ASEAN Cooperation in Food, Agriculture and Forestry

MANUAL

PRACTICAL GUIDELINES FOR

THE DEVELOPMENT OF HIGH HEALTH

Penaeus monodon BROODSTOCK

F i sher i es P u b l i c a t i o n S e r i e s No.2

FORWARD

The Manual on Practical Guidelines for the Development of High Health Penaeus monodon Broodstock has been prepared by the ASEAN Fisheries Sub – Working Group which is under the assignment of the ASEAN Sectoral Working Group on Fisheries since 1996.

Sincere thanks to the Fisheries Agencies of ASEAN Countries which have supported the work in the preparation of the guidelines. It is hoped that this guidelines would benefit ASEAN’s shrimp culture industry in terms of its sustainability.

For the improvement of the Guidelines, comments and suggestions are welcomed. The comments and suggestions could be submitted to the ASEAN Sectoral Working Group on Fisheries through the ASEAN Secretariat.

CONTENTS

Part A:GUIDELINE ON HOW TO PRODUCE HIGH-HEALTH Penaeus monodonBROODSTOCK 1

Background and Rationale

Selection of Wild Broodstock

Breeding Plan

Production Process of Primary Stock

Facilities Requirement

References

Part B: EVALUATION OF BROODSTOCK AND POST-LARVAL QUALITY

I.Broodstock

  1. Viruses

1. Monodon baculovirus (MBV) Disease

2. Hepatopancreatic parvovirus (HPV)

3. White Spot Disease

B. Bacteria

1. Vibriosis

II.Post-larvae

What is considered as ‘good quality Postlarval Shrimp’?

Pathogen Detection

Proper Management

References

Part C: CRITERIA FOR SELECTION OF OFFSPRING

I. Morphology

II. Stress test

Part D: IDENTIFICATION OF DISEASE

I.Virus

A. Wet mounts

B. Histology

  1. Polymerase Chain Reaction
  2. In situ Hybridization
  3. Immunochemistry

II. Bacteria

  1. Gram Staining
  2. Standard Culture Method, Identification by Classical Method and Rapid Test Kits
  3. Culture and General Tests
  4. Polymerase Chain Reaction
  5. Immunochemistry

* * *

A GUIDELINE ON

HOW TO PRODUCE HIGH-HEALTH Penaeus monodon BROODSTOCK

BACKGROUND AND RATIONALE

The production of certified high-health domesticated Penaeus monodon shrimp broodstock is important to sustain the shrimp culture industry in ASEAN countries. Hence, the development of a guideline on selective breeding program of domesticated P. monodon was proposed and discussed. Based on the last meeting held in Indonesia on 25 – 27 March 1998, the participants from ASEAN countries had agreed that it is more viable to produce a manual on the general guidelines in producing “high-health” (HH) broodstock instead of the original plan of producing disease-free broodstock which is difficult to be achieved. The purpose of this guideline is to develop a husbandry protocol for a sustainable supply of high-health domesticated shrimp broodstock. HH shrimp in this guideline is defined as those with improved survival and growth rates and specific pathogen free or SPR broodstock. Improvement of survival and growth rates to marketable size is the top priority to reduce production cost. Likewise, better-feed conversion efficiency and tolerance to various types of environment are important criteria to be considered.

Once a high-health shrimp breeding plan is established, selective breeding can be initiated to converse genetic integrity of the HH shrimp and avoid inbreeding. Current breeding procedures permit accurate identification of the maternal parent a cross but not the paternal parent. Therefore, pedigrees based on maternal families will be maintained for HH population. The breeding program developed for P. vannamei was adopted in this guideline with modifications. The major activities for the establishment of high-health stock are : quarantine and screening protocols, breeding strategies, facility requirements for each step, and management of breeding centers and multiplication stations.

SELECTION OF WILD BROODSTOCK

A genetic survey to assess the genetic diversity of different shrimp populations in each country will be done. A large amount of genetic variability is needed to ensure the success of any genetic improvement program.Allozyme electrophoresis and the more advanced DNA analysis will be used in the screening and genetic evaluation of the different populations. At least 3 populations should be selected and 10 to 12 individual families will be used in the breeding plan. The microchip, bird band and plastic home-made eye tags can also be used for tagging the broodstock. Wild female broodstock with a minimum weight of about 120 g and male weight 70g up will be selected. These brooders should have as perfect phenotype as possible with no signs of infectious cuticle and gonads. The unmated female is preferable. All selected broodstock have to pass the screening for important specific diseases such as SEMBV and MBV using protocol included here. A summary of the different production activities of high health (HH) Penaeus monodon broodstock for each population is shown in Table 1.

