitary homogenates and elicited ovulation, though the extent
of response was not described. Licht (1974) failed to induce in vitro
ovulation or maturation by exposing follicles considered to be sexually mature to homologous pituitary extracts, but recently
demonstrated an ovulatory response with a gonadotropin-releasing
hormone (Gn-RH) agonist (Licht et al. 1982).
Failure of animals to respond to standard laboratory methods could
result from a number of factors including (I) poor quality of anuransoften supplied to the researcher by the commercial dealer, (2) improper administration (dosage and frequency of injection) of thehormone(s) used as the ovulatory stimulus, (3) improper environmental regime under which the animals were held prior to treatment, (4) lack of feeding which is often indicative of asynchronous
ovarian development, (5) injection of hormones without regard toa possible circadian rhythm of sensitivity, and (6) inability to selectfrogs which possess a mature complement of oocytes capable ofundergoing maturation and ovulation in response to an appropriate
hormonal stimulus.
Several of these factors, including photoperiod, temperature, andthe circadian injection scheme, have been shown to have a regulatory effect on reproductive development and sensitivity of thebullfrog (Horseman et al. 1978; Penkala 1978; Easley et al. 1979).
Even subtle disturbances such as the time of day the animals are
fed or handled and their tanks cleaned can cause cumulative changes
in hormonal or physiological conditions of anurans (Meier et al.
1973; Horseman et al. 1976; Licht et al. 1983; Mbangkollo and
DeRoos 1983).
Studies by Horseman et al. (1978) indicated that an environmental
regime consisting of a 12L: 12D photoperiod and 25 ± I °C air andwater temperature prevented appreciable ovarian regression andatresia in either laboratory-reared or wild caught laboratoryconditioned bullfrogs. Furthermore, this regime was conducive tomaintenance of normal gonadal condition in females under longterm laboratory conditions (Culley et al. 1982). Apparently cyclic

Potential Ovulatory
Stimuli for the Bullfrog
Rana catesbeiana
DUDLEY D. CULLEYKIRK A. EASLEYLYNN DENSONBARBARA A. CARR
M. ARIF arHMAN
School of Forestry. Wildlife, and Fisheries
Louisiana Agricultural Experiment Station
Louisiana State University Agricultural Center
Baton Rouge, Louisiana 70803

ABSTRACf
Rana caJesbeiana pituitaries and progesterone and a synthetic gonadotropin releasing hormone (Gn-RH) agonist proved to be an effective and consistent ovulatory
stimuli for the buUfrog. Mammalian gonadotropins, pituitary homogenates fromother anurans, and gonadotropin-releasing hormone (LHIFSH-RH) failed to elicit
ovulation, but further documentation is warranted. BuUfrogs with mature ovaries
were observed throughout the year from south Louisiana populations and wereovulated by hormonal injection. Use of pituitaries taken from wild populations
captured during the spring, summer, and fall were equaUy effective in producing
an ovulatory response, as were pituitaries taken from laboratory-cultured females.
Female pituitaries appeared a more potent inducer of ovulation than malepituitaries. Females force-fed a prepared diet were successfully ovulated aller
8 and 12 weeks of feeding. Laboratory-reared females maintained under standard laboratory conditions (12L:12D, 2S± 1°C) were ovulated as early as 6 months
postmetamorphosis and as late as 34 months postmetamorphosis.

59environmental zeitgebers are not absolutely essential for normal
ovarian development, at least during the initial wave of reproductive development or recrudescence; we have consistently ovulated
females collected throughout the year and held in the laboratory
for several months before inducing ovulation, as well as females
cultured under cyclic temperature and photoperiod conditions.
This paper describes work completed on identifying environmental
factors that affect ovulation, and testing of potential ovulatory stimuli
other than bullfrog pituitaries and progesterone.
This work is part of a continuing effort by this laboratory todevelop standard techniques for laboratory or commercial cultureof the bullfrog. The depletion and poor quality of wild bullfrog
stocks threatens the continued use of these animals which haveplayed a very important role in biomedical research and teaching(Culley 1973; NAS 1974), and are widely utilized as human food.

MATERIALS AND METHODS

Table I-Oocyte stages in Rana caJesbeiana. Adapted from Penkala (1978).
Stage

_

Laboratory-reared female bullfrogs utilized in this study were either
collected as eggs or larvae from research ponds at the Louisiana
Agricultural Experiment Station Ben Hur Biological Research Area
(Baton Rouge, Louisiana), or from eggs fertilized within thelaboratory in accordance with methods outlined by di Berardino(1967) or McKinnell et al. (1976). The larvae were fed a prepareddiet and maintained in a rearing system as reported by Culley etal. (1977). After forelimb emergence, frogs were reared in fiberglass tanks designed by Culley (1976). Each tank was cleaned andfrogs fed daily, usually during the afternoon. Juvenile frogs were
fed almost exclusively mosquitofish (Gambusia affinis), whereas
the diet offrogs weighing more than 100 grams was supplemented
with other fish species, crawfish, and tadpoles. All animals (larvae,juveniles, and adults) were maintained on a 12-hour daily photoperiod (0600-1800 CST) with a light intensity within rearing containers of approximately 100 to 200 lux. Water and air temperature was 25 ± 1°C.
Wild collected female bullfrogs used in the tests were purchased
throughout the year from Gulf South Biological Inc. (Ponchatoula,Louisiana), Jacque Weil Co. (Rayne, Louisiana) and Davis Farms(Clovis, California). These animals were subjected to the same feeding and rearing regimes provided laboratory-reared stock when injections were not initiated immediately after purchase from a supplier.
Previous studies showed that ovarian condition influenced
ovulatory response, and females could not be selected at random
for determining the effect of hormone injections on ovulation (Culley
et al. 1982). These initial studies were reported by Othman (1978)
who showed that an ovulatory response could rarely be obtained
unless the oocytes were mature (Stage V) (Table 1). Egg condition
for most frogs in this study was determined prior to injections bysurgically removing a piece of the ovary (unpubl. data), along witha morphometric index which could be correlated to ovarian condition (Culley et al. 1982). It has been demonstrated that surgery does
not inhibit ovary development or affect ovulation (Carr and Culley,
1984a, b). Ovarian samples to be examined for developmental stage
and lor oocyte stage frequencies were fixed in 10% formalin. Eachsample was cut into three 5 x 5 mm fragments, to be analyzed
separately for oocyte counts and these values averaged for frequency
determinations for a given sample. Ovulation responses wererecorded as negative (no eggs released), partial (50-1,000 eggs),
or complete (all mature Stage V oocytes stripped from the female).
Staging of the ovary (Table 1) followed the schemes developed byDumont (1972), Kemp (1953), and Penkala (1978).

Diameter(mm)

I;'0.20IIA

0.20-0.35

Translucent (with nuclei visible) to pale white(early).

Clear oocytes; nuclei plainly discernible.

lIB

0.35-0.67

Solid white to cream color.

III

0.67-0.83

Light grey to darkly pigmented (melanin deposition
occurring) .

IV

0.83-1.25

Polarity evident; darkly pigmented animal hemisphere; greyish vegetal hemisphere.

