deals about bioenrichment and bioencapsulation in rotifers which are generally used as live feed for fishes(generally marine species)

2. ROTIFERS:-
• Rotifers are commonly called as Wheel animalcules
• 2200 species of which 90% inhabit in freshwater habitats.
• Their growth is assured by plasma increase and not by cell division.
• The filter-feeding nature of the rotifers facilitates the inclusion of specific nutrients essential
for the larval predators through bioencapsulation into their body tissues
• Rotifers could be used as a suitable live food organism for the early larval stages of marine
fish
Systematic position:
Phylum : Rotifera
Class : Monogononta
Order : Ploima
Family : Bcrachionidae
Genus : Brachionus
• important species:- Brachionus plicatilis, B.rotuniformis
• feed on algae and bacteria,Rotifers are an important part of the freshwater zooplankton,
being a major foodsource and with many species also contributing to the decomposition of
soil organic matter.

3. The rotifers are considered as an important live
feed in hatchery operation due to their :
– Planktonic nature,
– Tolerance to a wide range of environmental
conditions,
– High reproduction rate (0.7-1.4
offspring/female/day),
– small size
– Slow swimming nature.
– The possibility of rearing these animals at very
high densities (2000 animals/ml)

4. BIOENCAPSULATION :-
• bioencapsulation, also called enrichment or boosting is widely applied at
marine fish, crustaceans and other organism(live food organisms too)
hatcheries all over the world for enhancing the nutritional value with
essential fatty acid.
• Bioencapsulation or bioenrichment is the process involved in improving
the nutritional status of live food organisms either by feeding or
incorporating within them, various kinds of nutrients. Examples of
practical and experimental enrichment diets are unicellular algae,
emulsion, liposomes and microencapsulated diets.(CMFRI)
• Through bioencapsulation process we introduce essential and required
nutrients in the body of live feed which will be taken by the organism(fish
& other) of our interest

6. A. ALGAE (sole food material of rotifers)
B.FORMULATED FEEDS
C.OIL EMULSIONS

7. A.
• The high content of the essential fatty acid eicosapentaenoic
acid (EPA 20:5n-3) and docosahexaenoic acid (DHA 22:6n-3) in
some microalgae (e.g. 20:5n-3 in Nannochloropsis occulata and
22:6n-3 in Isochrysis galbana) have made them excellent live
food diets for boosting the fatty acid content of the rotifers.
• Rotifers submerged in these algae are incorporating the
essential fatty acids in a few hours time and come to an
equilibrium with a DHA/EPA level above 2 for rotifers
submerged in Isochrysis and below 0.5 for Tetraselmis.

8. MICROALGAE
ROTIFER
ROTIFER FEEDING ON MICROALGAE

9. • Short-term enrichment of rotifers with mlcroalgae
• Microalgae are used for short-term enrichment of rotifers before they are given to
the fish larvae to improve the content of n-3 fatty adds in the rotifers. Short-term
feeding with algae affects the protein content and dry weight of the rotifer.
• PReparation of enrichment For emulsified lipids, the (n-3) HUFA concentration in
the lipid source should be very high.
• 5g fish feed is homogenized for 2-3 min in a homogenizer or mixer or by vigorous '
shaking.
• Observing under microscope ensures proper emulsion
• Stored under refrigeration till use.
• Emulsifiers may be added to maintain the emulsion or a strong shaking prior to use
reforms the emulsion.
• - The enrichment media may be supplemental with water and fat-soluble vitamins
like vitamin A, D, E and K to homogenization.

10. • One potential risk in using fish oil for HUFA enrichment, the n-3 HUFA
particularly DHA in the very small triacyl glycerol (TAG) micelles generated in
enrichment procedures are prone to auto Oxidation, especially under
continuous aeration employed, for prolonged periods:' The addition of natural
antioxidants vitamin E and C (generally added as oil soluble 6 tocophenyt
acetate and ascorbyl palmitate) which are not effective until hydrolyzed in the
intestinal tract and absorbed and or synthetic antioxidants like ethoxyquine or
butylated hydroxyl anisole (BHA), minimizes peroxidation.
• Lecithin can be used to considerable advantage in enriching the nauplii with
22:6n3 HUFA rich fish oils (90:10; Fish oil: lecithin), lecithin acting both as
natural emulsifying agent and as a natural protectant against preoxidation.
Oils from marine organisms lik.e heterotrophic dinoflagellate
Crypthecodinium cohnii, which is mass cultured for a triacyl glycerol rich in
22:60-3. This oil is used to supplement infant formulae. Frozen-thawed cells
of C.cohnii have recently been used very successfully to supplement Artemia
nauplii directly with 22:6n-3. Spray dried Schizochytriumsp. a single celled
heterotrophic marine proteist similar to water molds of group
Labyrinthulomycota is also used.

