Although sperm cryopreservation has been carried

out successfully in a number of commercially

important aquatic species, particularly in some

teleost fish (see review of Rana in Muir & Roberts

1993) and also shellfish (Subramoniam 1993), the

technology is still not at the stage of advanced

commercial application that is seen in domestic

mammals. Cryopreservation of eggs and embryos,

of aquatic animals however, is a virgin field in

cryobiology and has not yet received any appreciable

amount of attention. The first successful attempt at

the cryopreservation of embryos of sea urchin was

reported by Asahina & Takahashi (1978). Later Zell

(1978) and Erdahl & Graham (1980) have reported

preliminary attempts to freeze the eggs of rainbow

trout

Diwan, A D

Kandasamy, D

CMFRI Digital Repository

Aquaculture Research. 1997. 28, 947-950Short communicationFreezing of viable embryos and larvae of marineshrimp, Penaeus semisulcatus de HaanA D DiwanCentral Institute of Fisheries Education. (ICAR. Deemed University) J.P. Road. Versova. Andheri (W). Bombay 400 061.IndiaK KandasamiRegional Centre of Central Marine Fisheries. Research Institute ICAR Marine Fisheries P.O.. Mandapam Camp 620 320.Tamilnadu. IndiaCorrespondence: Dr A D Diwan. Central Institute of Fisheries Education (ICAR Deemed University). Versova. Bombay 400 061. IndiaAlthough sperm cryopreservation has been carriedout successfully in a number of commerciallyimportant aquatic species, particularly in someteleost fish (see review of Rana in Muir & Roberts1993) and also shellfish (Subramoniam 1993), thetechnology is still not at the stage of advancedcommercial application that is seen in domesticmammals. Cryopreser\'ation of eggs and embryos,of aquatic animals however, is a virgin field incryobiology and has not yet received any appreciableamount of attention. The first successful attempt atthe cryopreservation of embryos of sea urchin wasreported by Asahina & Takahashi (1978). Later Zell(1978) and Erdahl & Graham (1980) have reportedpreliminary attempts to freeze the eggs of rainbowtrout. Studies have been carried out to cryopreservethe embryos of Japanese medaka Oryzias letipesSchlegel (Aril, Namai, Gomi & Nakazawa 1987),rainbow trout Oncorhynchus mykiss Walbaum(Nilsson & Cloud 1993) and zebra fish Brachydaniorerio Weber & de Beaufort (Zhang, Rawson & Morris1993). In recent years some attempts have alsobeen made to cryopreserve the embryos and naupliiof shrimp Penaeus indicus H Milne Edwards(Subramoniam & Newton 1993: Subramoniam1994; Simon, Dumont, Cuende, Diter & Aquacop1994). From a review of the literature it appearsthat studies on the cryopreservation of embryos andlarvae of fish and shellfish is still in a rudimentaryphase and much remains to be done. The presentstudy was carried out with a view to developing amethod for the successful freezing of viable embryosand larvae of marine shrimp, using the experimentalprotocol now described.Mature green tiger prawns, Penaeus semisulcatusde Haan were collected from Gulf of Mannar andbrought to the Central Marine Fisheries ResearchInstitute's hatchery at Mandapam Camp, Tamilnadu,South India. After acclimation to hatcheryconditions they were made to spawn in thelaboratory either naturally or by means of theeyestalk ablation technique. After spawning,the eggs were collected and their developmentalstages monitored carefully. Once the eggs entered theadvanced embryonic stage (8-10 h after spawning)they were used for cryopreservation studies.Experimental trials were also made simultaneouslyto cryopreserve freshly hatched larvae (nauplii). Thecryoprotectants used for freezing the embryos andnauplii were dimethylsulfoxide (DMSO; S.d. FineChemicals Ltd, Bombay), glycerol and mixtures ofDMSO and glycerol. The extender used to dilute thecryoprotectants was standard physiological saline.Its composition is given in Table 1. Differentconcentrations of the cryoprotectants DMSO andglycerol (5, 10, 15, 20, 25 and 30%) were preparedin physiological saline solution and tested for theirefficacy in preserving the viable embryos and larvaeat low freezing temperature. There was no significantchange in the pH of the cryoprotectants after diluting© 1997 Blaekwell Science Ltd. 947Freezing marine shrimp embryos and larvae A D Diwan & K Kandasami Aquaculture Research. 1997. 28. 