Acquisitions and
Bibliographic Services Brannn
395 Wellington Street Ottawa, Ontario K1A0N4
NOTICE
Direction des acquisitions e!
des services bibliographiquas
395, rue Wellington Ottawa (Ontario) K1A0N4
Your tiki Voire rdhiwricc
Our ttiti Notit> r&tetnrice
AVIS
The quality of this microform is heavily dependent upon the quality of the original thesis submitted for microfilming.
Every effort has been made to ensure the highest quality of reproduction possible.
If pages are missing, contact the university which granted the degree.
La qualite de cette microforme depend grandement de ia qualite de la these soumise au microfilmage. Nous avons tout fait pour assurer une qualite superieure de reproduction.
S'il manque des pages, veuillez communiquer avec I'universiti qui a confer e le grade.
Some pages may have indistinct print especially if the original pages were typed with a poor typewriter ribbon or if the university sent us an inferior photocopy.
La qualite d'impression de certaines pages peut laisser a desirer, surtout si les pages
originates ont ete dactylographies a I'aide d'un
ruban use ou si I'universite nous a fait parvenir une photocopie de qualite inferieure.
Reproduction in full or in part of this microform is governed by the Canadian Copyright Act, R.S.C. 1970, c. C-30, and subsequent amendments.
La reproduction, meme partielle, de cette microforme est soumise a la Loi canadienne sur le droit d'auteur, SRC 1970, c. C-30, et ses amendements subsequents.
Canada
Essential Fatty Acid Metabolism, Juvenile Requirements and Reproductive Performance of the Chinese Prawn (Penaeus chinensis)
By
Xueliang Xu
Submitted in partial fulfillment of the requirements for the
degree of Doctor of Philosophy
at
Dalhousie University Halifax, Nova Scotia, Canada
February, 1994.
© Copyright by Xueliang Xu, 1994
1*1
of Canada du Canada Acquisitions andBibliographic Services Branch
395 Wellington Street Ottawa, Ontario K1A0N4
Direction des acquisitions t»
des services bibliographiques
395, rue Wellington OUawa (Ontario) K1A0N4
Your Me Vcxre rtt&orKe
Our Me Nolle tiUtence
The author has granted an irrevocable non-exclusive licence allowing the National Library of Canada to reproduce, loan, distribute or sell copies of his/her thesis by any means and in any form or format, making this thesis available to interested persons.
L'auteur a accorde une licence irrevocable eit nor. exclusive permettant a la Bibliotheque nationale du Canada de reproduire, prefer, distribuer ou vendre des copies de sa these de quelque maniere et sous quelque forme que ce soit pour meftre des exemplaires de cette these a la disposition des
personnes interests.
The author retains ownership of the copyright in his/her thesis.
Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without his/her permission.
L'auteur conserve la propriety du droit d'auteur qui protege sa these. Ni la these ni des extraits substantiels de celle-ci ne doivent etre imprimes ou autrement reproduits sans son autorisation.
ISBN 0-315-93803-X
Canada
N a m e
>W/
0**1X.
K.Dissertation Abstracts Intemationafh arranged by brood, general subject categories. Please select the one subject which most nearly describes the content of your dissertation. Enter the c ^responding four-digit code in the spaces provided.
SUBJECT TERM SUBJECT C O D E
Subjed Categories
THE HUMANITIES AND SOCIAL SCIENCES
COMMUNICATIONS AND THF. ARTS
Architecture C729 Art History 0377 Cinema 0900 Danco 0378 Fine Arts 0357 Information Science..,. ,....,..0723
Journalism 039) Library Science 0399 Mass Communications 0708
Music 0413 Speech Communication 0459
Theater 0465
EDUCATION
General , 0515 Administration ,.,., .0514 Adult and Continuing ,...,,.,0516
Agricultural 0 5 1 7 Art 0273 Bilingual and Multicultural 0282
Business. 0688 Community College .,.,,...,...0275
Curriculum and Instruction 0 7 2 7 Early Childhood 0 5 1 8 elementary 0524 Finance 0277 Guidance and Counseling 0519
Health 0680 Higher 0745 History ot 0520 Home Economics ,,.,02.78 Industrial 0521 Language and Literature 0279
Mathematics 0280 Music 0522 Philosophy of 0998 Physical 0523
Psychology 0525 keading 0535 Religious 0 5 2 7 Sciences.,.,, 0714 Secondary...,.,,..,, „ ...0533
Social Sciences 0534 Sociology of 0340 Special 0529 Teacher Training 0530 Technology 0710 Tests anarVieasurements 0288
Vocational 0747
LANGUAGE, LITERATURE AND LINGUISTICS
Language
General 0679 Ancient 0289 Linguistics 0290 Modern 0291 Literature
General 0401 Classical.. 0294 Comparative 0295 M e d i a l 0 2 9 7 Modern 0298 African 0316 American.... 0591 Asian 0305 Canadian (English), , 0352
Canadian (French) 0355 English 0593 Germanic 0311 Latin American 0312 Middle Eastern 0315 Romance 0313 Slavic and Ea.',r Eu'opean 0314
PHILOSOPHY, RELIGION AND THEOLOGY
Phi'osophy 0422 Religion
General 0318 Biblical Studies 0321 Clergy 0319 Hi.toryof 0 3 2 0 Philosophy of 0322 Theology 0469
SOCIAL SCIENCES
American Studies 0323 Anthropology
Archaeology 0324 Cultural 0326 Physical... 0327 Business Administration
General 0310 Accounting 0272 Banking 0770 Management , 0454 Marketing 0338 Canadian Studies 0385 Economics
General 0501 Agricultural 0503 Commerce-Business 0505 Finance 0503 History 0509 Labor 0510 Theory 0511 Folklore , 0358
Geography 0366 Gerontology 0351 History
General 0578
Ancien' 0579 Medievol 0.531 Modern 05d2 Elack 0328 African 0331 Asia, Australia and Oceania 0332
Canadian 0334 European 0335 Latin American 0336 Middle Eastern 0333 United States 0337 History of Science 0585
Low 0398 Political Science
Generaj 0615 International Law and
Relations 0616 Public Administration 0 6 1 7
Recreation 0814 Social Work 0452 Sociology
Geneial 0626 Criminology and Penology ...0627
Demography 0938 Ethnic and Racial Studies 0631
Individual ond Family
Studies 0628 Industrial and Labor
Relations 0629 Public and Social Welfare ....0630
Social Structure and
Development 0700 Theory and Methods 0344 Transportation 0709 Urban and Regional Planning ..,.0999
Women's Studies 0453
THE SCIENCES AND ENGINEERING
BIOLOGICAL SCIENCES
Agriculture
General . . „ . . . , , 0473 Agronomy ..,.0285 A n i m d Culture and
Nutrition 0475 Animal Pathology 0476 Food Science and
Technology 0359 Forestry and Wildlife 0478 Plant Culture 0479 Hani Pathology 0480 Plant Physiology 0 8 1 7 Range Management 0777 W o o d Technology 0746
Biology