V

1.:!5-1.50

Last distinct class prior to maturation; polarity
highly resolved, with animal hemisphere lighter incolor and vegetal hemisphere very white.

VI

Highly variable

Atretic, follicles regressing; evidenced by swollen
oocytes with irregular, discolored surfaces, whichbecome highly vascularized and granularized; earlystage oocytes become yellow to orange in color, latestages become brown to black; in late phase,follicles reduced to condensed clump of pigment
granules.

Experiment 1:
Condition affecting ovulation
With an effective ovulatory stimulus identified (a combined injection of 2 + R. catesbeiana pituitaries and 5 mg progesterone ondays 1 and 3), studies were undertaken to define management techniques to enhance reproductive control. Injection procedures
followed Culley et al. (1982). Monitoring of ovarian condition was
as described above. Controls received injections of distilled water(carrier). The following studies were concluded:(I) Wild female bullfrongs were collected at various times ofthe year to determine if ovulation could be induced (normal breeding
season for all collection sites was April through August, with thepeak occurring in April through June)_ Arrangements were madewith a supplier to collect the animals within a given time (one night)
and frogs were either picked up the next day or were shipped to
our laboratory within 48 hours. Injection was accomplished within
48 hours after arriving in the laboratory.
(2) Wild bullfrogs not containing a mature complement of oocytes(Stage V) were placed under standard laboratory conditions(l2L:12D, 25°C) (Horseman et al. 1978) and provided a continuous supply of crawfish for 8 weeks, or force fed a pelleted diet
(Liao 1980) for periods of 4, 8, or 12 weeks.
Final ovarian condition was ranked in accordance with the following scheme: (12) Stage V eggs resulting in complete ovulation;(11) Partial ovulation (50-1,000 eggs ovulated with remaining
oocytes mature (Stage V); (10) Stage V oocytes but no ovulationand no atresia; (9) Stage V oocytes with one lesser stage containing atretic oocytes; (8) Stage V oocytes with two egg stages withatretic oocytes; (7) Stage IV oocytes and no atresia; (6) Stage IV
oocytes and one stage with atretic eggs; (5) Stage III oocytes; (4)Stage III and atresia; (3) Immature vitellogenic ovaries (Stage II
A and II B) without atretic oocytes; (2) Immature vittelogenic ovaries
with atresia; (1) Immature previtellogenic ovaries (Stage I).
Bullfrog pituitaries and progesterone were then injected as outlined above.
(3) Laboratory-reared bullfrogs of various ages and sizes were
injected to further define the flexibility with which ovulation could
be induced. All animals used in the study were cultured from the60

RESULTS AND DISCUSSION

egg under conditions as already described. Because growth rates
vary, the age from metamorphosis to a condition of mature oocytes
varies from 6 to 12 months. However, several frogs undoubtedly
had developed a mature complement of oocytes several months
before injections (as determined by the morphometric index) (Culley
et al. 1982). Four days after injection, the frogs were opened andthe most advanced oocyte stage identified (including any ovulatedeggs).

_

Experiment 1
Table 2 shows that ovulation was not limited to wild bullfrogs collected during the peak of the natural breeding season (April throughJune). Bullfrogs with mature ovaries were observed throughout theyear, and eggs were obtained by hormonal injection for all months
attempted. Hormone dosages necessary to induce ovulation did notvary seasonally as long as a mature complement of oocytes waspresent within the ovary (frogs with Stage IV oocytes did not
ovulate). The pituitaries used for injections were taken from wild
females collected in March, July, August, and September and fromlaboratory-cultured females (12L: 12D regime). The fact that wild
bullfrogs with mature oocytes could be obtained all year indicatesthat some reproductive-related hormonal activity continues, althoughseasonal changes of gonadotropins and steroids in wild populations
are evident (Licht et al. 1983). Laboratory-cultured females held
under light conditions other than 12L: 12D may also show variation in plasma gonadotropins, as Horseman et al. (1978) demonstrated significant rates of oocyte atresia under light regimes greaterand less than 12 hours.
Laboratory-reared females, maintained under standard laboratory
conditions from hatching, ovulated in response to injections ofbullfrog pituitaries and progesterone as early as six months postmetamorphosis (Table 3). Age of the animal did not appear criticalas ovulation was achieved in laboratory animals from 6 to 34 months
postmetamorphosis. Offspring from parents with rapid growth
characteristics (II through 18 of Table 3) were maintained on acontinuous feeding regime from metamorphosis, attained reproductive maturity, and ovulated 6 to 14 months after metamorphosis.
Jorgensen (1967) and Jorgensen et al. (1978) suggested that nutrition is critical in the development and maintenance of vitellogenic
oocytes in Bufo bufo bufo. Horseman et aI. (1978) added that nutritionally healthy animals under proper environmental conditions
could maintain a population of mature oocytes for a considerable
length of time, which possibly accounts for the wide age interval
over which the bullfrog can be ovulated. Our results in this andother studies (Penkala 1978; Othman 1978) suggest that withouta stimulatory environmental regime and consistent feeding activity
by the animals, ovarian patterns can become acyclic, deviating into
patterns of alternating atresia and recrudescence with few of thedeveloping oocytes reaching a postvitellogenic state of maturity.

Experiment 2:
Testing of other potential ovulatory stimuli
Having established that bullfrog pituitaries and progesterone induce
ovulation when the females were placed under, or were received
from, a variety of environmental conditions, efforts turned tolocating other stimuli for ovulation because bullfrog pituitaries are
difficult to obtain. Commercially available preparations wereselected in most cases. Treatment schedules and dosages are shownin Table 5. Tests were conducted with both wild and laboratory
bullfrogs as in Experiment I. Controls consisted of carrier solutions, and bullfrog pituitaries and progesterone were also used to
document effectiveness. The environmental regime under which
the animals were held was as reported above, unless specificallystated otherwise.
All injections were given one hour after the onset of light, withthe exception of multiple daily injections of a synthetic mammalianleutinizing hormone/follicle stimulating hormone-releasing hormone(LH/FSH-RH) (National Institute of Health, NIAMDD-ABBOTT
Lot 19-192-AC) and a gonadotropin releasing hormone agonist (GnRH agonist) (im Bzl-D-His6, Pro9-Net) (McCreery et aI. 1982).
Rana catesbeiana leutinizing hormone (LH), Mammalian LH (NIHLH-520), human chorionic gonadotropin (HCG) (Sigma ChemicalCo., St. Louis, Missouri), and mixed male and female acetonedried Rana pipiens pituitaries (NASCO, Fort Atkinson, Wisconsin) were injected intraperitoneally. Also injected intraperitoneallywere male and female Rana catesbeiana pituitaries extracted fromlaboratory-cultured frogs and purchased heads (from Gulf South
Biological Supply Co., Ponchatoula, La, July, August 1979; July.
1980; November 1980; March 1981) and progesterone (Sigma
Chemical Co., St. Louis, Missouri), using methods reported byde Berardino (1967). LH/FSH-RH was injected into the dorsal
lymph sacs.
Attempts to strip eggs were made 24 hours after the initial injection and at subsequent 12-hour intervals for the duration of the injection period. Forty-eight hours after the final injection, frogs weresacrificed by spinal pithing to access ovarian condition.