947-950with physiological saline solution. The embryos andnauplii at the required stage were collected byfiltering them through nylon bolting silk (400 mesh)and approximately 200 embryos per 100 ml vialliable 1 Composition of extender used. The pH of extendersolution 7.5Chemical ingredientsNaCIKCICaCl2MgCl2NaHCOaKHCO3Glucose(gm %)0.6500.0140.0120.0050.0200.0010.100were placed in different grades (5-30%) of DMSO,glycerol and a mixture (1 :1 ) of DMSO and glycerol.The embryos and nauplii were transferred to the100 ml plastic vials as quickly as possible. Thedifferent grades of cryoprotectants were pre-chilledat 5 °C before use. After transferring the embryosand nauplii into the different grades ofcryoprotectants, all the vials were immediatelytransferred to a programmable cryostat (AmericanOpticals, USA) held at -10 °C. The overall coolingrate was adjusted to produce a temperature dropof-1 °C per 5 min. The viability of the cryopreservedembryos and nauplii was monitored at regularintervals of 1. 2, 4 and 6 h. At each intervalfrozen embryos and nauplii were thawed to roomtemperature, washed throughly in physiologicalsaline solution and then transferred in glass troughs(10 litre capacity) containing aerated sea waterTable 2 Hatching rate (%) of embryos (freeze-thawed) of shrimp P.semisulcatus frozen (-10 °C) in different cryoprotectants.When Dimethylsulfoxide (DMSO) and glycerol were used Independently, the hatching rate of embryos was nil at theconcentrations 5-30%. Values are given as means ± SDConcentrations (%) of cryoprotectants used10 15 20 25 30Duration ofcryopreservation (h)DMSO + Glycerol 55 ± 3 50 ± 240 ± 2 50 ± 340 ± 2 50 ± 340 ± 1 50 ± 250 ± 2 40 ± 250 ± 3 50 ± 250 ± 2 50 ± 250 ± 3 50 ± 3Ihble 3 Revival rate (%) of nauplii (freeze-thawed) of shrimp P.semisulcatus frozen (-10 °C) in different cryoprotectants.When DMSO alone was used as a cryoprotectant the revival rate of nauplii was found to be nil at concentrations 5-30%.Values are given as means ± SDConcentrations (%) of cryoprotectants used10 15 20 25 30Duration ofcryopreservation (h)GlycerolDMSO + GlycerolGlycerolDMSO + GlycerolGlycerolDMSO + GlycerolGlycerolDMSO + Glycerol10 ± 155 ± 35± 155 ±25± 155 ± 25± 140 ± 1-55 ±3-55 ±3-55 ±3-50 ± 1-50 ± 2-50 ±3-50 ± 2-50 ± 2-50 ±2-50 ±2-50 ±2-50 ±2948 © 1997 Biacicweil Science Ltd. Aquaculture Research. 28 , 947-950Aquaculture Research. 1997. 28 . 947-950 Freezing marine shrimp embryos and larvae A D Diwan & K Kandasami(salinity 35%) to study their viability. The viabilityof the embryos was determined by allowing themto hatch out successfully as nauplii. In the case of thecryopreserved nauplii, viability was demonstrated byreviving their activity on thawing. All experimentswere repeated three times and the results ohtainedare pooled and given in Tables 2 and 3.It was found that when DMSO and glycerolwere used independently as cryoprotectants, theviability of the freeze-thawed embryos and naupliiin all the grades was nil as the embryos did nothatch out and the nauplii were dead. However,40-50% of the embryos preserved in concentrationsof 5-20% of the mixture of DMSO and glycerol,hatched out successfully to nauplii after 1-6 h ofpreservation. In the higher concentrations ofcryoprotectant, i.e. 25-30%, however, the embryosdid not show any signs of hatching.The nauplii preserved in the different grades ofDMSO (5-30%) did not revive at all. Thosepreserved in glycerol, however, did show a limitedcapacity for revival, although it was only 5-10%in the 5% concentration of glycerol after 1 and2 h of preservation. No nauplii survived in theremaining grades of glycerol. In the mixture(DMSO + glycerol) the revival rate of freeze-thawednauplii was 50% or more up to 6 h in the 5 -20% grades. However, the revival rate was nil inthe 25 and 30% concentrations. The growth inthe case of both the nauplii of the freeze-thawedembryos and the revived frozen nauplii wasmonitored until they grew to the size of juvenilesand was found to be normal when compared withtheir control counterparts.Limited success has been claimed in thecryopreseri'ation of viable eggs/embryos and larvaeof finUsh and shellfish. The reason given is thatin higher animals, due to the large size of theeggs and embryos, there will be interference inthe penetration of the cryoprotectants and thusprevention of uniform cooling during thecryopreser\'ation process. Sometimes the large yolkpresent in the eggs and embryos tends to developcrystals while freezing and damage the eggstructures (Seymour 1994). In shrimps, althoughthe size of the eggs and embryos is small, theeggs naturally absorb water soon