General, , 0306 Anatomy 0287 Biostatistics 0308 Botany 0309 C e l l . . . 0379 Ecology 0329 Entomology „ , 0353
Genetics 0369 Limnology , 0793 Microbiology ...0410 Molecular 0307 Neuroscicnce 0317 Oceanography 0 4 1 6 Physiology 0433 Radiation 0821 Veterinary Science 0778 Zoology 0472 Biophysics
General 0786 Medical 0 7 6 0
EARTH SCIENCES
Biogeochomistry Geochemistry
0425 0996
Geodesy 0370 Geology 0372 Geophysics 0373 Hydrology 0388 Mineralogy..,.,., 0411 Paleobotany 0345 Paleoecalogy 0426 Paleontology 0418 Paleozoology 0985 Palynology , 0427 Physical Geography 0368 Physical Oceanography 0415
HEALTH AND ENVIRONMENTAL SCIENCES
Environmental Sciences 0768 Health Sciences
General 0566 Audiology 0300 Chemotherapy 0992 Dentistry 0 5 6 7 Education 0 3 5 0 Hospital Management 0769
Human Development 0758 Immunology 0982 Medicine and Surgery ...,,,..,0564
Mental Health 0347 Nursing 0569 Nutrition 0570 Obstetrics and Gynecology ,.0380
Occupational Health ana
Therapy 0354 Ophthalmology 0381 Pathology..,. 0571 Pharmacology 0419 Pharmacy 0572 Physical Therapy 0382 Public Health 0573 Radiology 0574 Recreation , 0575
Speech Pathology 0460 Toxicology.., 0383 Home Economics 0386
PHYSICAL SCIENCES Pure Sciences
Chemistry
General 0485 Agricultural 0749 Analytical 0486 Biochemistry 0487 Inorganic 0488 Nuclear 0738 O r g a n i c . . . 0490 Pharmaceutical 0491 Physical 0494 Polymer 0495 Radiation 0754 Mathematics 0405 Physics
Gentral 0605 Acoustics 0986 Astronomy and
Astrophysics 0606 Atmospheric Science 0608
Atomic 0748 Electronics and Electricity 0607
Elementary Particles and
High Energy 0798 Fluid and Plasma 0759 Molecular 0609 Nuclear 0 6 1 0 Optics 0752 Radia!;on 0756 Solid State 0611 Statistics 0463
Applied Sciences
Applied Mechanics 0346 Computer Science 0984
Engineering
General 0537 Aerospace 0538 Agricultural 0539 Automotive 0540 Biomedical 0541 Chemical 0542 Civil 0543 Electronics and Electrical 0544
Heat and Thermodynamics... 0348
Hydraulic 0545 Industrial 0546 Marine 0 5 4 7 Materials Science 0794 Mechanical 0548 Metallurgy 0743 Mining 0551 Nuclear 0552 Packaging 0549 Petroleum 0765 Sanitary and Municipal 0554
System Science 0790 Geotechnolcgy 0428 Operations Research 0796 Plastics Technology 0795 Textile Technology 0994
PSYCHOLOGY
General 0621 Behavioral 0384 Clinical 0622 Developmental 0620 Experimental 0623 Industrial 0624 Personality 0625 Physiological 0989 Psychobiology 0349 Psychometrics 0632 Social 0451
To my parents, Xu Fan and Xiong Wanghc
and my wife, Si Xiuying, whose support and encouragement made this thesis possible.
*2
IV
TABLE OF CONTENTS
List of Figures viii List of Tables xi
Abstract xiv List of abbreviations xv
Acknowledgements xvi
Chapter I: Background 1.
1-1 The Status of World Shrimp Production
and The Importance of Shrimp Culture 1.
1-2 Shrimp (Prawn) Culture in China:
Achievements and Problems 2.
!-3 The Goal of The Research and Anticipated Benefits 5.
Chapter II: Literature Review 8.
II—1 Fatty Acid Composition of Crustaceans 11.
II—2 Essential Fatty Acid Metabolism in Crustaceans 14.
II—3 Essential Fatty Acid Requirements of Crustaceans 20.
II—4 The Importance of EFA in Reproduction of Crustaceans 24.
Chapter III: Fatty Acid Composition of the Chinese Prawn, P.chinensis 31.
Ill—1 Introduction 31.
Ill—2 Materials and Methods 34.
III-3 Results 36.
Chapter IV: Effect of n-3 and n-6 Fatty Acids on Survival, Molting
and Growth Rate of Juvenile Chinese Prawn 56.
rV-1 Introduction 56.
IV-2 Materia's and Methods 58.
IV-3 Results 60.
IV-4 Discussion 61.
Chapter V: Essential Fatty Acid Requirement of the Juvenile Chinese Prawn 71.
V - l Introduction 71.
V-2 Materials and Methods 73.
V-3 Results 75.
V-4 Discussion 77.
Chapter VI: Effect of Dietary Lipid Sources on Fecundity, Egg Hatchability
and Fatty Acid Composition of Broodstock of the Chinese Prawn 100.
VI-1 Introduction 100.
VI-2 Materials and Methods 101.
VI-3 Results 104.
VI--4 Discussion 106.
Chapter VII:Influence of Dietary Lipids on Ovary, Hcpatopancrcas and Muscle Fatty Acid Composition of Cultured Chinese Prawn
Broodstock 121.
vi
i
Vll-1 Introduction 121.
VII-2 Materials and Methods 122.
VII-3 Results 124.
VII-4 Discussion 125.
Summary and Conclusions 134.
Bibliography 138.
vn
Fig. II—1 Three families of unsaturated fatty acids derived from dietary
fatty acids or de. novo synthesis 19.
Fig. Ill—1 Variation in total lipid content during the early
developmental stage of P. chinensis 50.
Fig. Ill—2 Variation in saturated fatty acid content during the early
developmental stages of P. chinensis 51.
Fig. III-3 Variation in 16:ln-7 and 18:ln-9 content during the early
developmental stages of P. chinensis 52.
Fig. IU-4 Variation in monocnoic acids content during the early
developmental stages of P. chinensis 53.
Fig. Ill—5 Variation in 18:2n-6 and 18:3n-3 content during the early
developmental stages of P. chinensis 54.
Fig. Ill—6 Variation in n-6 and n-3 HUFA content during the caily
developmental stages of P. chinensis 55.
Fig. rV-1 Effect of n-3 and n-6 faity acids on weight gain of
P. chinensis (with the control diet) 69.
Fig. IV-2 Effect of n-3 and n-6 fatty acids on weight gain and weight
gain per molt of P. chinensis (with the control diet) 70.
viii
1 I
Fig. V - l Regression plot of dietary 18.2n-6 and 20:2n-6 in body
lipid of P. chinensis 88.
Fig. V-2 Regression plot of dietary 18:3n-3 anu 20:3n-3 in carcass
lipid of P. chinensis 89.
Fig. V-3 Effect of dietary n-3 and n-6 fatty acids on growth
of P. chinensis 90.
Fig. V-4 Effect of n-3 and n-6 fatty acids mixtures on growth
of P. chinensis 91.
Fig. V-5 Effect of dietary n-3 and n-6 fatty acids on survival
of P. chinensis 92.
Fig. V-6 Effect of n-3 and n-6 fatty acids mixtures on survival
of P. chinensis 93.
Fig. V-7 Effect of dietary n-3 and n-6 fatty acids on body lipid
content of P. chinensis 94.
Fig. V-8 Regression plot of dietary 20:4n-6 and 20:4n-6 in carcass
lipid of P. chinensis 95.
Fig. V-9 Regression piot of dietary 18:2n-6 and 18:2n-6 in carcass
lipid of P. chinensis 96.
Fig. V-10 Regression plot of dietary 22:6n-3 and 22:6n-3 in carcass
lipid of P. chinensis 97.
IX
I
nof P. chinensis 98.
Fig V-12 Effect of dietary n-3 fatty acids on survival
of P. chinensis 99.
Fig. VI-1 Regression plot of 22:6n-3 content in egg lipid
and hatch rate of P. chinensis 11.6.
Fig. VI-2 Regression plot of 20:5n-3 content in egg lipid
and fecundity of P. chinensis 1.17.