Table 2-0vulation responses of wild bullfrogs to R. caJesbeiana pituitaries
and progesterone.
Month
No. animals
and
Mean
with
Ovulation response
yearCollection No.
body wt.
Stage V3
collected(g)site
animaJs
oocytes
Neg. Panial Complete

Experiment 3:
Ovulatory response to a Gn-RH agonist

March 78
April 78
June 78
July 78Aug. 78
Sept. 78
Oct. 78
Nov. 78
March 79
Oct. 79

Work by McCreery et aI. (1982) demonstrated the effectivenessof a synthetic gonadotropin releasing hormone agonist [(im Bzl)
DHIS6, Pro9 , Net]-Gn-RH. TFA #97-74-10. Daily injections for
3 to 4 days resulted in ovulation of female bullfrogs, and single
injections caused spermiation in males.
This experiment was designed to test animals from another population (Louisiana). Ovarian samples were taken prior to injections,and test and control procedures were the same as previously
described. Dosages are shown in Tables 3 and 4.

R
R
R
R
P
R
R
R
C
P

6
4
7
5
6
5
4
10
4
8

aR = Rayne, Louisiana;
P = Ponchatoula, Louisiana;
C = Clovis, California.

61

624
710
483
533
597
693
556
580
649
586

2
2
4
2
4
0
0
I
3
4

4
I
3
3
2
4
4
9
I
4

2
I

0
I
0
I
0
0
0
I

0
2
4
I
4
0
0
I

3
3

Table 3-0varian condition and ovulation responses oflaboratory-reared buUfrogs to R. caJesbeiona pituitaries andprogesterone.
Froga.bAge'(months)

Total bodywI. (g)Most advanced
oocyte stage

Ovulation
response

I
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18

10
7
11
12
12
34
11
II
II
II
6
6
16
16
16
14
14
14

476
456
309
290
246
686
336
253
256
382
381
365
370
362
280
412
431
296

V
IV
IV
IV
IV
V
IV
V
IV
IV
V
V
V
V
IV
V
V
V

Complete
Negative
Negative
Negative
Negative
Partial
Negative
Negative
Negative
Negative
Complete
Complete
Complete
Complete
Negative
PartialPartialPartialTable 4--Ovulatioo responses and ovarian condition of laboratory-reared cooditioned wild bullfrogs subjected to a daily feeding of live crawfish for 8 weeks(Treatment D) or force-fed a pellet diet daily for 4, 8, or 12 weeks (Treatments
A, Band C, respectively).
Body weight (g)after (wks)

aFrogs 1-10 were laboratory-reared animals from field collected eggs.
bFrogs 11-18 were F , laboratory-reared animals from parents
selected for rapid growth.
'Postmetamorphosis.

Preovulatory wild-collected bullfrogs were brought into a mature
ovarian condition with minimal atresia when held under laboratory
conditions for eight weeks, either fed crawfish or force fed artificial
pellets (Table 4). Growth rates were excellent under both treatments,and ovulation was achieved in all animals injected with bullfrog
pituitaries and progesterone after eight weeks.
After only four weeks of force feeding under laboratory conditions, animals were undergoing recruidescence and vitellogenesis(Stage III and IV oocytes). Furthermore, the ovaries contained agroup of black, late atretic bodies (Rankings 4 and 6) which probably represented an earlier wave of ovarian recrudescence. After
12 weeks of forced feeding several bullfrogs had apparently completed the ovarian cycle (Le., the ovary was ready to undergo
maturation and ovulation), but when the proper ovulatory stimulus
was not received the ovaries evidently began to regress, as was indicated by the swollen oocytes with discolored surfaces (Rankings
9 and 6). The atretic response could have also been enhanced byexcessive handling.

Initial

4

458
410
400
389
390
355
400
350
335
370
371
451
420
417
465
349
403
360
468
388
497
420
415
355
402
375
412
373
472
364
504
418
402
397

467
430
408
391
421
382
438
352
357
392
379
464
412
425
503
360
411
390
466
390
502
424
432
385
410
378
440
405
501
410
528
533
412
424

8

463
478
525
482
540
551
434
492
472
574
481
586
500
555
462
484
464
486
444
590
490
579
601
471
454

12

506
600
540
682
546
614
492
498
504

Treatment
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
B
C
C
C
C
C
C
C

C
C
0
0
D
D
D
LJ
0
D

Animals
injected

Ovulation
responsea
N

P

X

X

X
X

X
X
X
X
X
X

X
X
X
X

C

X
X

X
X
X
X
X
X

X
X
X
X

Ranking of
fmal ovarian
condition
7
6
6
6
4
4
7
7
4
10
10
12
10
11
12
7
10
9
9
8
12
6
9
6
6
9
7
6
12
12
12
12
7
7

aN = Negative; P = Partial; C = Complete.

tivity. supporting similar findings by Wright (1961) and Wrightand Flathers (1961). In most cases, ovulation was complete 24 to48 hours after the second injection.
Of the 59 wild-collected bullfrogs injected with female bullfrog
pituitaries and progesterone, 24 had mature Stage V oocytes. All
24 of these animals ovulated in response to the injections, with 18
frogs yielding complete ovulation and 6 responding with partial
ovulation. Laboratory-reared and laboratory-conditioned wild
bullfrogs also responded favorably to the ovulatory stimulus.
No ovulation responses were obtained with HCG, mammalian
LH, R. catesbeiana LH, or pituitary homogenates from otheranurans (Table 5). However, the amphibian LH was available in
only small amounts; therefore only nine females, each receiving
20 /-Ig of this hormone/injection, were evaluated. Large doses of
HCG (500-1,000 IV) and mammalian LH (2.5 mg) were injectedin an attempt to overcome suspected phylogenetic differences in
specificity of the mammalian gonadotropins, but without success.
These phylogenetic differences are not so distinct in Bufonidae(Houssay 1947; Kjaer 1969; Kjaer and Jorgensen 1971) and Pipidae(Redshaw 1972) where LH and HCG actively induce ovulation,
both in vitro and in vivo. Furthermore, although amphibians appear to respond more readily to homozooic pituitary injections thanExperiment 2
Both male and female Rana catesbeiana pituitaries and progesterone
proved to be an effective ovulatory stimulus for the bullfrog (Table
5). Consistent ovulation was achieved with pituitaries fromlaboratory frogs, and wild frogs collected in March, July, August,and November. These findings concur with the early investigationsof Wolf (1929) on Rana pipiens and Houssay (1947) on Bulo
arenarum where anuran ovulation was elicited only by homoplastic
pituitary injections of implantations. Simultaneous injections of two
female bullfrog pituitaries and 5 mg progesterone on day I and day
3 produced consistent ovulatory responses. Two or four malepituitaries with progesterone produced partial ovulation. Progesterone was apparently effective in enhancing pituitary ovulatory ac62

Table S-Summary of ovulation responses of bullfrogs to various hormonal stimuli.

