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  1. 1. CULTURE OF ROTIFERSZooplankton used for feeding marine fish larvae, FW.The success of rotifers as a culture organism are manifold,1. Planktonic nature,2. Tolerance to a wide range of environmental conditions,3. High reproduction rate (0.7-1.4 Small size and slow swimming velocity make them a suitable prey for fish larvae that have just resorbed their yolk sac but cannot yet ingest the larger Artemia nauplii.5.Possibility of rearing these animals at very high densities (i.e. densities of 2000 animals./ml have been reported by Hirata (1979). Even at high densities, the animals reproduce rapidly and can thus contribute to the build up of large quantities of live food in a very short period of time.6. Last, the filter-feeding nature of the rotifers facilitates the inclusion into their body tissues of specific nutrients essential for the larval predators.
  2. 2.  Reproduction by sexual method( fertilisation of eggs) leads to formation ofresting eggs , to tide over unfavorable conditions . By asexual method- parthenogenetic reproduction occurs – males aredeveloped - at optimum conditions of temp, light and pH-, which are provided forthe mass culture of Rotifers.
  3. 3. Different strains - Two different morphotypes, namely Brachionus rotundiformis or small (S-type) rotifers and Brachionus plicatilis or large (L-type) rotifers. The differences among the two types can be clearly distinguished by their morphological characteristics- The S- and L- morphotypes differ in their optimal growth temperature. The S-type = 28-35°C, L-type = 18-25°C. Salinity S type- 18-20ppt L type- 30pptSuitable for larvae withsmall mouth opening
  4. 4.  Life span of rotifer depends on temperature. At 25 OC – 0.5 to 1.5 days. Female starts laying eggs once every 4 hrs. Optimum conditions for growth- Temp- 25-30 OC pH- 7.5-8.5 Salinity- 20-30 ppt. Ammonia- <1mg/L D. O> 2.2 mg/LTurbulence given by aeration- keeps rotifers and food particles in suspension.Culture systems for Rotifers3 UNITS1. Unit for culturing of food for Brachionus- Primary requirement for culture of rotifers is the feed.- Chlorella, bacteria and yeast.2. Unit for Brachionus stock culture3. Mass culture Unit
  5. 5. 2. Unit for Brachionus stock culturea. Collect rotifers from Brackish water bodies- 50 L of pond water filtered through filter of mesh size- 50-100mm .b. Examine under microscope., pick out rotifers using fine dropper andplace in cavity block with 3.5 ml distilled water.c. Chlorella medium is put into cavity blocks.d. One rotifer is introduced into this cavity block, covered with glass plate , keptIn diffused light.e. At every 12 hours, the medium is replaced with fresh chlorella and add live rotiferWith eggsf. Gradually increase the culture volume to 25 ml in a 50 ml beaker and change the culture daily once.g. Use 50-70 mm size mesh to separate the rotifers , continue the process till the density reaches 50 numbers of rotifers per ml, and culture vol. is 500ml. Chlorella density may be 3-4 million cells/ml.
  6. 6. 3. Mass culture UnitMass culture methods are of three types Batch culture – Thel population of culture vessel is entirely harvested at or near its peak density.Used as inoculum for other culture vesels, useed for feeding in LRT. Small to medium sized containers are used, kept indoors. After harvest the entire batch is discarded, then a fresh batch is started all over again. Filtered sea water is enriched with fertilisers such as urea,/ amm.sulphate and super phospahte, it is inoculated with Chlorella. Medium aerated, inoculated with rotifers once the suitable density of algae is reached. Rotifers grow and multiply, at density of 100-300 ind./ml ,they are harvested. Semi- continuous culture : Partial harvest is done at intervals. The harvested volume replaced by fresh medium to keep the growth going on. Large amount of algae produced in outdoor tanks. Risk of contamination by other organisms and building up of metabolites. Feedback culture: Faecal and other particulate matters settled at the bottom of rotifer culture tanks are allowed to undergo bacterial decomposition which is used as a fertiliser for algae cultured in a separate tank.