Table 1.Production scheme for high health Penaeus monodon broodstock

Working group 1 / Working group 2
1). Selection of wild broodstock from many locations based on the population genetic evaluation.
2). Selection of larvae HH-P15 for grows out. Before the first grow-out the high environmental variance in growth rate should be minimized. Screening protocol for PL15 should also be incorporated.
3). Selection of 120 days HH-F0 (large size and photogenic screening)
4). Selection of 7 months HH-F0 (large size and photogenic screening) / 1)Set up nuclear breeding centers (NBC)
2)Preparation of facilities for spawners and offspring in NBC
3)Assignment of multiplication centers (MC) for grow out of improved HH- P15
4)Pond preparation for grow out of HH- P15 (HH-F0)
5)Rearing of HH-F0 to 120 days (size sampling every 30 days, growth rate determination)
6)Rearing of HH-F0 (mixed and individual family) 120 days to 7 months female : male = 1:2
5). Selection of 10 months (or 1 year) HH-F0 (HH-F0 broodstock or the primary stock) for production of HH-F1 larvae
6). Repeating step 2) to 5) for production of HH-F2 larvae
7). Repeating step 2) to 5) for production of HH-F3 larvae
8). Unselected control line will be simultaneously developed for the proper comparison of selected offspring’s after three generations of breeding / 7)Rearing of 7 months HH-F0 to 10 (or 12) months, female : male = 1:2 (obtain HH-F0 broodstock)
8)Repeat step 2) to 7) for HH-F1, HH-F2 and HH-F3 broodstock to be cultured in the MC
Breeding plan

The breeding plan must consider several genetic parameters such as breeding value of individuals, parent-offspring relationships, and genetics gain from progeny testing of full sib and half sib. The following mating plan should be applied for each generation:

  1. Stock four females from each of 10 to 12 families and four males from each of 10 to 12 families into maturation tanks (recommended 3 tons).
  1. After 7-10 days period, collect all females mated (to eliminate multiple mating by a single male). In families with unsuccessful spawning, artificial insemination will be used with randomly chosen males.
  1. Spawn at least one female from each family and stock the offspring in individual larval rearing systems.
  1. The reproductive performance of each female such as spawn size, fertilization and hatching rates, and nauplii count and quality should be recorded.
  1. Nauplii from each family will be reared to PL 15-20 in separate 1-3 ton tanks. The PL 15-20 from each family will then be reared separately in outdoor tanks or earthen ponds up to 4 months or 120 days.
  1. The 120 days shrimp 5-20% of fast growth male and female in each family will be selected and tagged, smaller shrimp will be discarded.

Production process of primary stock

P. monodon (PL 15-20) from pond-reared healthy broodstock F0 (wild parent) are cultured for 120 days in earthen pond at 10 – 15 pcs/m2. The fast growers are selected and transferred to other ponds while the densities are reduced twice until the final density is 0.5 – 1.0 ind/m2. They are selected as breeders for the production of F1 by natural mating and artificial insemination technique. Forty pairs of broodstock collected from different localities are acclimated in separate maturation indoor tanks for 7 days.

During this period, broodstock are fed with formulated and fresh feeds. The feeds are monitored for the presence of pathogens. Females are unilaterally eyestalk ablated. When gravid females are obtained they are allowed to spawn separately in 500-L black cylindrical tanks. Spawners are taken out the following morning after monitoring the number of females that spawned. Eggs are washed with chlorinated seawater and transferred to the hatching tank of the same size with disinfected water at 28-30 ppt. Parameters (fecundity, % fertilization and hatching rates, and nauplii count and quality) for reproductive performance of each female are taken. Nauplii from each female from each family are reared in 3 - 6 ton tank at 50 ind/L until PL 15-20 Survival rate from each batch are recorded. Post larvae are transferred to other 6 – 10 tons concrete ponds or tanks for future observation of growth until they can be tagged. Then the tagged juveniles are pooled in concrete or earthen ponds for 120 days. Body weight (BW) are measured to the nearest 0.01 g and 0.1 cm for body length (BL) from post orbital edge to tip of telson. Samples are taken for morphometric and non-morphometric characters on the 30th , 60th and 120th day. Survival rate, feed conversion ratio, coefficient of variation in size, gonad and mating conditions, and male and female ration of offspring from each family and population are determined on the 120th day. Significant growth differences among shrimp progeny derived from different broodstock will be monitored.

The best growing stock is used as the “primary stock” for the subsequent production of the next generation of domesticated. P. monodon (F2) (Fig. 1).

Pathogenic screening for non-carrier viruses is done from each culture phase (broodstock, hatchery and grow-out).