N

Animals'

Mean bodywI. (g)

6
3

WC
WC

632
601

4
9
4
7
7
15
15
II
12
7
13
9
15
20

WC
WC
WC
WC
WC
WC
WC
WC
LR
WC
LR
WC
LR
WC

639
523
616
460
451
490
525
530
277
641
286
436
236
585

10

WC

410

9
10
13
10

WC
WC
LR
WC

420
523
427
486

6

WC

624

63

WC

585

16

LR

413

WC

444

10

WC

441

8

F, Straine362

8Lab-cond. fwild bullfrogs
Lab-cond. 8
wild bullfrogs

423

8

437

DosageTreatment

Amphibian LH
2Ol'g
Amphibian LH
20l'g
5 mg
Progesterone
2.5 mgMammalian LH
500 IV
HCG
1000 IVHCG
HCG (l4L:IOD)
500 IVHCG (8L:16D)
500 IV
LH/FSH-RH
l0l'g
LH/FSH-RH
5Ol'g
LH/FSH-RH
100 I'g
LH/FSH-RH
10 I'g
LH/FSH-RH
100 I'g
LH/FSH-RH
10 I'g
LH/FSH-RH
21'g
LH/FSH-RH
21'g
LH/FSH-RH
10 I'gProgesterone
51'g
LH/FSH-RH
21'g
R. catesbeiana pituitaries 2 <;>5 mg
Progesterone
R. catesbeiana pituitaries 2 <;> glands
10 glands 0", <;>R. pipiens pituitaries
10 glands 0", <;>R. pipiens pituitaries
5 mg
Progesterone
10 glands
R. pipiens pituitaries
R. catesbeiana pituitaries 2 <;> glands
R. catesbeiana pituitaries 2 <;> glands
Progesterone
5 mg
R. catesbeiana pituitaries 2 <;> glands
5 mg
Progesterone
R. catesbeiana pituitaries 2 <;> glands
Progesterone
5 mg
R. catesbeiana pituitaries 40"
Progesterone
5 mg
R. catesbeiana pituitaries 2 <;> glands
Progesterone
5 mg
R. catesbeiana pituitaries 2 <;> glands
Progesterone
5 mg
R. catesbeiana pituitaries 2 <;> glands
5 mg
Progesterone'wc = Wild Collected; LR = Lab-rearedbN = Negative; P = Partial; C = Complete'Injection time: 0700, 1000, and 1300 hrs.
dInjection time: 0900 and 1500 hrs.

Ovulation
response b

No.

Frequency

No. animals
with Stage V
oocytes

N

P

C

2
2

Alt. days
All. days

4
2

6
3

0
0

0
0

2
3
2
10
10
3
3
3
21
9
63
28
28
3

Alt. days
Daily
All. days
Daily
Daily
Daily
Daily
Daily
Daily
3 X Daily'
3 X Daily'
2 X Daily
2 X Daily"
Daily

2
3
3
3
3
9
8
4
4
3
5
8
4
6

4
9
4
7
7
15
15

9
15
20

0
0
0
0
0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0
0
0
0
0

4

Daily

6

7

3
3
5
2

Daily
All. days
All. days
All. days

8
6
4
3

9
10
13
10

0
0
0
0

0
0
0
0

2

All. Days

2

4

2

0

2

All. days

28

35

9

19

2

All. days

9

9

4

2

All. days

2

All. days

4

2

Alt. days

2
2

Injections

11

12
7

13

0

7
7

0
3

0

7

3

4

Alt. days

7

0

7All. days

6

6

0

2

eLaboratory-reared animals from parents selected for rapid growth.
fHeld and fed in laboratory two months before injection.
8Second attempt to ovulate the laboratory-conditioned wild bullfrogs(same animals as in f) 8 weeks after the first attempl.

to mammalian gonadotropins, there often appears to be a gonadotropic specificity between genera of anurans. For example, ovulation can be induced readily by injecting homoplastic pituitaryhomogenates in Bufo, but less readily by material from Leptodactylus oce/latus and not at all by extracts from Xenopus pituitaries(Houssay 1947). The specificity may be more acute in Rana as
injection of Rana pipiens pituitaries and progesterone was not aneffective ovulatory stimulus in the bullfrog under conditions of thistest (Penkala 1978) (Table 5).
Various combinations of dosage and circadian injection schemesof LH/FSH-RH failed to elicit ovulation from the bullfrog (Table
5). Likewise, simultaneous injection of LH/FSH-RH and progesterone produced negative results. These findings were somewhat
surprising in view of the fact that LH/FSH-RH has proven to be
an extremely effective stimulus of spermiation in the bullfrog (Easley
et al. 1979) and in other anurans (Licht 1974). Although LH/FSH-

RH stimulates gonadotropin secretion in all classes of vertebrates(Peter 1978), there are questions concerning its specificity. Nanogram doses register overwhelming responses in mammals (Schalley
et al. 1973); but in studies with fishes and amphibians, massive
and frequent doses of LH/FSH-RH were required to induce anovulatory response (Vellano et al. 1974; Thornton and Geschwind
1974; Lam et al. 1975; Lam et al. 1976). Daniels and Licht (1980)
reported that a single injection of LH/FSH-RH stimulated several
hundred-fold increases in plasma LH and FSH levels in adultbullfrogs. However, females were less responsive than males tothe hormone, and the half-life of the molecule was only 10-20
minutes suggesting that only constant profusion or very frequent
injections of the hormone is required to provide an effective
ovulatory stimulus. This was demonstrated recently with R.
catesbeiana (P. Licht, Zoology Dep., Dniv. Calif., Berkeley, pers.
commun. 1985).
63

Table 6 points out the importance of injecting bullfrogs with StageV oocytes. All frogs tested (Table 5) had either Stage IV or V
oocytes predominating, and females with Stage IV oocytes were
not ovulated by any of the hormones.

Table 6-00cyte stage and ovulatory response of bUllfrogs injected withvarious hormone schemes, irrespective of dose and origin of the frog.
Ovulation response

Experiment 3
Tables 7 and 8 show the female ovulatory response to the synthetic
Gn-RH agonist that was effective in sperrniating male and ovulating
female bullfrogs collected in California (McCreery et al. 1982).
The hormone also caused ovulation of atretic, stage ill, and IV eggs.
Attempts to fertilize the stage III, IV, and atretic oocytes were unsuccessful, although Stage V eggs were successfully fertilized. Apparently the agonist is a potent stimulator for ovulation, but notfor promoting egg maturation with the dosages used. The agonist
is, at present, the most promising material for inducing ovulationin bullfrogs. Currently it is commercially available from Bachem,
Inc., Torrance, CA, as LH-RH [Des-GlylO, im-Benzyl-D-His6 ,Pro9] Nhet (Code PLHRI40).

Stage IV

Stage V

HormoneinjectedNo.
animalsNo.

Ovulation

No.

Ovulation

LH/FSH-RH
LH/FSH-RH, progesterone
LH/FHS-RH,
R. catesbeiana pimitaries
HCG
R. pipiens pimitaries
R. pipiens pimitaries
and progesterone
R. pipiens and R. catesbeiana
pimitaries
R. catesbeiana pimitaries
and progesterone
Mammalian LH
Amphibian (R. catesbeiana)
LH

97
20

52
14

0
0

45
6

0
0

10
27
13

4
15
9

0
0
0

6
12
4

3
0
0

10

7

0

3

0

6

4

0

2

2

122
4

58
2

2
0

64
2

59
0

9

3

0

6

0

Table 7-0vulatory response of wild-caught bullfrogs to various dosages ofa synthetic Gn-RH agonist.