Fig. VI-3 Regression plot of 20:4n-6 content in egg lipid
and fecundity of P. chinensis 118.
Fig. VI-4 Effect of dietary lipid sources on hatch rate
of P. chinensis 119.
Fig. VI-5 Effect of dietary lipid on egg production
of P. chinensis 120.
^•atT'flS^
List of Tables
Table 1-1 Total annual cultured prawn production in China (1980-1990) 3.
Table III—1 Conditions used in Gas Chromatography 44.
Table III—2 Variation in lipid concentrations of ovary, hepatopancreas
and muscle during the ovarian maturation of Chinese prawn 45.
Table III—3 Variation in major fatty acid composition of ovaries of wild
and cultured Chinese prawn 46.
Table III—4 Variation in major fatty acid composition of hepatopancreas
of wild and cultured Chinese prawn 47.
Table III—5 Variation in major fatty acid composition of muscles
of wild and cultured Chinese prawn 48.
Table III—6 Variation in fatty acid composition of total lipids during
the larval development of Chinese prawn 49.
Table IV-1 Basal diet ingredients for diets used in the study of EFA
requirements of P. chinensis 65.
Table IV-2 Purified fatty acids added as triglycerides to experimental
diets used in EFA study of P. chinensis 66.
Table IV-3 Fatty acid analysis of experimental diets 67.
xi
and growth of P. chinensis 68.
Table V - l Basal ingredients for diets used in the study of EFA
requirements of P. chinensis 82.
Table V-2 Fatty acid profile of 12 experimental diets for juvenile
Chinese prawn 83.
Table V-3 Fatty acid analysis of 12 experimental diets 84.
Table V-4 The total lipid analysis of experimental diets and prawn
carcasses 85.
Table V-5 Effect of dietary n-3, n-6 and their mixture fatty acid on
body fatty acid composition 86.
Table V-6 Effect of n-3 and n-6 fatty acids on survival, molting and
growth of juvenile Chinese prawn 87.
Table VI-1 The composition of experimental diets for Chinese prawn
broodstock 111.
Table VI-2 The fatty acid composition of the four experimental diets and
the control diet (clam, Ruditapes philippinum) 1.12.
xu
Table VI-3 Effect of different lipids on fecundity and hatchability
of Chinese prawn broodstock 113.
Table VI-4 Effect of different dietary lipids on egg fatty acid
composition of Chinese prawn broodstock 114.
table VI-5 Fatty acid composition (percent of total fatty acids) of the post spawning and stage III gonadal tissue of broodstock
P chinensis, fed diets containing different sources of lipid 115.
Table VII-1 The composition of experimental diets for Chinese prawn
broodstock 129.
Table VII-2 The fatty acid composition of the four experimental diets and
the control diet (clam, Ruditapes philippinum) 130.
Table VII-3 Effect of different dietary lipids on fatty acid composition
of ovary of Chinese prawn broodstock (at stage V) 131.
Table VII-4 Effect of different dietary lipids on fatty acid composition
of hepatopancreas of Chinese prawn broodstock (at stage V) 132.
Table VII-5 Effect of different dietary lipids on fatty acid composition
of muscles of Chinese prawn broodstock (at stage V) 133.
xiii
Feeding trials on both juveniles and broodstock of the Chinese prawn, Penaeus chinensis were conducted using highly purified n-3 and n-6 fatty acids (l8:2n-6, 18:3n- 3, 20:4n-6 and 22:6n-3) as well as using various lipid sources. In the absence of n-3 or n-6 fatty acids, juvenile Chinese prawns suffered high mortality (96-100%). EFA- deficiency resulted in poor growth and very low total body lipid (0.0071% of body wt.).
Body lipid levels were also extremely low when either 18:2n-6 or 18:3n-3 was fed as the sole dietary unsaturated fatty acid supplements; only a mixture of both 18:2n-6 and 18:3n-3 or longer chain highly unsaturated fatty acids (20:4n-6 or 22:6n-3) added individually or in mixtures were effective in raising carcass lipid levels.
Juvenile prawns fed diets containing a mixture of 18:2n-6 and 18:3n-3 had better weight gains than did those fed on either 18:3n-3 or 18:2n-6 alone. With respect to growth response, the long chain n-3 fatty acid 22:6n-3 had the greatest EFA value among these four n-3 and n-6 series fatty acids, whereas the EFA value of 20:4n-6 was between 18:3n-3 and 22:6n-3 for juvenile Chinese prawns. The results indicate that n-3 and n-6 fatty acids are essential for survival and normal growth of Chinese prawns and EFA values ot the fatty acids in the diet of juvenile Chinese prawns increased in the order: 18:2n-6<18:3n-3<20:4n-6<22:6n-3.
Fatty acid analyses showed that juvenile prawns possess the ability to elongate dietary 18:2n-6 and 18:3n-3 to 20:2n-6 and 20:3n-3, respectively, however, no further desaturation of either n-3 or n-6 of 18 carbon fatty acids was observed, indicating that Chinese prawn juveniles may lack delta-6 desaturase. In its wild habitat most foods ingested by prawns are rich in longer chain n-3 HUFA. Therefore, the results confirm that long-chain n-3 and n-6 HUFA are essential in the diet of Chinese prawn.
Different dietary lipid sources had a great effect on fecundity of broodstock as well as egg hatchability and egg fatty acid composition of Chinese prawn. The present data indicates that the fecundity and hatchability of prawn broodstocks are related to their dietary HUFA content; the highest correlation coefficients were found between 20:5n-3 and fecundity (r2 = 0.844) and between 22:6n-3 and percentage hatch (r2 = 0.852).
Though other explanations of the results are possible, I hypothesize that 20:5n-3 may play a specific role in oogenesis (possibly in combination with 20:4n-6) and that 22:6n-3 plays a critical role in embryonic development leading to successful hatching of the Chinese prawn.
xiv
AA BF3
DHA EFA EPA FA GSI HUFA PUFA PC PE PG PL PS Wt.
16.0 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:6n-3
Abbreviation Arachidonic acid Boron trifluoride
Docosahexaenoic acid (22:6n-3) Essential fatty acid
Eicosapentaenoic acid Fatty acid
Gonadosomatic index
Highly unsaturated fatty acid Polyunsaturated fatty acid Phosphatidylcholine Phosphatidylethanolamine Prostaglandin
Phospholipid Phosphatidylserine Weight
Palmitic acid Linoleic acid Linolenic acid Arachidonic acid Eicosapentaenoic acid Docosahexaenoic acid
XV
The auther is deeply indebted to my supervivors Drs. John D. Castell (cxt.) and Ronald K. O'Dor (int.), for their expert guidance, encouragement, kindness and full support; I would like to also express my sincere thanks to my supervisory committee members, Drs. Gary F. Newkirk, for his help and Robert G. Ackman, for his valuable comments which improved the thesis, and to Dr. Christopher Parrish for undertaking the role of external examiner.
I thank Drs. J. Pringle and S. Lall for their help and providing working space in the Halifax Fisheries Laboratory, Department of Fisheries and Oceans, Canada.
I also would like io express my heartfelt gratitude to the International Development Research Centre (IDRC) of Canada for the financial support, especially to Dr. Brian F. Davy, Mr. Andrew McNaughton, and other officers for their understanding and encouragement during my PhD studies. !, especially, have to thank the Lett Bursary Committee and the Department of Biology, for their timely help when I had financial difficulties in completing my study.
Finally, I am very grateful to my wife, daughter, brother and sisters for their understanding and support during the long haul of completing my PhD studies.