after theirrelease in order to get swollen and activated forfertilization. After fertilization, a strong hatchingenvelope (protective extra-cellular matrix) formsaround the egg (Clark & Griffin 1993). Thepresence of water and the thick protective envelopesurrounding the eggs makes the freezing of viableeggs and embryos problematic (Simon et al, 1994).Subramoniam & Newton (1993) were the first tosuccessfully preserve nauplii of P. indicus. Theyheld them at -30 °C using liquid nitrogen andreported survival of 82% for nauplii frozen to -30 °C and 63% at -40 "C. They further reportedthat the toxicity response of the variouscryoprotectants (glycerol, ethylene glycol,methanol, DMSO, formamide) to nauplii wassimilar but while cryopreserving the embryos usingthe cryoprotectants detailed above they found thatglycerol was more toxic to the morulae stageembryos. They could not achieve any success withregard to the cryopreservation of embryos.Zhang etal. (1993), while working oncryopreservation of zebra fish embryos, studied thetoxicity response of different cryoprotectants suchas methanol, DMSO, glycerol, ethenediol andsucrose on different stages of embryonicdevelopment. They found that DMSO andethenediol are more toxic to fish embryos of heartbeat stage. They obtained the best embryo survivalrate at -10 °C and the least was at -30 °C. Iceformation within the egg was found to be themain factor affecting the survival of the embryos.In the present study, when DMSO and glycerolwere used independently they proved to be moretoxic. Similarly, higher concentrations of thecryoprotectants (25 and 30%) were found to beunsuitable for cryopreservation at -10 °C. Furtherinvestigation is warranted to refine the technique,once the exact nature of the damage to theembryos and nauplii by various factors duringcryopreservation is known.AcknowledgmentThe authors wish to express their sincere thanksto Dr M. Devaraj, Director, Central Marine FisheriesResearch Institute, Cochin for providing facilitiesand encouragement.ReferencesAril N., Namai K.. Gomi F. & Nakazawa T. (1987)Cryopreservation of medaka embryos duringdevelopment. Zoologicai Science 4(5), 813-818.Asahina E. & Takahashi T. (1978) Freezing tolerance inembryos and spermatozoa of the sea urchin. Cryobiology15, 122-127.© 1997 Blackwell Science Ltd. AquacuiUm Research. 28 , 947-950 949Freezing marine shrimp embryos and larvae A D Diwan & K Kandasami Aquaculture Research. 1997, 28, 947—950Clark W.H. & Grifiin F.J. (1993) Acquisition andmanipulation of Penaeoidean gametes. In; CRC-HandBook of Mariculture (ed. by ].P. McVey). Vol. 1, 2ndedn. pp. 133-151. CRC-Press, London.Erdahl D.A. & Graham E.F. (1980) Preservation ofgametes of freshwater tish. In: Proceedings ofInternational Conference of Animal Reproduction andArtificial Insemination, Madrid, pp. 317-326.Muir J.E. & Roberts R.J. (1993) Conservation andpreservation of genetic variation. In: Recent Advancesin Aquaculture. Vol. IV, pp. 297-337. Blackwell ScienttflcPub.. London.Ntlsson E. & Cloud J.G. (1993) Cryopreservation ofrainbow trout (Oncorhynchus mykiss) blastomers.Aquatic Living Resources 6(1). 77-80.Seymour J. (1994) Freezing time at the zoo. New Scientist1, 21-23.Simon C. Dumont P., Cuende F. & Diter A. (1994)Determlnatron of suitable freezing media forcryopreservation of P. indicus embryos. Cryobiology31(3), 245-253.Subramoniam T. (1993) Spermatophores and spermtransfer in marine crustaceans. In: Advances in MarineBiology (ed. by J.H.S. Blaxter & A.J. Southward), Vol.29. pp. 129-214. Academic Press. New York.Subramoniam T. (1994) Cryopreservation of crustaceangametes and embryos. Proceedings Indian NationalScience Academy, B 60(3). 229-236.Subramoniam T. & Newton S.S. (1993) Cryopreservationof embryos of the penaeid prawn Penaeus indicus.Current Science 65, 176-177.Zell S.R. (1978) Cryopreservation of gametes and embryosof salmonid fishes. Annals Biology Animal Biochemistryand Biophysics 18. 1089-1099.Zhang T., Rawson D.M. & Morris G.J. (1993)Cryopreservation of pre-hatch embryos of zebra fish(Brachydanio rerio). Aquatic Living Resources 6(2),145-153.950 1997 Biackwetl Science Ltd, Aquocuiture Research. 28, 947-950

Freezing of viable embryos and larvae of marine shrimp, Penaeus semisulcatus de Haan

A D  Diwan

K K Kandasami

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Freezing of viable embryos and larvae of marine shrimp, Penaeus semisulcatus de Haan

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