No.

Mean bodywt. (g)DosageNo.
injections

Time(days)

Preinjection aoocyte stage

Ovulation
response

3

475

21'g

I/day

2

V

Two partial
One complete

3

435

4/day

2

V

All complete

2

483

4/day

3

V

All complete'Most advanced oocyte stages. All frogs contained Stage I eggs, and a few StagellA eggs, a normal condition.

Table 8-0vulatory response of wild-caught bullfrogs' with different ovarian conditions injected with 3 I'g of a synthetic Gn-RH agonist 3 x /24 hr for 5 days.
Preinjection boocyte stage

Ovulatory responseby Stage'

Postinjection ovary conditionand ovulated oocytes

Frog
No.

Body
wt. (g)414

IV some atretic

No change evident

2

432

III, IV, V normal

No change evident

482

III, IV, V some alretic

P

P

490

III, IV some atretic

P

P

475

IV normal
V some atretic

P

P

524

IV atretic
V few present, normal

4

6

II

III

IV

V

P

Atretic and normal III, IV, V oocytes
ovulated; III, IV oocytes in ovary atretic;
no Stage V oocytes in ovary.
Stage III, IV atretic in ovary, coelomic
cavity, oviduct, and ovisac.

P

Ovulated normal and atretic Stage IV and
V eggs, most atretic. Ovary contained
normal and atretic Stage IV and V.
Ovisac fJ.lled with jelly and nuid.

P

Ovulated a few eggs, Stage V. Ovary
contained Stage IV atretic eggs.

'Collected June, 1981 near Ponchatoula, LA.
bSee Table I. Stage I always present; Stages IIa, lib normally present, but may be absent in recently ovulated females;
Stages ill, IV, V indicate more advanced stages of ovary development. Normal refers to eggs as defined in Table I, i.e.,
diameter, polarity, uniform coloration, spherical shape. Atretic refers to incorrect diameter, poorly defined polarity, mottlingcoloration, vascularization, non-spherical shape.
'Dashes = no ovulation; P = partial ovulation.

64

CONCLUSIONSCITATIONS_This study demonstrates the effectiveness of bullfrog pituitaries andprogesterone and a synthetic Gn-RH agonist in stimulation of ovulation in bullfrogs. The almost total lack of ovulatory response byfemales with Stage IV eggs in this study and atretic eggs (Othman
1978) indicate that the injected materials had little effect on
stimulating oocyte development. The Gn-RH agonist was a potent
ovulator, as even immature and atretic eggs were ovulated. Other
materials failed to elicit ovulation, but the lack of response may
be related to improper injection schemes.
The ovulation response of females from three different populations (but similar climatic environments) collected at different timesof the year indicates that ovulatable females can be obtained fromwild populations throughout the year. However, each individual
must be evaluated for ovarian condition.
Laboratory-reared females, under proper temperature and photoperiod conditions, can be brought to sexual maturity and maintainedin mature condition for an extended period. Wild females withimmature ovaries can be brought to full maturity using living or
prepared diets within 8 weeks. Use of prepared diets appears toalleviate the need for living food, but the ovulatory response may
not be as consistent as when living food is used. The stress of force
feeding the frogs daily may have contributed to the reduced
response, and was evident for females force-fed for 12 weeks.

ACKNOWLEDGMENTS_

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_

The authors wish to thank Gail Marshall, Joe Penkala, and Tom
Graham for technical assistance. This work was supported by the
Research Resources Division of the National Institutes of Health(Grant RR(0635) and the Louisiana Agricultural Experiment Station. The Gn-RH agonist was provided by Paul Licht, Dept. of
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65

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1974. The effect of synthetic luteinizing hormone-releasing hormone on ovulation in the crested newt. Gen. Compo Endocrinol 24:338-340.
WOLF, O. M.
1929. Effect of daily transplants of anterior lobe of the pituitary on reproduction of the frog (Rana pipiens Shreber). Anal. Rec. 44:206.
WRIGHT, P. A.
1961. Induction of ovulation in vitro in Rana pipiens with steroids. Gen. Compo
Endocrinol 1:20-23.
WRIGHT, P. A., and A. R. FLATHERS.
1961. Facilitation of pituitary induced frog ovulation by progesterone in earlyfall. Proc. Soc. Exp. BioI. Med. 106:346-347.

66

INTRODUCTION

Treatment of Fish Hatchery
Water for Solids andTurbidity Removal_

Hatchery systems are primarily of two types: Single pass and recirculating or reconditioning systems. In single-pass systems the water
enters the hatchery, is used once and discharged. In recirculating
systems the used water is filtered and purified in one or more ways
and reused for such purposes as broodstock maintenance or rearing of juveniles. From 90 to 95% of the water is reused in recirculating systems, the other 5-10% made up from fresh water.
In site selection for a fish hatchery, one of the prime considerations is a reliable year-round source of good quality water: freeof pollutants and disease organisms, high in oxygen, within thetemperature range of the organism being cultured, and containing
a minimum of solid and organic material. The purity of the water
is important in reducing the costs of treating incoming water anddecreasing overall operating costs of the hatchery. With single-passsystems the water may only need to be treated once or twice if treatment of effluent is required. With recirculating systems the problem is compounded. Incoming as well as reused water may require
treatment and, in many cases, the effluent may require treatment.
Ever increasing pollution problems make it more difficult to locate
sources of good quality water for hatcheries. For site developmentof cold-water fish hatcheries (trout and salmon), one may still findsources of relatively pure mountain water in the Pacific Northwest
region of the United States. These water sources may require little
or no treatment for once-through systems. For most hatchery-reared
species, however, particularly brackish water and warm-water
species, locating a source of good quality water may be a difficult
task. Coastal waters are becoming more polluted from industry,
sewage treatment plants, power plants, marine oil spills, andagricultural runoff. Freshwater bodies such as lakes and rivers, often
suffer from one or more pollution sources.

THOMAS B. LAWSONAgricultural Engineering Department
Louisiana Agricultural Experiment Station
Louisiana State University Agricultural Center
Baton Rouge, Louisiana 70803

WATER SOURCES_

Water for hatcheries may come from one source or from a combination of several sources including coastal seawater, lakes, rivers,
wells or ponds.

Seawater
The hatchery culture of marine or brackish water species is often
hindered by the fact that it is difficult to locate a coastal site thatis not affected by some type of pollution. Once-productive coastal
areas in the United States are affected more and more by pollution
in the form of industrial wastes, sewage, and oil spills. The location of a suitable coastal hatchery site is further complicated by thesystem of permits and bureaucratic "red tape" that one must go
through in order to locate a hatchery in a coastal region.
It is possible that a hatchery could be developed inland for theculture of marine organisms such as oysters and clams through theuse of commercially available artificial sea salts; this, however,would be feasible only on a small scale. The expense of using
artificial sea salts on a large scale would be prohibitive.