Fig. 1.Protocol for Production of High-Health Broodstock

F0 WildPop 1Family 1 2 3 ……..10 females

BroodstockRandomFamily 1 2 3 …… 10 males

Offspring

F1 offspring

Phatogenic screening forSelection for growth

Non-carrier of virusesand survival rate

Photogenic screening forSelection of the top

non-carrier of viruses5-20%

HH-F1 Brookstock

Photogenic screening for

Non-carrier of viruses

Repeat all steps for next generation evolution for genetics gain in growth and survival rate from each family after the third generation

* In case individual tagging cannot be done, the progeny from the mating of each maternal line are reared in separate tanks until marketable size or until 120 days to ensure continuity of the maternal pedigrees. The families are then be mass reared in broodstock ponds after tagging with bird band at the eyestalk.

Facilities requirement

Implementation of the HH broodstock shrimp-breeding program requires a network of facilities for successful operation. Facilities for maturation, spawning, larval rearing, nursing, grow-out, quarantine and pathogenic screening are required. The most important is the nucleus breeding centers (NBC) where the selective breeding activities are conducted under the strict quarantine procedures. Post larvae from the best strains obtained from the selection process are distributed to multiplication centers (MC). MC will produce HH post larvae to HH broodstock. Hence, the center must have full quarantine capabilities to maintain HH status and to exclude pathogens throughout 10 months or one-year broodstock cycle. Each facility should be capable of maintaining at least 40 families from each four populations. The resulting HH broodstock are then transferred for testing at government and private commercial hatcheries for seed production (Fig.2).

Fig.2.Facilities Required for this Programme

Population 1,2, If possible more than

3,4 PQA for each Family ..10 (or PopulationMore)

More than 1 NBC for Each population

Improved genetics selection families

More than 1 MC for Each population

REFERENCES

Gall, G.A.E. 1990. Basis for Evaluating Plans. Aquaculture. 85:125-142.

Gjeddrem, T. 1985. Improvement of Productivity through Breeding Schemes. Geojournal. 10(3): 233-241

Gjeddrem, T. and E. Fimland. 1995. Potential Benefit from High-Health and Genetically Improve Shrimp Stocks. Proceeding of Special Session on ShrimpFarmingBatonRange. Lousiana USA

Kasornchandra, J.,S. Boonyaratpalin, R. Khongpradit and U. Aekpanithanpong. 1995. Mass Mortality Caused by Systematic Bacilliform Virus in Cultured Penaeid Shrimp, Penaeus monodon, in Thailand. Asian Shrimp News. 5:2-3

Lannan, J.E. and J. Wyban. 1990. Broodstock Management Program for SPF Shrimp. Unpublished manuscript. The Oceanic Institute. 24p.

Lightner, D.V., R.M. Williams, L.L. Mohney, J.P.M. Clerx, T.A. Bell and J.A. Block. 1985. Recent Advances in Spenaeid Virus Disease Investigations. J. World Mariculture Soc. 16:267-274.

Menasveta, P., A.W. Fast, S. Piyatirativorakul, and S. Rungsupa, 1991. An Improved, Closed Seawater Recirculation Maturation System for Giant Tiger Prawn (Penaeus monodon Fabricius). Aquaculture Engineering 10:173-181

Millamena, O.M., R.A. Pudadera and M.R. Catacutan. 1985. Effect of Diet on Reproductive Performance of Wild Ablated Penaeus monodon Broodstock. In Proceedings of the First International Conference on the culture of Penaeid Prawns/Shrimps. Aquaculture Department, Southeast Asian Fisheries Forum. Asian Fisheries Society, pp 178-179. Y. Taki, J.H. Primavera and J.A. Llobrera (eds.), Manila, the Philippines.

Quinition, E.T., R.M. Caballers and L. Gustilo. 1993 Ovarian Development in Relation to Changes in the External Genitalia in captice Penaeus monodon. Aquaculture 114-:71-81

Refstie, T,.1990. Application of Breeding Schemes. Aquaculture. 85:163-169

Sano, T,.T. Nishimura, H. Fukuda and T. Hayashida, 1984. Baculovirus Mid-gut Gland Necrosis (BMN) of Kuruma Shrimp (Penaeus japonicus) larvae in intensive culture systems. Helgolander Meeresunters, 37:225-264.

Sugama. K. Haryanti and Samuel, L. 1998. Breeding and Genetic Study in Tiger Shrimp, Penaeus monodon. P. 33-40 In Report of the Workshop on Formulation and Assignment of Activities for the Development of Disease Free Penaeus monodon Broodstock in ASEAN Countries. 23-25 March 1998, Bumi Wiyata Hotel and TrainingCenter Depok, West Java, Indonesia.

Sunden, S.L.F. and S.K. Davis. 1991. Evaluation of Genetic Variation in a Domestic Population of Penaeus vannamei (Boone): A Comparison with Three Natural Populations. Aquaculture. 97:131-142.

Wyban, J.A., J.N. Sweeney and R.A. Kanna. 1998. Shrimp Yields and Economic Potential of Intensive Round Pond System. J. World Aquaculture Soc. 19(4): 210-217.

