Viable nauplii of the tiger prawn Penaeus monodon have been produced by artificial insemination.

A technique of implanting electroejaculated spennatophores from the males into the thelycum of

newly moulted, eyeablated females is described. The implanted spennatophores are retained by the

female till the next moult. Three females were artificially inseminated 10 times and every time they

matured and spawned. One of the spawnings yielded healthy normal larvae which were reared to

postlarval stage in the laboratory

Laxminarayana, A

Muthu, M S

CMFRI Digital Repository

Current Science, October 20, 1984, Vol. 53, No. 20 1075 ARTIFICIAL INSEMINATION OF PENAEUS MONODON M. S. MUTHU and A. LAXMINARA YANA Central Marine Fisheries Research Institute, Cochin 682018, India. ABSTRACT Viable nauplii of the tiger prawn Penaeus monodon have been produced by artificial insemination. A technique of implanting electroejaculated spennatophores from the males into the thelycum of newly moulted, eyeablated females is described. The implanted spennatophores are retained by the female till the next moult. Three females were artificially inseminated 10 times and every time they matured and spawned. One of the spawnings yielded healthy normal larvae which were reared to postlarval stage in the laboratory. INTRODUCTION A RTlFICIAL insemination (AI) in the freshwater .t\..caridean prawns has been fairly successfuI1•2• A similar technique of AI has been patented3 in the U.S.A. for the American species of Penaeus with open thelycum. Among penaeid prawns with closed thely-cum AI has been tried in the case of Penaeus japo-nicus4.5. These methods are not applicable to the commercially important species of marine prawns in India as they differ from the species referred to above in the shape of the thelycum and the structure of the spermatophore. Prawn breeding work at the Marine Prawn Hatchery Laboratory of the Central Marine Fisheries Research Institute, Cochin, has resulted in the development of an AI technique for the giant tiger prawn Penaeus monodon. The method is described in this paper. MATERIALS AND METHODS Adult specimens of P. monodon were collected from the sea as well as from the brackishwater ponds at Narakkal. Three immature adult females were sub-jected to unilateral eyestalk ablation using an elec-trocautery apparatus and were kept individually in circular plastic lined pools containing 250 I of filtered seawater. The pools were aerated with oil free air from an air blower. Everyday about 90 % of the water in the pools, was siphoned out along with the sediments and faecal strands and replaced with fresh filtered seawater without disturbing the prawns. The pools were co-vered with black cloth to avoid stress to the animals. Three mature males which were not eyeablated were kept in a 10 m3 plastic lined pool fitted with a subgraval biological filter. The prawns were fed ad libitum with clam meat. The temperature in the pools varied from 28S'C to 30SC, the salinity from 32.0 to 33.5 ppt and the pH 8.1-8.2. The daily change of seawater in the 250 1 pools stimulated frequent moulting of females at intervals of 10-15 days. Whenever the female moulted the sper-matophores stored in the thelycum were lost along with the moulted cuticle. Moulting took place around midnight and the female was artificially inseminated with spennatophores from a male around 6 o'clock in the morning when the newly formed cuticle was still flexible. The spermatophores were extracted without injuring the male by electroejaculation, a method described in detail by Sandifer and Lynn2• For insemination the moulted female was caught from the pool with a soft hand net and held with the left hand, ventral side up, on a slab of rubber foam kept in a trough of filtered seawater. The pair of spermato-phores electroejaculated from a male were received on a sterilised spatula held in the right hand and quickly inserted into the thelycum of the female. The female was allowed to recover in the trough of seawater which was continuously aerated. The trough was gently lowered into the 250 I of seawater in the plastic pool and the female was allowed to swim out. The im-planted spermatophores were retained inside the the-lycum of the female. Every morning the bottom water in the pool was sampled to see if spawning had taken place. When eggs were seen in the samples the female was transferred to another pool and the eggs were allowed to develop undisturbed. An estimate of the number of eggs spawned was made by counting the eggs in aliquot samples. RESULTS The details regarding the dates of moulting, insemi-nation and spawning and the number of eggs laid by the three