Lakes
Lakes are usually a good source of water, although they are often
affected by pollution sources, either urban, industrial or agricultural.
67

CLASSIFICATION OF SOLIDS

In addition, lakes and reservoirs often harbor fish disease organisms
and contain enough nutrients to promote excessive algal growth.
They also present temperature problems in many cases, due to therise and fall of a thermocline at different times of the year. Intakes
from lakes should be screened to remove trash, small fish, and eggs.

_

The subject of this paper is the removal of solids and turbidity fromfish hatchery water. Removal of various types of solid particles andturbidity from water before it enters the hatchery is important in
reducing the expense involved in providing a good quality environment in which cultured organisms can grow.
In order to better understand the processes involved in removing
solids and turbidity from water, a discussion of the classificationof solids is necessary.

Rivers and Streams
Rivers and streams are often subject to extreme fluctuations in flow,
and may carry considerable amounts of debris which must be
screened out at the intake. The water also may contain large amountsof silt which must be settled out before it reaches the hatchery. Waterfrom rivers and streams may also contain pollutants, excess
nutrients, and disease organisms, thus requiring extensive screening and filtering in addition to disinfection.

Screenable solids
For lack of a better tern, those solids such as twigs, branches, andstones which are large enough to be removed by some type ofscreening device are referred to as "screenable solids." Sticks,
stones, and other forms of large solid materials may damage pumping equipment and clog pipes and water lines. These objects should
be removed at the water intake.

Wells
Water for hatcheries may come from deep or artesian wells, or
springs. Deep wells require pumping and often contain nitrogengas, hydrogen sulfide, carbon dioxide or other gases which must
be removed before the water can be used. Groundwater may contain pollutants or toxic substances such as arsenic, iron, insecticides,
and herbicides. In addition, groundwater is usually very low, orcompletely devoid, in oxygen and must be aerated before use.

Total solids
Analytically, the total solids content of water is defined as the matterthat remains as residue upon evaporation at 103 to 105°C (Metcalf
and Eddy 1972). The material referred to as screenable solids is
not generally determined by analytical methods; therefore, thismaterial is not included in the category of total solids. Classification of particle sizes for analytical determinations are summarized
in Figure 1.

Ponds
Ponds are often a good source of high quality water since the user
generally has control over what goes into the pond. However, ponds
may sometimes suffer the same fate as lakes and may receive pollution in the form of silt from shoreline erosion or chemicals fromagricultural runoff. Nutrients from agricultural areas often cause
excessive algal blooms. In addition, if fish are present in the watersupply pond, they may keep the water stirred up so that the wateris excessively turbid. High levels of turbidity are generally notharmful to fish except in exceptionally high concentrations; but
turbidity does limit spawning somewhat, and it also hinders egg
development.

Colloidal

10- 5

10- 4

'r

I

I

I

Suspended10- 3Size of Particle.mm
Removable By Coagulation

Recycled water

Settleable

Figure I-Classification and size range of particles found in water (Metcalf and
Eddy 1972).

In a strict sense, water which has been reconditioned and recycled
may be considered a water source. It is often feasible to justify theadded expense of reconditioning water for reuse, and in many cases
the same water may be recycled as many as ten times before it isdischarged (Piper et al. 1982). Water may be recycled for a varietyof reasons: The quantity of source water may be insufficient, or
the quality may not come up to the desired standards. The cost oftreating hatchery effluents is reduced considerably when the samewater is used several times before discharge. Continuous quality
control and sterilization of fresh incoming water may be expensive(Piper et al. 1982).

Suspended solids
Suspended solids are those having a particle diameter >0.001 mm(Metcalf and Eddy 1972). This includes solids which will settle out
in an Imhoff cone within a 60-rninute time period. Settleable solids
are those having a particle diameter >0.01 mm (see Fig. 1). Thosesuspended solids having a particle diameter between 0.01 and 0.001
mm will not settle and must be removed from water by other means.

68Colloidal solids

Behavioral changes occur in fish exposed to clay turbidities >20,000
mg/L, and appreciable mortalities result when clay turbidity reaches
175,000 mg/L (Boyd 1982).
Other harmful effects of high turbidity are the diminished oxygen-holding capacity of the water and the difficulty of water disinfection. This second point is especially apparent where ultraviolet (UV)radiation is used to disinfect water. The lethal dosage of UV radiation required to kill microorganisms in water is a function of several
factors: microorganism species, water depth (or thickness of waterfilm), turbidity, and exposure time (Wheaton 1977). Turbidity not
only reduces light penetration into the water, it also sheltersorganisms from exposure to UV light.

Colloidal solids are those classified as having a particle diameter
between 0.001 and 0.000001 mm (Fig. 1). The colloidal fraction
cannot be removed by settling and must be removed from waterby coagulation, chemical precipitation, biological fIltration, or othermeans.

Dissolved solids
Dissolved solids are those classified as having a particle diameter<0.000001 mm (Fig. I). The dissolved solids consists of bothorganic and inorganic ions and molecules that are present in solution in the water. These may include metals such as copper, iron,and zinc, toxic organic molecules of herbicides and insecticides,
un-ionized ammonia, and other substances harmful to fish.

SOLIDS AND TURBIDITY REMOVALA variety of treatment processes are available to the fish culturist
for the removal or reduction of solids and turbidity from water.
The choice depends largely on such factors as water quality before
treatment, degree of required cleanliness, site limitations, and capitol
investment required.

Turbidity
Turbidity is a measure of the light-scattering properties of water.
Colloidal particles, matter suspended in the water, and algae generally all contribute to turbidity. Turbidity may be present in water fromany source, while larger particles such as silt may be present only
in rivers and streams.
The most common method of reporting turbidity is in Jackson
Turbidity Units (JTU's). One JTU = 1 mg/L of turbidity as
measured on a silica scale. Silica was chosen as the means to express
turbidity with 1 mg/L of Si0 2 equivalent to one unit of turbidity(Sawyer and McCarty 1978).

EFFECTS OF
SOLIDS AND TURBIDITY_

Screening
Large objects and particles such as twigs, branches, and stones may
easily be removed by a screening process. The screen may be placed
over the intake pipe (Figs. 2a and 2b), or a screen or bar rack may
be placed within a channel coming into the hatchery (Fig. 3). The
screening device may be a simple mesh screen or bar rack which
requires periodical hand cleaning, or it may be a more elaborate
self-cleaning mechanical bar rack such as used in water treatment
or sewage treatment plants. For a complete discussion of screensand bar racks, the reader is referred to Metcalf and Eddy (1972)
and Wheaton (1977).
Stationary screens are seldom used to trap particles < 1. 5 to 3
mm diameter (Wheaton 1977). Rotating microscreens may be used
to remove particles as small as 5 1.1 (Piper et al. 1982).