I wish I could spend more words on appreciations to the people who used to help me in different ways during my PhD studies, but it is believed that helping others will be rewaided with good.
xvi
I
Chapter I. Background
1-1 The Status of World Shrimp Production and the importance of shrimp culture
The total harvest of wild shrimp from the oceans increased steadily from 1.349 million MT in 1974 to 2.507 million MT in 1989. Since then it has remained constant. It is clear that, due to over-fishing pressures and limits to the natural recruitment, shrimp production from wild stocks will not increase in the future and may be subject to collapse as has been the case of many other commercially fished species such as cod in eastern Canada.
In addition, there is a widespread opinion that the demand for shrimp is very strong and that it will continue to increase in the future. This idea is supported by the dramatic increase in the price of shrimp in recent years. Shrimp culture is expected to fill the gap in meeting the world demand by consumers. The rapid expansion of shrimp culture in Ecuador, China, South-East Asia and Latin America tends to confirm this viewpoint. In 1990, the world's shrimp farmers produced a record crop; a total of nearly 633,000 metric tons (whole shrimp), up 12% from the previous record harvest of 565,000 metric tons in 1989 (FAO, 1991). Their one million hectares of ponds yielded over 630 kilograms per hectare. Shrimp farmers now produce 25% of the shrimp placed on world markets. In 1980, only ten years ago, shrimp farmers produced about 2% of the world's shrimp total supply.
In fact, there has been a rapid expansion of shrimp farming throughout the v/orld
1
1. Rapid growth rate of many cultured penaeid species. (6 mo to 1 yr to market size).
2. High market price (up to $28.G0US/kg in some markets).
3. Worldwide demand, especially in developed western countries.
4. High nutritional value and good consumer acceptance.
5. Development of technology for hatching and rearing of larval stages.
6. Comparatively simple culture technology.
The future for shrimp culture is quite bright; however, there is one significant restriction - the limitation of seed stock due to a shortage of mature shrimp.
1-2 Shrimp (Prawn) Culture in China: Achievements and Problems
China has thousands of years experience in aquaculturc, and is especially famous for fresh water fish farming. The current expanding prawn culture started in the early 1960's because of shortages caused by over-fishing. When the landings of one of the most important species (Penaeus chinensis) began to decline, the Chinese Government developed a long-term strategic policy to stimulate the advance of China's prawn culture.
Through this policy, the shortages caused by over-fishing have been increasingly replaced with cultured prawns.
Since the 1970's, prawn experimental biology and ecology studies have expanded along with the rapid development of the prawn farming industry in China. Great strides have been made in the mass production of prawn fry and their grow-out to market sized prawns since 1978. The total production of hatchery reared prawn fry and cultivated prawns has increased dramatically in the past decade (from 5,180 MT in 1980 to
3 approximately 200,000 MT in 1990, Table 1-1). Prawn farming has spread from the north coast (where P. chinensis are harvested from the wild) to the south coast of China (where the higher temperatures lead to even faster growth rates). This rapid expansion in the number of prawn farms around the coast has made China the world's top prawn producing country.
Table 1-1, Total annual cultured prawn production in China (1980-1990)
Year Annual Production (MT)
1980 1981 1982 1983 1984 1986 1987 1990
5,180 6,704 14,158 17,950 38,600 80,000 153,300 200,000
P. chinensis (Osbcck) is also known as P. orientalis Kishinouye or the Chinese prawn. This species is grown at higher latitudes than other cultured shrimp. P. chinensis
is naturally distributed in water 90-180 m deep; much deeper than the areas in which other cultured species live. These observations suggest that P. chinensis can tolerate colder temperatures than other cultured shrimp. The natural distribution of P. chinensis is limited to the Asian coast from the mouth of the Pearl River in China to Bohai Bay in the northern Yellow Sea.
P. chinensis reportedly has high growth rates under culture conditions (optimun growing temperature is about 25° C), and tolerates very low salinities (0.86 ppt). P.
chinensis needs to molt approximately 50 times during its one year life circle (its early developmental stages include 6 developmental stages for nauplii, 3 stages for zoca and 3 stages for mysis before becoming postlarvae). Its fast-growing characteristic enable farmers to culture them from postlarvae to market size (about 12-15 cm in length and 30-50 g in weight, however, the wild broodstock females are bigger than cultured prawns, usually, 18-24 cm in length and 80-100 g in weight) within 4-5 months. In addition, its small egg size (half that of P. japonicus) enables nauplii to be reared at high densities (100,000-150,000 per cubic meter sea water, Chavez, 1989). The biological characteristics of P. chinensis can be found in "The Culture of Cold-Tolerant Shrimp" edited by Main and Fulks (1990).
Prawn juveniles are frequently fed nutritionally unbalanced formulated diets consisting of minced fish, mussel, soybean cake, peanut cake and rice bran, depending on the resources locally available. Not all portions of feeds are consumed by the prawns.
Sometimes, feeds are lacking in essential nutrients. At other times, excessive feeding results in pollution of the rearing water. These conditions lead to slow growth, poor feed
5 efficiency, disease conditions and high mortality. More research must be done on the nutritional requirements of prawns, as well as their physiology and biology, in order to improve the quality of the formulated diets and enhance the growth rate and disease resistance. Though some research has been done on the protein and amino acid requirements of P. chinensis (He, 1988), virtually nothing is known of the fatty acid requirements of this species.
The limited supply of wild and pond-reared broodstock remains one of the major constraints in the further expansion of the prawn culture industry in China. In order to solve this problem, many fisheries research laboratories and hatcheries in China have been developing certain techniques for maturing P. chinensis under captive conditions. So far, however, the results are inconsistent. One possible cause for the inconsistency may be related to variability in the type and quality of the broodstock diets used. Research on the effects of nutrition on fecundity and egg quality is vital to this important aspect of prawn culture in China as well as in most other shrimp and prawn producing countries. Since lipids play an important role in ovarian maturation as well as embryonic development, the lipid and essential fatty acid (EFA) requirements in prawn broodstock diets are given a high order of priority in current research.
1-3 The goal of the research and anticipated benefits
The goal of the research can be divided into two main aspects. The first aspect includes determining EFA requirements for the juveniles of Chinese prawn, evaluating the
relative importance of n-3 and n-6 fatty acids, and assessing the prawn's ability to chain elongate and desaturate n-3 and n-6 fatty acids; to see if the chain length of EFA in the diet will affect the survival, molt, growth and body EFA compositions and to establish what is the optimum dietary EFA level for the juveniles of Chinese prawn.
These experiments will not only enable us to better understand basic lipid metabolicm in prawns, but also could benefit prawn culture. The available space for continued expansion of extensive and semi-intensive prawn culture is becoming a limiting factor in China. These culture techniques rely on natural feed production or feeding only to supplement natural feed. Further expansion and improvement of the prawn culture will increasingly depend on the development of intensive culture technology which relies on nutritionally complete formulated feeds. Therefore, the qualitative and quantitative requirements of uietary EFA for the prawn will provide extremely valuable information to the prawn feed producers for making high quality feeds and thus, to enhance the growth and survival rate of the prawn. This will assist in the continued development of prawn culture.
A second aspect of this research program is to determine the importance of HUFA in the Chinese prawn broodstock diet, and to better understand the physiological role of the HUFA in reproductive process of Chinese prawn. Recent studies on nutritional requirements for penaeid maturation have focused on lipids which provide energy, as well as essential nutrients such as sterols, phospholipids and essential fatty acids (Tcshima et al., 1988; Teshima et al., 1988; Millamena, 1989; O'Leary and Matthews, 1990; Lytle et al., 1990). In fact, egg quality and supply are the major bottlenecks in prawn culture. The
7 usual practice, for most penaeid species, is the production of larvae and post-larvae from wild broodstock or wild females matured in captivity. It is very important to improve reproductive performance of captive broodstock and to define the nutritional value of lipids for maturation and reproduction. However, the effect of dietary lipids on biochemical composition of their important organs such as gonad and hepatopancreas, as well as fecundity and hatchability of crustaceans, has not been previously reported.