  7. 7. ENRICHMENT OF ROTIFERS Micro algae rich in PUFA , EPA, DHA and vitamins Rotifers ( Upgraded nutritional quality) feed for fish larvae.Rotifers fed with Isochrysis and Nannochloropsis and Chlorella.Artificial diet is added to the rotifer culture medium for 6- 12 hrs.D.O should be at least 4ppm.Enriched rotifers are stored at 5 to 10OC for upto 14 hrs, to avoid loss of PUFA.
  9. 9. Reproduction in Artemia Artemia is ovo viviparous, fertilised eggs undergo dev’t inside theuterus of female , nauplii are set free into the water. If living conditions deteriorate, artemia lays eggs – cysts, which have aharder shell and an exceptional ability to survive. Shell glands located in the uterus produces a shell around the embryo,when it is in the Gastrula stage and embryo enters into diapause. One adult – 300 cysts or nauplii in every 4 days, cysts can survive for upto50-60 days. Cysts are 200- 400 microns in diameter, biconcave, with 3 layered shell. Thickest layer is Chorion – lipoprotein impregnated with chitin andhaematin, protecting the cyst from radiation and mechanical stress. Outer cuticular membrane prevents penetration of molecules larger thanCO2, inner cuticular membrane, highly elastic, forms the hatchingmembrane.
  10. 10. Biconcave Artemia cysts
  11. 11. Conditions for hatching of cystsStored cysts when soaked in sea water, hatches out within 24-36 hrs. Salinity – to be maintained between 15-35 ppt . Density – one gm cyst in one L. Aeration – Vigorous.@ 10-20 litre air per minute. Oxygen level nearer to saturation . Temperature – 25-32 OC. pH- 8-8.5. (1 gm Na bicarb in One L of water.) Ilumination- 2000 lux at water surface, not less than 1000 lux.
  12. 12. Hatchingof cysts
  13. 13. The cyst swells, assumes a spherical structure within 1-2 hrs. After 15- 20 hrs, Cells burst, prenauplius surrounded by the hatching membrane becomes visible Embryo comes out, hangs underneath empty shell- umbrella stage. Hatching membrane breaks, free swimming nauplii emerges. Newly hatched nauplius – brownish orange , 3 pairs of appendages- antennule antenna and mandibles Ocellus/ naupliar eye present. Donot feed for abt 12 hrs. then starts to feed on algae, bacteria and detritus of 1-40 micron size. After 15 moults, nauplii becomes adult.Hatching percentage = No. of nauplii hatched per 100 cysts.Hatching efficiency = No. of nauplii hatched per gram of cyst- 3 lakhs/ gm.Hatching output = Naupliar biomass (dry wt in mgs) per gram of cyst .600 mg/gram.Hatching rate = rate at which nauplii hatch out .
  14. 14. Decapsulation of cystsArtemia cyst shells – contaminated with bacteria , fungi/ micro organisms.Empty shells, unhatched cysts or residues from the hatching mediumshould be avoided.Fish larvae might get infected, heavy mortality may occur.
  15. 15. Decapsulation of cysts Artemia cysts Soaked in SW for 1 hr, aerated and cooled. Cysts filtered and transferred to the decapsulation medium.(Hypochlorite soln.) Sea water - 1400 ml. Bleaching powder – 71 gm Na carbonate - 64 gms
  16. 16. Medium cooled using ice water ,agitated for 5-10 min. Till the color of cysts become orange. Cysts filtered and washed with sea water to remove free chlorine Cysts washed with 0.1% Sodium thiosulphateCysts filtered and washed with fresh sea water Incubated for hatching.
  17. 17. Bioencapsulation of Artemia Conc. of EFA – 20: 5n-3 eicosapentenoic acid (EPA) – determines the nutritional quality of Artemia . Emulsified concentrates (HUFA) is added at 300 ppm conc. in rearing medium with vigorous aeration. The nauplii are non selective feeders, ingest the HUFA HUFA are not metabolised by nauplii. They are stored at 10 OC . Antibiotics, and growth promoters can also be delivered to the larvae thru bio encapsulation.