_

Relatively large quantities (500 to WOO mg/L) of suspended material
may be tolerated by fish for short periods of time with minimal
effects; however, long-term exposure to certain types of suspended
material may cause clubbed gills in some species (Bell 1973). Fishculturists have noted evidence of gill irritation in trout and salmon
fingerlings held in turbid water, and this is considered a common
avenue of infection by fungi and pathogenic bacteria (Bell 1973).
Fine materials such as silt are detrimental to hatcheries since they
may coat eggs, thus reducing or eliminating the necessary oxygen
exchange. Salmonid eggs may suffer mortalities up to 85 % if
15-20% of the void spaces between eggs are mled with sediment(Bell 1973). This smothering effect may also promote developmentof fungus on the eggs. Silt and other fine settleable material must
be removed from hatchery water before it enters the hatchery.
Turbidity may result from industrial or domestic waste discharges
or it may be caused by living organisms including phytoplankton
and zooplankton. Turbidity caused by plankton is generally desirable
in growout ponds, but algae should not be allowed to enter thehatchery since it will clog pumping and piping systems or fIlters.
Turbidity resulting from humic substances is generally not harmful to fishes, but such waters usually have low pH, low nutrient
levels, and limited light penetration ability for photosynthesis (Boyd1982).
Turbidity resulting from suspended clay particles is extremely
undesirable. This type of turbidity occurs to a large extent in ponds,
such as in South Louisiana, due to the high clay content of the soil.

(a)
SCREEN(b)

Figure 2-Screens over intake to (a) pump and (b) pipe.

69

-D
..
---.

+f-

--------------

Hopper.-~ Sludge Removal
Figure 3-Fixed screen across a channel.

Figure 4-Horizontal rectangular sedimentation basin.

Sedimentation--

Particles too small to be screened, such as pebbles and sand, having
a density greater than water may be removed readily by allowing
them to settle by gravity in still or slowly moving water in asedimenation basin or "grit chamber." There is an unlimited
number of basin design configurations from which the culturist may
choose, but the horizontal rectangular basin, with or without selfcleaning devices (Fig. 4), is probably the most widely used (Wheaton1977). Grit chambers, as they are known in the wastewater treatment industry, are basins in which larger, more dense particles will
settle out within a short period of time. Generally a retention timeof 15 to 60 minutes is sufficient to settle out the larger particles(Bell 1973; Liao et al. 1972). Therefore, basins must be designed
to provide the particle with the proper amount of time to settle.
For example, if the water flow rate is 100 m3/minute, then a basinhaving a volume of 6,000 m 3 is required to provide a retention
time of 60 minutes.
For very small particles, a settling time of several days or weeks
is often required. Therefore, land space must be available to provide large enough settling basins, or else some other means ofremoval must be available.
Sedimentation basins are generally divided into four zones or areas(Fig. 5): inlet area; settling area where particles settling occurs;sludge area where settled particles accumulate; and outlet area. The
inlet and outlet areas are separated from the settling area by baffles
to provide a quiet zone in which particles can settle out with aminimum of disturbance. Sedimentation basin effectiveness isstrongly influenced by each of the four areas and by interaction
between areas. For example, if the water flow rate through the basin
is too high, sedimentation will not occur.
Particles with a density less than the water in which they aresuspended will not settle out regardless of the time allowed. These
particles may be removed by one of two methods: coagulation or
chemical precipitation. Coagulation refers to a process in whicha gentle mixing action brings several particles into contact with one
another. As they clump together, the density of the mass increases
until it becomes heavier than water and then the particulate mass
will settle. The time required may be several hours or days, depending on the size and density of the particles.

--

Inlet
Area

-

-

Settling Area

--

-

--

-

Outlet
Area

- -

__
Sl2dg~e~---- - -- -- ----Figure S-Four areas within settling basin.

In chemical precipitation, a chemical is added to the water whichcauses the particles to clump together in a mass called a "floc."
The floc then settles out of solution or may be filtered out. The
degree of clarification depends upon the amount of chemicals added
and the care with which the process is controlled. Through the useof chemical precipitation it is possible to obtain water essentially
free from solids in suspension or in the colloidal state (Metcalf and
Eddy 1972). In wastewater treatment processes, from 80 to 90%
of total suspended solids, 50 to 55 % of total organic matter, and
80 to 90% of bacteria may be removed by chemical precipitation(Metcalf and Eddy 1972).
The most commonly used chemicals for precipitation in fishculture water are lime (Ca(OHh) and alum, or aluminum sulfate(AI 2 (S04h ·14H2 0). However, use of these chemicals may alter
the pH of the water somewhat; therefore some control must be maintained on the pH.

70

Sand filters
Influent

Sand filters consist of a layer of sand or other particulate material
through which water flows. Filtering is a mechanical process bywhich particles too large to pass through the spaces between sandparticles are filtered out. The maximum particle size which may
pass through the ftlter is detennined by the sand grain size. Generally
sand particle sizes vary from 0.02 to 2 mm (Wheaton 1977). By
using gravel, rocks, and various shaped plastic media, the particle
size removed may be extended upwards.
Sand ftlters may be classified as slow or rapid (Spotte 1970; Muir
1982; Wheaton 1977). In slow sand filters, the hydraulic loading
rate is usually about 0.2 m 3/m 3 of filter bed per day (Muir 1982).
The sand grain size normally varies from 0.3 to 0.4 mm. The
smallest particle size removed with the filter is about 30 J.I (Spotte1970), and the depth of the filter bed is usually no more than 0.7
m (Muir 1982). The filter must be cleaned by periodic backwashing or by scraping off the top layer of sand and replacing it with
washed sand.
In rapid sand filters, there is generally more than one layer offilter material. Three to five layers of various size particles are commonly used, with the layers arranged in increasing size from thebottom of the filter, with the finest material on top (Fig. 6). The
smallest size particle removed by the rapid sand filter is determinedby the sand grain size of the uppermost layer, but generally thesmallest particle removed is the same as for the slow-rate sand filter,i.e., 30 J.I (Spotte 1970).
In rapid sand filters the hydraulic loading rate ranges from 60
to 200 m 3/m 3 of filter bed per day (Muir 1982). The depth of thefilter bed varies from 0.7 to 1.0 m (Muir 1982). These filters must
be cleaned periodically by backwashing and some means of disposalof the backwash must be provided.

FilterTank

Sand Layer
Small Gravel
Large Rock

Collection Hain For Effluent Discharge

and Backwash Inflow.

Figure 6-Rapid sand filter utilizing layers of different sized filter media (Wheaton1977).