Therefore, the improvement of reproductive performance, including fecundity, hatchability and larval quality from captive broodstock, is essential. To sum up, these experiments not only fill in the gaps in the fields of nutritional and physiological function of the reproduction of crustaceans in general but also facilitate prawn culture development.
Experimental objectives for juvenile prawn studies.
1. To investigate relative EFA values of 18:2n-6, 18:3n-3, 20:4n-6, 22:6n-3.
2. To determine the optimum dietary level of 18:3n-3 for the prawn.
3. To test effects of n-6 and n-3 mixtures. Possibly both series are essential and some combination may be superior to either type of the fatty acid alone in the diet.
4. To assess relative values of mixtures of long chain (20 and 22-carbons) versus short- chain (18-carbon) n-6 and n-3 fatty acids.
Experimental objectives for prawn broodstock.
1. To investigate the nutritive value of four lipid sources for prawn broodstocks.
2. To evaluate the importance of HUFA for prawn egg development.
i,
3. To determine the relationship between dietary HUFA level and fecundity, and the relationship between egg hatch rate and dietary HUFA level as well.
4. To compare lipids of eggs, ovaries and hepatopancreas of wild versus cultured broodstock and investigate their possible impacts on reproductive- performance.
CHAPTER II: Literature review
Fat is actually a subset of the class of nutrients known as lipids, but the term fat is often used to refer to all the lipids. The lipids include triglycerides (fats and oils), phospholipids, and sterols; all are important to nutrition. The triglycerides provide the body with a continuous fuel supply, keep it warm, and protect it from mechanical shock;
their component fatty acids serve as starting materials for important hormonal regulators.
The phospholipids and sterols contribute to the cell structures, and cholesterol serves as the raw material for hormones, vitamin D, and bile.
Recent advances in nutritional studies on mammals and human (Innis, 1991; Lands, 1987, 1990; Mead, et al., 1986), and early work on fish (Castell et al., 1972; Cowey &
Sargent, 1977; Sargent, 1989; Takeuchi, 1978; Yone, 1978; Watanabe, 1985) and crustaceans (Teshima, 1978; Castell, 1981; Kanazawa, 1985) have demonstrated the importance of lipid nutrition, especially essential fatty acid requirements and their physiological role in humans and other mammals, as well as in marine animals.
Two families of fatty acids, the n-6 and n-3, are known to be essential for normal cell function. The n-6 and n-3 fatty acids accepted as essential in marine fish and crustaceans diets arc linoleic (18:2n-6), linolcnic (18:3n-3), eicosapentaenoic (EPA) (20:5n-3) and docosahcxaenoic (22:6n-3) (DHA) (Castell et al., 1972; Kanazawa et al., 1977, 1979; Watanabe et al„ 1975; Fuji: et al., 1976; Read, 1981; Martin, 1980; Cowey
9
and Sargent, 1977; Xu et al., 1993).
The most commonly accepted shorthand designation used to identify fatty acids involves two numbers separated by a colon. The first number gives the number of carbons. The number following the colon is the number of double bonds. A new notation, the n-, is increasingly replacing the Greek Ji.tga (o>) to indicate the position of the first double bond counting from the methyl end of the fatty acid, (e.g., 18:2n-6 indicates a fatty acid with 18 carbons and two double bonds with the first double bond located between the sixth and seventh carbon atoms counting from the methyl end of the chain).
Many marine crustaceans contain relatively low levels of total lipid (about 1-2% total wet weight); a high proportion (> 50%) of which is made up of polar lipids. These polar lipids, principally phospholipids, are vital components of their biomembiancs.
Phospholipids are important components of cell membranes, comprising 40-50% of the dry weight of these membranes, and function to maintain structural and physiological properties of the membranes. Fatty acids occur in nature; as esters in these lipids. The unsaturated nature of the EFAs is crucial to their role in these complicated process.
There is sufficient evidence that essential fatty acids (EFAs) arc involved in many complicated processes, directly or indirectly. In the absence of dietary EFAs, mammals developed numerous deficiency symptoms including increased membrane permeability, elevated respiration rates and skin lesions. The deficiency has also led to a fatty liver in mammals (Burr and Burr, 1930; Fiennes, et al., 1973), and a high mortality of larvae and juveniles was observed in fish and crustaceans (Castell et al., 1972, 1976; Takeuchi and Watanabe, 1977; Kanazawa et al., 1978; Xu et al., 1993).
11 The unavailability of 18:2n-6, or particularly 20:4n-6, in terrestrial animals' diet results in a cessation of growth that is quickly restored when these fatty acids are made available as reviewed by many authors (Aaes-Jorgensen E. 1961; Alfin-Slater, R. B. and L. Aftergood, 1968; Holman, R. T.1968). Similar results have been found in marine crustaceans (Kanazawa et al., 1977).
Lipids, especially n-3 and n-6 long chain highly unsaturated fatty acids (HUFA), are not only essential for survival and growth of juvenile fish and crustaceans as mentioned above, but are also important for normal maturation in fish and crustaceans (Shimma et al., 1977; Lawrence et al., 1979; Middleditch et al., 1979; Brown et al., 1980; Mourente et al., 1989, 1990; Harrison, 1990; Xu et al., 1993). The subject of lipid nutrition and fatty acid metabolism of fish and crustaceans is complex, but several comprehensive reviews have been published (Sargent et al., 1989; Cowey and Sargent, 1979; Castell, 1982; Kanazawa, 1985).
In recent years, concern has increased that research on nutritional requirements of marine fish and crustaceans, especially on the essential fatty acid requirements and their physiological roles in currently cultured marine animals in most developing countries, is not keeping up with the rapid development of world aquaculture. Because of the differences that exist among species of marine animals, for example, 18:3n-3 can meet the dietary requirement of rainJx.w trout (Castell et al., 1972; Watanabe et al., 1974) and carp (Takeuchi and Watanabe, 1977) for essential fatty acid, but cannot satisfy red sea bream (Fujii and Yone, 1976; Yone et al., 1978). It is thus important not to assume that results of research with one species can be directly applied to feed formulation for
different species. Therefore, studies of qualitative and quantitative EFA requirements of important cultured prawns and on the roles of EFA in reproductive processes will not only provide extremely valuable information to the prawn farming industry, but also enable us to better understand the basic lipid biosynthetic pathway of crustaceans in general. Though some research has been done on EFA requirements of some pcnacids (P.
japonicus, P. monodon), virtually, nothing is known of the EFA requirements of Chinese prawn, P. chinensis.
The purpose of this chapter is to bring together infoi/nation on the nutritional essentiality, content and metabolism of n-6 and n-3 fatty acids of the crustaceans during early development, the growth period and reproduction. The importance of n-3 long chain HUFA supplementation on survival and growth, as well as of n-3 and n-6 fatty acids in the composition of the animal tissue, are also reviewed.