Wyban, J.A., J.S. Swingle, J.N. Sweeney and G.D. Pruder. 1992. Development and Commercial Performance of High-Health Shrimp using Pathogen-free (SPF) broodstock Penaeus vannamei. In Proceeding of the Special Session of Shrimp farming. World Aquaculture society, Baton rouge, L.A., USA. Pp 254-260.

Yano, I. 1993 Ultraintensive Culture and Maturation in Capacity of Penaeid Shrimp. In CRC Handbook of Mariculture, 2nd Edition, Volume 1: Crustacean Aquaculture, J.P. Mc Vey (ed), pp 289-313. CRC Press, Boca Raton, Ann Arbor, LondonTokyo

Yashiro, R. and S. Songtawontawe. 1996. Cultivation of Black Tiger Shrimp, Penaeus monodon Broodstock by Reducing Stocking Density and Transferring Pond Tech. Paper No. 14/1996. National Institute of Coastal Aquaculture, Songkhla, Thailand. 9pp.

Yashiro, R. and P. Na-anant. 1997. Reproductive Performances, Moulting Interval and Artificial Insemination of Penaeus monodon from the AndamanSea. Tech. Paper No. 7/1997. National Institute of Coastal Aquaculture, Songkhla, Thailand. 17pp.

EVALUATION OF BROODSTOCK AND

POST-LARVAE QUALITY

  1. BROODSTOCK

In the last several years, shrimp diseases have had a devasting effect on world shrimp farming. Diseases increase risk, deterring investment and commercial development. Most of these disease-associated problems are caused by viruses, for which there are no therapeutic cures. Like the livestock and poultry industries, the shrimp aquaculture industry will require certified high health broodstock in order to overcome these problems. Thus, shrimp broodstock quality is considered as one of the key factors in influencing successful shrimp culture.

Shrimp pathogens

The main reason for detecting and identifying pathogens in shrimp broodstock is to be able to detect pathogens in the carrier state before the onset of disease. Carrier detection is most useful for diseases associated with obligate pathogens such as viruses that may be transmitted vertically or horizontally. Viruses are intracellular pathogens that may be transmitted vertically through the egg or ovarian fluid during spawning, or they can be transmitted horizontally from host to host. Therefore, viral detection in shrimp is important in controlling the spread of the disease in evaluating the health status of a population or culture facility. At present, there are three (3) viruses considered significance for shrimp broodstock.

Techniques currently used to demonstrate viral infections in shrimp include examination of epidemiological features and gross structural changes, microscopy of stained and wet mount preparations, histopathology, electron microscopy, immunological methods and DNA-based protocols.

A.Viruses
  1. Monodon baculovirus (MBV) disease

MBV is the most common virus of Penaeus monodon. It has been reported that the disease is latent until the shrimp are faced with stressors, leading to the development of disease. MBV has been observed in all life stages of P. monodon. Shrimp larvae seem to be more susceptible to MBV than postlarvae and adults. Despite massive MBV infections in cultured shrimp populations, no serious impact has been reported. MBV infections may significantly reduce growth, crop value, and over-all health status of the shrimp. MBV, however, may pre-dispose infected shrimp to other pathogens resulting in the increase of the mortality rates.

Gross signs accompanying MBV infection may include reduced feeding and growth rates and increased gill and surface fouling organisms. Whitish midgut line through the abdomen has been found in severely affected larvae and postlarvae shrimp. The principal clinical signs of MBV infection is the presence of single or multiple spherical occlusion bodies in the hepatopancreas and midgut epithelial cells. Histological examination of MBV infections is dependent upon the demonstration of prominent, single or multiple eosinophilic spherical bodies within hypertrophied nuclei of hepatopancreatic tubule or midgut epithelial cells.

Vertical and horizontal transmission of MBV are likely to occur. Presumably, MBV is transmitted per os by ingestion of free virus, occlusion bodies and by cannibalism. It has been suggested that wild-caught broodstock may be a source of MBV giving rise to contaminated nauplii to hatcheries and finally to the shrimp ponds.

DNA probes are available for MBV diagnosis and primer sequences for PCR amplification have been published recently. Spreading of MBV is possible via horizontal and vertical transmission.

2. Hepatopancreatic parvovirus (HPV)

HPV is spherical, 22-24 nm in diameter and occurs within an intranuclear inclusion body. Signs of HPV disease are not specific, including poor growth rate in appearance, reduced preening activity as evident by increased gill and surface fouling organisms. HPV infects hepatopancreatic epithelium commonly in the distal portions of hepatopancreatic tubule, the E-cells. HPV infection results in the formation of prominent nuclear inclusion bodies showing intense basophilic reaction by H&E staining. These distinctive inclusion bodies are useful for diagnosis of HPV infection. The mode of transmission for HPV is unclear.DNA probes and primer sequences for PCR amplification have been published and available for diagnostic procedure.