females are given in table 1. All the females were implanted with a fresh set of spennatophores after each moult. Every implantation was followed by 1076 Current Science, October 20, 1984, Vol. 53, No. 20 Table 1 Details of experiments in artificial insemination of Penaeus monodon Specimen No. 2 3 Source Sea Sea Pond Total length of female mm 264 237 221 Carapace length rom 70 S8 57 Date of eyestalk ablation 9.2.84 9.2.84 18.3.84 Date of 1 st moult and implantation 23/24.2.84 16/17.2.84 20/2l.3.84 Date of 1st spawning 9.3.84 26.2.84 28.3.84 (No. of eggs) (75,600) (86,800) (648,400) Date of 2nd moult and implantation 11/12.3.84 2/3.3.84 30/31.3.84 Date of 2nd spawning 20.3.84 12.3.84 8.4.84 (No. of eggs) (74,200) (81,500) (580,600) Date of 3rd moult and implantation 21/22.3.84 17/18.3.84 10/1l.4.84 Date of 3rd spawning 29.3.84 28.3.84 20.4.84 (No. of eggs) (71,600) (72,200) (90,600) Date of 4th moult and implantation 1.4.84 Date of 4th spawning 10.4.84 (No. of eggs) (69,600)* * 1686 nos. nauplii hatched out. a spawning 7-15 days later. The 264 mm long female moulted and spawned 4 times, the other two females each moulted and spawned 3 times. Totally there were 10 spawnings which yielded 1,930,800 eggs. However only the 4th spawning of the 264 mm female on 10 Apri11984 resulted in the production of 1686 viable nauplii. The eggs in the other nine spawnings did not undergo normal development.. Although the hatching rate was low (2.4 %) in the successful implantation, the very fact that at least some of the eggs developed into normal, healthy nauplii shows that the AI was successful. Since the prawn had earlier moulted 4 times the eggs should have been fertilized only by the sperms from the implanted spermatophores. Some of these nauplii were collected from the 250 1 pool for further rearing in 3 large beakers. In each beaker 100 nauplii were kept in 5 I of seawater and reared on a diet of mixed phytoplankton cultured separately. The larvae were healthy and normal and metamorphosed into postlarvae on 19 April 1984. The number ofpostlarvae (PLl) obtained was only 37. But this low rate of survival of the larvae was due to frequent current failures leading to stoppage of aer-ation for extended periods. DISCUSSION Although only one out of the 10 artificial insemi-nations was successful, there is no ambiguity about the source of the sperms that fertilized the eggs in the successful experiment, as the sperrnatophores were implanted after the female had moulted. The sperms from any natural impregnation undergone by the female in the sea would have been lost along with the moult. Lumare' implanted the spermatophores into the thelycum of P.japonicus females with fully developed gonads during the intermoult stage. In 22 implan-tations 994,460 eggs were produced and the average hatching rate was 3.3 %. But, eventhough the females were said to be free of spermatophores, the possibility of some residual sperms remaining inside the seminal receptacle of the thelycum is not ruled out in Lumare's experiments. In P. monodon non-viable eggs are frequently produced even by naturally impregnated spawners collected from the sea. So the low percentage of success in these implantation experiments is not surprising. However, efforts are beings continued to improve the fertilization rate in artificially insemi .. nated P. monodon. The present work has opened new vistas in selective breeding and genetic manipulation of commercially very valuable species of penaeid prawns such as P. monodon for aquaculture purposes. The technique will be invaluable for future work in hybridization of penaeid prawns. r i Current Science, October 20, 1984, Vol. 53, No. 20 ACKNOWLEDGEMENTS The authors are grateful to Dr E. G. Silas, Director and Dr M. J. George, Senior Scientist, CMFRI, Cochin for their encouragement and guidance in the prepar-ation of this article. They are also indebted to Mr. K. H. Mohamed, retired Senior Scientist, for his advice and suggestions during the course of this work. 4 May 1984 1. Sandifer, P. A. and Smith, T. 1. J., Proc. World 1077 MaricuJ. Soc., 1979, 10, 403. 2. Sandifer, P. A. and Lynn, J. W., in Advances in invertebrate reproduction, (eds) W. H. Clark, Jr. and T. S. Adams, Elsevier North Holland, New York,1981, p. 271. 3. Persyn, H. 0., United States Patent 4,031,855, June 28, 1977, 4p. 4. Ponticelli, A., Rivista ltaliana di Piscicoltura e lttiopath%gia, A XVI, 2, 125. 5. Lumare, F., J. World Maricul. Soc., 1981, 12, 335. 

Artificial insemination of Penaeus monodon

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Artificial insemination of Penaeus monodon

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