II—1 Fatty acid composition of crustaceans
It is well known that lipids of marine animals generally contain large amounts of long chain n-3 series fatty acids, whereas terrestrial animals contain relatively large quantities of n-6 series of fatty acids. Several reports have been published on lipids and fatty acid composition of marine crustaceans; for example, shrimps, Pandalus borealis (Ackman &
Eaton, 1967), and Crangon septemspinosus (Ackman & Hooper, 1973), lobsters, Jasus lalandii (de Koning & McMullan, 1966) and Homarus americanus (Brockcrhoff ct al., 1968; Harrison, 1991), and prawns, Penaeus japonicus (Guary,1973). These reports
13 indicate that the fatty acid composition of marine crustaceans is similar to that of marine fish (Sargent et al., 1989).
The primary producers in marine ecosystems are the unicellular algae. The fatty acids in the total lipid from growing and dividing phytoplankton contain up to 50% of total fatty acids as n-3 PUFA.
The last decade has witnessed significant advances in the culture of shrimp and prawns (Wickins, 1976). These developments have stimulated interest in the biochemistry of the cultured animals, particularly the effects of diet on animal composition and the identification of essential dietary requirements (New, 1976). Clark and Wickins (1980) analyzed the cultured prawn, Penaeus merguiensis and found that this prawn contains more polyenoic acids (20:4n-6, 20:5n-3 and 22:6n-3) than any other penaeid except Parapenaeopsis stylifera; although this sample was muscle only and hence mostly phospholipid, it indicates the importance of these long-chain n-3 and n-6 HUFAs in the growth and development of these marine crustaceans.
Lipid composition of shrimp may vary with season (Guary et al., 1975). The seasonal changes have been attributed to one or more of the following factors; water temperature, food, stage of development, sex and photoperiod. It has been demonstrated that the fatty acid composition of membrane lipids is significantly affected by temperature (Cossins et al., 1978) and that higher levels of short-chain (18 carbon) PUFA occur at higher environmental temperatures (Martin and Cecaldi, 1977), whereas lowering the environmental temperature enhanced the desaturation activity of fish liver microsomes (Ninno et al., 1974).
Bottino et al. (1980) investigated the effects of two environmental factors on fatty acid composition of three penaeid shrimp, P. setiferus, P. aztecus, and P. duorarum. They indicated that temperature induced fatty acid alterations arc considered to be an attempt to maintain membrane fluidity. On the other hand, variations in environmental temperature are also known to affect the fatty acid composition of phytoplankton (Holton et al., 1964). It is possible that the changes in fatty acid composition observed in shrimp reflects phytoplankton composition transmitted directly or indirectly to the shrimp through their marine food chain (Sargent et al., 1989). It is evident that marine fish and crustaceans (fish such as turbot, halibut, and red sea bream, and crustaceans like American lobster and Chinese prawn, etc.,) contain high levels of long chain n-3 fatty acids, no matter whether they come from cold water or warm water areas. Therefore, it can be deduced that in addition to the important factor of temperature, the type and content of dietary fatty acids may be the direct cause for changing the fatty acid profile of the animals.
The fatty acid composition of marine fish and crustaceans vary according not only to many physical and environmental factors, but also to physiological factors such as stage of development (Mourente and Odriozola, 1990 a, b, c). The fatty acid composition of P. setiferus has been found to change greatly during larval development, from the four major fatty acids (16:0, 16:ln-7, 18:ln-9 and 20:4n-6) in the egg to four major fatty acids (16:0, 18:ln-9, 20:4n-6 and 22:6n-3) in the postlarvae. There was a 66% increase in the amount of 22:6n-3 on a per-unit dry weight basis from egg to postlarvae (Ward et al., 1979). This might indicate the importance of 22:6n-3 in early larval development.
15 It has been suggested that 22:6n-3 may have a physiological role in the nervous system related to the biological properties of membranes (Neuringer et al., 1988; Bazan, 1990), as a modulator of lipid-protein interactions and neural enzyme activities (Neuringer et al., 1988), and as a precursor of lipoxigenase products (Bazan, 1990). The evidence indicates that, in mammals, large amounts of DHA are required during brain development, and DHA is most highly concentrated in synaptic membranes and in the disk membranes of photoreceptor cells of the retina (Bazan, 1990). Therefore, during development and differentiation of the central nervous system, DHA is required especially for synaptogcnesis, biogenesis of photoreceptor membranes and vision in general. This could be one of the most important and essential roles that DHA plays during the early developmental stages of marine animals. Teshima and Kanazawa (1982) reported that, for total lipids, the proportions of highly unsaturated fatty acids such as 20:5n-3, 22:6n-3 and 20:4n-6 increased during the period from ovary to zoea stage in P. japonicus, whereas those of saturated and monoene fatty acids, especially 16:0 and 16:ln-7, decreased during the same period. These results indicated that HUFA of n-3 and n-6 series are essentials and must be supplied in the diet to ensure successful growth and survival of prawns.
II—2 Essential fatty acid metabolism of crustaceans
The n-6 and n-3 fatty acid composition of animal tissue is dependent on the content and type of dietary lipid, the activity of the elongase and desaturase enzymes responsible for synthesis of long chain highly unsaturated fatty acid from their 18 carbon precursors,
and the partitioning of the various n-6 and n-3 fatty acids among oxidation, desaturation ano acylation processes. It is well established that fatty acids are both chain elongated and desaturated in the microsomal fraction of the cell. Thompson (1973) reported that liver microsomes possess a system for deacylation-reacylation in phospholipid biosynthesis.
This is consistent with the theory that a significant proportion of the newly formed fatty acid' was esterified, mainly into neutral lipids and into the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) fractions (Mud, et al., 1992). The latter suggested, therefore, that eel liver microsomes also possess an active deacylation-reacylation mechanism by which they are able to modify the fatty acid composition of their own phospholipid.
Teshima and Kanazawa (1980) pointed out that lipid resynthesis in the intestinal cell of crustaceans probably differs from that of mammals. It has been shown that although marine fish and crustaceans lack the ability to synthesize linoleic and linolcnic acids de novo, many are able to convert such parent acids to the longer chain and more unsaturated fatty acids (Mead et al., 1960; Kayama et al., 1963). Sinnhuber and his co-workers found that linolenic acid has a more essential role than linoleic acid in rainbow trout (Sinnhuber et al., 1969, 1972; Castell et al., 1972). Similarly, several fish and crustaceans rcquiicd n-3 series fatty acids more than n-6 fatty acids, and n-3 highly unsaturated fatty acids exhibited higher nutritional values (Watanabe et al., 1974, 1976; Takeuchi et al., 1976;
Yone and Fujii, 1975; Fujii and Yone 1976; Castell and Budson, 1974; Castell and Trider, 1974).
Holman (1964) and Mead (1971) summarized the evidence of the effectiveness among
17 the various polyunsaturated fatty acids in terms of competition among their acyl-CoA intermediates for desaturation and elongation enzymes. The major pathway for formation of long chain polyunsaturated fatty acids commences with delta-6 desaturation at carbon 6-7 of 18:2n-6 and 18:3n-3 and then elongation to 20:3n-6 and 20:4n-3, respectively, followed by desaturation at carbons 5-6 by the delta-5 desaturase. The resulting 20:4n-6 and 20:5n-3 are elongated to 22:4n-6 and 22:5n-3 and further desaturation at carbons 4-5 by delta-4 desaturase (Brenner 1974, 1981). The pathway gives rise to endogenous formation of a series of carbon 20 and 22 n-6 and n-3 fatty acids. As shown in the flow chart Figure 1.
Recently, some work has been conducted on delta-4 desaturase in animal lipid nutrition in order to clarifying whether long chain (20-22 carbons) n-3 or n-6 fatty acids are essentials for normal survival and growth of the animals. However, there are still some controversies concerning delta-4 desaturase pathway (Voss et al., 1992; Mimouni et al., 1991).