A discussion of biological fIltration was included in solids
removal, since the nitrification process just described begins withorganic solids dissolved into solution. As previously mentioned,
natural waters contain very little ammonia, the nitrification process being triggered by conversion of organic nitrogen to ammonia.
However, water that has been previously used for fish culture may
contain appreciable amounts of ammonia.
In biological filter design, the major operating parameters to be
defined are fIlter configuration, flow rate and distribution, wasteloading, surface area of the fIlter media, residence time, and suchenvironmental variables as temperature, pH, oxygen concentration,and organic loading (Muir 1982).
Many types of fIlter media have been tried, the object being toincrease the surface area available for bacterial growth. The rateof ammonia removal can be directly related to surface area in thosesystems where the flow through the media has been evenly distributed (Muir 1982). Some types of media include sand, gravel, stones,
plastic media of various shapes and sizes, styrofoam shapes, wooden
pallets, and plastic netting material. As water passes over the media,
bacteria extract their nutrients, oxygen, and other life needs fromthe water.
There does not appear to be a general system or set of equations
available to design biological fIlters. Most design efforts in the past
have been based on the results of equations used to design submerged
and trickling fIlters in sewage treatment plants. Many researchers
in the past two decades have attempted to describe mathematicallythe design of biological filters for aquaculture use, with varying
success. Their efforts are described at length by Wheaton (1977)
and Muir (1982). Spotte (1970) recommended, as a rule of thumb,that a hydraulic loading rate of at least 42 L/(rnin· m2) of fIlter bed
surface be maintained through the filter. Muir (1982) emphasizedthat for proper nitrification to take place within the fIlter, oxygenmust be available throughout the fIlter bed. At least 1-2 mg/L ofoxygen is required for nitrification to occur normally (Muir 1982).
A simple biological filter consists of a box filled with rocks (Fig.
7). Water enters the top, flows downward through the fIlter, andpasses out through underdrains in the bottom of the box. This typeof configuration is referred to as a downflow filter. Filters of thesame basic configuration may be operated as upflow fIlters, where
the water is forced upwards through the bottom of the box and flows
out the top. Both of these filters generally perform the same, andsince the rate of ammonia removal is related to surface area, theupflow and downflow regimes are equivalent (Muir 1982). One

Biological filters
In biological filtration, the organic nitrogenous compounds in thewater are converted aerobically into nitrate. Simply put, the process is a two-step conversion of ammonium nitrogen (NH: - N)
to nitrate as shown by the kinetic reactions in Equations 1 and 2(Wheaton 1977):(1)NO; + 0.502 = NO)(2)

In Equation 1, ammonium nitrogen is utilized as an energy sourceby autotrophic bacteria of the genus Nitrosomonas and converted
into the relatively toxic nitrite. In the second stage of the process(Equation 2), bacteria of the genus Nitrobacter convert the nitrite
to the nontoxic nitrate. This process is called "nitrification" andis known by every fish culturist.
Although the nitrogen cycle begins with the conversion of organic
nitrogen into ammonia, this stage is usually completed before thewater reaches the biological filter. Ammonia occurs at very low
concentrations in natural waters, or it may be present in fairly highconcentrations in fish culture water that is to be recycled. Un-ionizedammonia (NH 3) and nitrite (NO;) are toxic to fish and otheraquatic animals at very low concentrations (Bell 1973; Boyd 1982;
Wheaton 1977; Spotte 1970), while nitrate (NO) appears to havevery little, if any, toxicity even at concentrations as high as 400
mg/L (Boyd 1982; Wheaton 1977; Spotte 1970).
71

Influent

Water

Collectio
Area
Underdrain
Collection
SystemEffluent

Figure 8-Trickling tilter.

Figure 7-Typical downOow biologicattilter.

general advantage of upflow filters is that they seem to be less affected by solids clogging than downflow filters (Wheaton 1977).
Filters may also be classified as submerged or trickling filters.
Submerged filters are those in which the filter media is kept completely under water. In trickling filters, the water level is maintained below the bottom of the filter (Fig. 8). An advantage of thetrickling filter is that oxygen is supplied by natural air convection,and clogging by solids does not seem to be a problem (Wheaton1977). In both types of filters, the particle size removed depends
upon media particle size.
Another type of biological filter is the rotating biodisc or biodrurn(Figs. 9 and 10). In these systems, fixed media are rotated throughthe water and nitrificiation proceeds as in other previously describedsystems, with nitrifying bacteria growing on the surfaces of therotating media. Here again the emphasis is on designing the media
with a maximum surface area. The discs or drums normally arerotated at about 2-6 revolutions/minute through the water medium,
both aerating and allowing contact with the bacteria growing on
the discs (Muir 1982). Suspended solids are normally separated andrecycled through the tanks at the bottom of the discs (Muir 1982).
Solid material sloughing off the discs are generally more easily
settleable or removed by additional fIltration. Fifty percent settling
occurs within 10 minutes residence time, and 100% settling within
30 minutes (Muir 1982).
The disc surface area required is 0.5 m2/kg of fish for channel
catfish (Muir 1982). A BOD and NH 3 -N removal of more than
95 % occurs at a hydraulic loading of 0.06-0.1 m3/(m2 • day) (Muir
1982). Surface area requirements and loading rates may vary between species. Biodiscs or biodrums may be operated singly or in
multiple stages.

Rotating
Biodiscs

Shaft

Drive
Motor

LiquidLevel-

Influent---

Effluent

Tank(a)

Rotating
Disc(b)

Figure 9-Rotating biodisc system (a) side view and (b) end view.

Other systems
Diatomaceous earth filters
The filtering systems described in this paper are by no means the
only options available to the fish culturist. Other methods exist forremoving dissolved organic material, gases, ions, and moleculesfrom solution. Some of these methods are activated carbon, foam
fractionation, and ion exchange. Because of the complexity of theseprocesses, discussion of each was considered out of the scope of
this paper. For further discussion of these processes, the reader
is referred to Spotte (1972), Wheaton (1977), and Muir (1982).

Diatomaceous earth (DE) filters may be used to remove extremely
fine particles (0.1-5 11) (Muir 1982). In these filters a layer of gradedskeletal remains of diatoms is held against a porous sleeve byvacuum or pressure, and particulate matter is removed as the water
passes through. Because of the expense involved, these systems are
only practical where water of extreme clarity is required. Typicalloading rates are 43-130 m3/(m2 ·day) (Muir 1982).

72

___>>- Eff luent

Figure to-Single-stage biodrum system.

CITATIONS_BELL, M. C.
1973. Fisheries handbook of engineering requirements and biological criteria.
u.s. Anoy Corps of Engineers, Portland, OR.
BOYD, C. E.
1982. Water quality management for pond fish culture. Elsevier Sci. Pub\. Co.,New York, 708 p.
LIAO, P. B., R. D. MAYO, and S. W. WILLIAMS.
1972. A study for development of fish hatchery water treatment systems. U.S.
Anoy Corps of Engineers, Walla Walla, WA.
METCALF and EDDY, Inc.
1972. Wastewater enginering: Collection, treatment, disposal. McGraw-Hili,
New York, 782 p.
MUIR, J. F.
1982. Recirculated water systems in aquaculture. In: Muir, J. F., and R. J.
Roberts (eds.), Recent advances in aquaculture. Croom Helm Pub!., London,
453 p.
PIPER, R. G., I. B. McELWAIN, L. E. ORMA, J. P. McCRAREN, L. G. FOWLER,
and J. R. LEONARD.
1982. Fish hatchery management. U.S. Dep. Int., Fish Wild!. Serv., Wash.,
D.C. 517 p.
SAWYER, C. N., and P. L. McCARTY.
1978. Chemistry for environmental engineering. McGraw-Hill, New York, 532p.

SPOTTE, S. H.
1970. Fish and invertebrate culture. Wiley-Interscience, New York, 145 p.
WHEATON, F. W.
1977. Aquacultural engineering. Wiley-Interscience, New York, 708 p.

73