The competitive interaction among fatty acid substrates for desaturation is generally explained in terms of the affinity of the delta-6 desaturase in the order 18:3n-3>18:2n- 6>18:ln-9 (Brenner, 1974,1981). This provides an explanation for the decrease in tissue 20:4n-6, 22:4n-6 and 22:5n-6 which occurs with increased dietary intakes of 18:3n-3, and for the similar inverse relationship between tissue levels of 22:6n-3 and dietary 18:2n-6 (Anding and Hwang, 1986; Garg et al.,1988; Lands et al., 1990). In the absence of cither n-3 or n-6 in the diet, 20:3n-9 generally will increase in the tissue lipids of EFA-dcficient fed animals (Holman, 1968; Mead et al., 1986).
Kayama et al.(1980) showed that the prawn P. japonicus, fed on a fat-free diet for a 50-day period, had lower levels of HUFA, especially in the nonpolar lipids. However, dietary supplementation with the fatty acids 18:ln-9,18:2n-6 and l8:3n-3, increased the levels ofthese respective fatty acids but not the HUFA that would result from elongation and desaturation in the prawn lipids. At the same time, prawns fed the diet containing pollack liver oil increased the percentage of 20:5n-3 and 22:6n-3 acids in the polar lipids and 22:6n-3 in the nonpolar lipids. They found that although the conversion of dietary linoleic acid to arachidonic acid and linolenic acid to eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) in prawn is observed, the conversion ability is not as strong as in the rainbow trout (Castell et al., 1972; Watanabe et al., 1974).
Recent studies with P. kerathurus have shown that 65% of the fatty acids of the total ovarian lipids are incorporated into egg and embryos during spawning (Mourcntc et al., 1991). Bell and Dick (1990) demonstrated that EPA and DHA are major components of the phospholipids of eye membranes of the prawn, P. borealis, indicating that these fatty acids may also play an important physiological role in neural tissues of marine crustaceans.
Crustacean tissues are particularly rich in phospholipids and this phenomenon is well demonstrated in hemolymph. The large amount of phospholipid (about 65% of total lipid) in the blood seems to be characteristic of crustaceans. In many organisms, the predominant plasma lipid serves a transport function and it is reasonable to assume that phospholipids are the principal lipid transport moiety in crustaceans (Chapcllc, 1986).
Studies with fish and crustaceans have demonstrated that linolcnic acid, 18:3n-3 had
Diet or de novo synthesis
Diet 18:3n-3
i
i A-6
1 8 : 4 n - 3 " ^ 20:4n-3
; I I
A-6
18:3n-6-*" 2 0 : 3 n - 6
i
18:0
i
[ A-9
f
18:1n-9
', A-6
f
1 8 : 2 n - 9
A-5 A-5
20:5n-3 - ^ 2 2 : 5 n - 3 20:4n-6 -*• 22:4n-6
2 0 : 2 n - 9
i
' A-5
2 0 : 3 n - 9
A-4?
T
22:6n-3i A-4 ?
T 2 2 : 5 n - 6
Figure II-l. Three families of unsaturated fatty acids derived from fatty acids or de novo synthesis.
v©
greater EFA value than linoleic acid 18:2n-6, for the prawns, P. japonicus (Kanazawa et al., 1977, 1979) and P. chinensis (Xu et al., 1993). Although 18:3n-3 and 18:2n-6 were equally effective in carp, Cyprinus carpio (Watanabe et al., 1975) and eel, Anguilla japonica (Takeuchi et al., 1980), 18:2n-6 was more effective than 18:3n-3 in Tilapia
zillii (Kanazawa et al., 1980) and Tilapia nilotica (Teshima et al., 1982; Takeuchi et al., 1983). In contrast to these animals, neither 18:2n-6 nor 18:3n-3 exerted a growth promoting effect for red sea bream, Chrysophrys major (Yone and Fujii, 1975; Fujii and Yone, 1976). However, marine animals fed diets containing highly unsaturated fatty acids, such as EPA (20:5n-3) and DHA (22:6n-3), yielded higher weight gains than diets with either 18:2n-6 or 18:3n-3 in all of the above mentioned animals (Yu and Sinnhuber, 1972; Fujii et al.,1976; Takeuchi and Watanabe, 1977; Kanazawa et al.,1978; Tcshima, 1978).
Recent studies with marine fish have suggested that they accumulate more DHA in neural tissues than in non-neural tissues (Tocher and Harvie, 1988; Henderson and Tocher, 1987). Mourente et al. (1991) demonstrated that DHA specifically accumulated in juvenile turbot brain during development, indicating a DHA requirement for nonnal early development of the brain. Proportionately higher growth and development of neural rather than other tissues occur after hatching, therefore, n-3 HUFA, especially DHA, must be provided in the broodstock diet to ensure successful embryonic development and healthy offspring.
There is sufficient evidence to postulate that marine crustaceans have a dietary requirement for n-3 and n-6 series fatty acids. The presence and effectiveness of the
21 biochemical pathway to chain elongate and desaturate 18:3n-3 or 18:2n-6 fatty acid would ultimately determine whether there would be a specific dietary need for HUFA (20:4n-6, 20:5n-3 and 20:6n-3); certain species of marine fish and crustaceans do have a specific requirement for 20:5n-3 and 22:6n-3 because of the absence of this pathway (Cowey and Tacon, 1983).
Evidence for a specific metabolic function for 18:3n-3, other than as a precursor of n-3 HUFA, has not been published. Results to date indicate that 18:3n-3 is the essential dietary n-3 fatty acid in species with de.ta-6 desaturase activity, although 20:5n-3 and 22:6n-3 are metabolically more active n-3 fatty acids with regard to normal cell membrane composition and function. It is reasonable to infer the n-3/n-6 ratio used as an index of fatty acid status in marine fish and crustaceans fatty acid studies is probably improper; therefore, 22:6n-3 and 20:4n-6 content should be accepted as the critical indices to assess fatty acid status of marine animals.
Most work on EFA metabolism has been done on mammals and little is known about marine crustaceans. Therefore, much more needs to be learned about the four different desaturases that act at the 9-10, 6-7, 5-6, and supposedly at the 4-5 carbon positions of the acyl-CoA during the fatty acid metabolism of crustacean.
II-3 Essential fatty acid requirements of crustaceans
Although studies on lipid metabolism in crustaceans were conducted in the early 60's (Zandee, 1962), significant studies on the essential fatty acid requirements of marine
crustaceans were not initiated until the mid-to-late 1970's. Kanazawa et al., (1979) demonstrated that, unlike rainbow trout, marine crustaceans exhibited only limited ability to elongate and desaturate 18 carbon polyunsaturated fatty acids of the n-3 and n-6 scries to the longer-chained, more highly unsaturated forms, which were physiologically essential, and that these had to be supplied in the diet. In studies with P. japonicus (Kanazawa et al., 1977), P. indicus (Read, 1981), P. serratus (Martin, 1980) and P.
chinensis (Xu et al., 1992), it has been shown that 18:3n-3 has greater essential fatty acid (EFA) value than 18:2n-6 and, with P. japonicus, and that longer chain n-3 highly unsaturated fatty acids such as eicosapentaenoic acid 20:5n-3 (Kanazawa et al., 1978) and docosahexaenoic acid 22:6n-3 (Kanazawa et al., 1979) have greater EFA value than 18:2n-6 or 18:3n-3.
Linoleic acid (18:2n-6) is an important essential fatty acid for mammals, and it is converted in vivo to arachidonic acid (20:4n-6) which possesses higher EFA activity (Lands, 1986). However, investigations of the nutritional requirements of marine fish and crustaceans have shown that fatty acids of n-3 family are of greater EFA value than those of the fatty acids of the n-6 family (Castell et al., 1972; Kanazawa et al., 1.979; Xu et al.,
1992).
Colvin (1976) fed P. indicus with selected seed oils and found that there appears to be a limited capacity for biosynthctic change of assimilated fatty acids to polycnoic forms of longer chain length. Thus the conversion of n-3 family fatty acids to 20:5n-3 or 22:6n-3 did not appear to be superior in prawn fed a 5% linseed oil (rich in 18:3n-3) diet, despite the high dietary and tissue depot levels of the precursor, 18:3n-3. Similarly,
23 it was apparent that the high levels of linoleic acid in all diets did not result in the synthesis of longer chain polyunsaturates of the same fatty acid series (ie. 20:4n-6 or 22:5n-6).
Kanazawa and his co-workers (1977, 1978) studied the biosynthesis of fatty acid in the prawns, P. japonicus, P. monodon and P. merguiensis using radioactive acetate and found that radioactivity was mainly associated with palmitic (16:0), stearic (18:0), palmitoleic (16:1), oleic (18:ln-9) and 20:ln-9 acids, but only slightly with linoleic (18:2n-6), linolenic (18:3n-3), EPA (20:5n-3) and DHA (22:6n-3). These results were also supported by EFA requirement feeding trials (Kanazawa et al., 1977, 1978, 1979, 1980; Guary et al., 1976; Shewbart et al., 1974; Colvin, 1976).
In the mid-70s, many workers concentrated studies on EFA requirements of crustaceans and demonstrated that, like marine fish, marine crustaceans lack the ability for dc novo synthesis of n-6 and n-3 fatty acids, which perform essential biological and physiological functions and must be supplied in the diet (Castell et al., 1972, Fujii and Yone, 1976, Kanazawa et al., 1979, Watanabe et al., 1974 and Yu and Sinnhuber, 1977).
Kanazawa et al. (1978, 1979) reported that n-3 fatty acid requirements of P. japonicus was 1% of the diet, and elevation of 20:5n-3 or 22:6n-3 levels from 1% to 2% gave no further improvement in the weight gains of prawns. Since these two fatty acids play important roles in fish and crustacean nutrition and physiology, it is important to better understand their physiological role and nutritional requirement.
Some studies on the nutrition of fish and crustaceans have produced evidence implying a general requirement for long chain (>20C) polyenoic acids. Among these, the
fatty acids of the n-3 variety which alleviate EFA deficiency symptoms in fish (Lee et al., 1967; Castell et al., 1972; Owen et al., 1972) and prawns (Shewbart and Mies, 1973;
Sick and Andrews, 1973) are most important.
Studies on EFA requirements of crustaceans have shown that the nutritional value of lipids is largely a function of the type and content of unsaturated fatty acids. Lipids such as pollack liver oil, rich in n-3 highly unsaturated fatty acids (HUFA) have been reported to have high nutritional value for P. duorarum (Sick and Andrews, 1973). Similarly, Guary et al. (1976) showed that sardine oil and short-necked clam oil had a beneficial effect when added to the diets of P. japonicus, AQUACOP (1978) reported that cod liver oil (also high in n-3 HUFA) produced the best growth and survival of P. merguiensis as compared with other dietary lipid supplements tested.
Recently, it has been demonstrated that a deficiency in n-3 fatty acids influences the fatty acid composition of the brain of rat, in particular by reducing the levels of 22:6n-3 (Bourre et al., 1984). Changes in fatty acid profiles of the brain following recovery from n-3 fatty acid deficiency in the rat take place at a very slow rate compared to other tissues (Youyou et al., 1986). The deficiency of n-3 PUFA is correlated with reduced visual performance in rhesus monkeys (Neuringer et al., 1986), with effects on rat learning ability (Bourre et al., 1989). Xu et al., (1992) conducted EFA requirement studies on P. chinensis using purified 18:2n-6, 18:3n-3, 20:4n-6 and 22:6n-3, and found that the Chinese prawn is unable to desaturate 18 carbon n-3 or n-6 fatty acids to longer chain HUFAs and that the best weight gain was obtained with the prawn fed a diet containing 1% of 22:6n-3. In marine animals and especially in crustaceans, the
25 information on the origin and the role of n-3 fatty acids so far remains scanty. It is evident that marine crustaceans lack delta-6 desaturase to convert 18 carbon acids to HUFA, therefore, 18:2n-6 and 18:3n-3 which are essential for mammals might not meet the EFA requirement of marine crustaceans. The essentiality of 18:2n-6 and 18:3n-3 is questionable and HUFA of both n-3 and n-6 series (22:6n-3 and 20:4n-6) are probably essential for marine crustaceans. Clearly the dietary EFA requirement of any crustacean is a result of the sum of the requirements of all the organs and tissues (such as brain, eyes etc.) that make up that animal.
II—4 Importance of EFA in reproduction in crustaceans
There have been many attempts to attain a successful maturation of various penaeid shrimps in captivity by controlling endocrinological and environmental conditions. The more common techniques include eyestalk ablation, hormonal dispensation, regulation of temperature and photoperiod, and combinations of these factors (Kanazawa, 1982). In addition to these factors, successful maturation of ovaries possibly depends on the nutritional status of broodstocks (Brown et al., 1980; Lawrence et al.,1980; Chamberlain and Lawrence, 1981).
It is known that the X-organ-sinus gland complex of the eyestalk controls both molting and gonad development via processes of hormonal inhibition. Molting-inhibiting hormone (MIH), secreted by the sinus gland, actively inhibits Y-organ synthesis of crustecdysone (Skinner et al., 1985). Gonad-inhibiting hormone (GIH), also secreted by the sinus gland (Adiyodi and Adiyodi, 1970; Charniaux-Cotton and Payen, 1988) appears
to inhibit the reproductive process by mechanisms which are as yet unidentified.
However, it has been well documented that removal of eycstalks accelerates both maturation and molting rate in crustaceans (Adiyodi and Adiyodi, 1970; Skinner ct al., 1985; Koshio et al., 1991).
Several studies which document fatty acid compositions of the organs of different shrimp species have demonstrated that, throughout ovarian maturation, ovarian lipids contained higher proportions of 20:5n-3 and 22:6n-3 than the hepatopancreas (Tcshima and Kanazawa, 1983; Jeckel et al., 1989; Ji and Xu, 1992). Research on nutrition of P.
monodon broodstock showed that ovarian lipid levels increased from 5.8% in immature prawns to 17% in fully mature wild females and from 7.5% to 21% in wild ablated females (Millamena et al., 1984). The content of highly unsaturated fatty acids, HUFA (20 and 22-carbon) ranged from 12 to 25% of total fatty acids of both unablatcd and ablated wild prawns. The high content of these HUFA in the broodstock prawns and eggs probably indicates their importance in the prawn reproductive process. Harrison (1990) published a comprehensive review of reproduction of crustaceans and emphasized that excess 18:3n-3 may compete for available enzyme and result in competitive inhibition of the conversion of 18:2n-6 to n-6 family fatty acid metabolites. This may particularly reduce the levels of arachidonic acid (20:4n-6) which is an important precursor of prostaglandins (PGs) in insects and vertebrates. This fatty acid has also been identified as the precursor for prostaglandin in higher vertebrates (Kinsclla, 1987). PGs have been demonstrated to play a critical role during the ovulatory process in tclcosts (Goctz, 1983).
Therefore, low levels of 20:4n-6 in the broodstock diet or in ovary may lead to low