11. • Rotifers grown on the Culture Selco (CS®) have an excellent HUFA
composition: 5.4, 4.4 and 15.6 mg.g-1 dry matter of EPA, DHA and (n-3)
HUFA respectively which is significantly higher than for cultures grown on
algae/baker’s yeast but comparable in case the latter cultures are subjected
to an additional enrichment treatment.
• The level of total lipids is approximately 18%.
• Since the use of CS® allows direct enrichment of the rotifers without the
need of a cumbersome bioencapsulation treatment, complementary diets
such as Protein Selco® (PS) and DHA Culture Selco® (DHA-CS) are in use
in order to incorporate higher levels of protein and DHA.
• The advantage of direct (or long term) enrichment are multiple; in that. the
fatty acid profile obtained is stable and reproducible, the lipid content is
comparable to that obtained in wild zooplankton, rotifer losses are lower.

12. • One of the cheapest ways to enrich rotifers is by using oil
emulsions. Although home-made emulsions can be
prepared with egg lecithin and fish oils, Commercial
emulsions are generally more stable and have a selected
HUFA composition.
a.Home-made emulsions
b.Commercial emulsions

13. types of oil emulsions

14. • The first emulsions were made from (n-3) HUFA rich fish oils (i.e.
cuttlefish oil, pollack liver oil, cod liver oil, menhaden oil, etc.) and
emulsified with egg yolk and seawater (Watanabe et al., 1982,
1983). Recently, more purified oils containing specifically high
levels of the essential fatty acids 20:5n-3 and 22:6n-3 have been
used. Since the stability and storage possibility of these products
is relatively low they are usually made on the spot and used
immediately.
• For very specific applications, or when the requirements of the
fish can not be fulfilled with commercial emulsions, this technique
may also be used to incorporate lipid extracts from zooplankton,
fish, fish roe, or other sources

15. • Several emulsified diets are commercially available and based on well-
defined formulations. Very popular are the self-emulsifying concentrates
(Selco®, Inve Aquaculture NV, Belgium) which can boost the HUFA content
of the rotifers in a few hours. In this technique a rotifer suspension containing
200-300 individuals.ml-1 is immersed in a diluted oil-emulsion for 6 h,
harvested, rinsed and concentrated before being fed to the predators.
• In view of the importance of DHA in marine larviculture, considerable efforts
have recently been made to incorporate high levels of DHA and/or high ratios
of DHA/EPA in rotifers. To date the best results have been obtained using the
self-emulsifying product DHA-Super Selco®. Compared to the results
obtained with Super Selco®, the boosting of CS-rotifers with this product
under standard enrichment practices results in a threefold increase of DHA
and total (n-3) HUFA.

16. • Furthermore, the evolution of the concentrations of EFA within enriched
rotifers after being administered to the predator tanks has been investigated.
Results reveal that EFA levels remain rather constant for at least 7 h under
clear water culture conditions at 20°C; with only a 30% drop in DHA being
noted after 12 h
• Most commercial emulsions are rich in triacylglycerols and/or methyl esters
and no emulsions have been formulated with phospholipids and/or wax ester
• t is interesting to note that after enrichment the composition of the rotifers did
not differ more than a fraction of 30 to 45% in (n-3) HUFA (Fig. 3.13).
Moreoever, the lipid composition of the rotifers was also little affected by the
composition of the diet. However, when the efficiency of DHA and (n-3) HUFA
incorporation in rotifers is analyzed it is obvious that better results are
obtained with the extraction products. Since all diets are consumed with
approximately equal efficiency it means that phospholipids (present in the
extraction products) were more easily assimilated and metabolized by the
rotifers

17. 2.Techniques for vitamin C enrichment
• Rotifers cultured on instant baker’s yeast contain 150 mg vitamin C/g-1 DW, while for Chlorella-fed
rotifers contain 2300 mg vitamin C/g-1 DW.
• Problems of operculum deformities can occur due to reduced vitamin C levels.
• Enrichment of rotifers with AA is carried out using ascorbyl palmitate (AP) as a source of vitamin C
to supplement the boosters.
• AP is converted by the rotifers into active AA up to 1700 mg.g-1 DW after 24 h enrichment using a
5% AP (w/w) emulsion.
• Bioencapsulation is done using the lipophilic ascorbyl palmitate (AP) as a stable and bioavaitable
source of Ascorbic acid (AA) in emulsions and particulate boosters for the live food prior to their
feeding to fish larvae. Applying experimental self-emulsifying concentrates supplemented with 10
and 20% AP, high levels of AA could be obtained in 24 h enriched Artemia nauplii. Four fold increase
in AA is obtained with 20% AP

18. • To our knowledge Protein Selco® is the only enrichment
diet especially designed for protein enrichment in rotifers.
• The high levels of proteins allow the cultures to continue
to grow and to develop during the enrichment period.
• Normally it is used in the same way as an oil emulsion
(blended in a kitchen blender) and distributed in the tank
at a concentration of 125 mg.l-1 seawater at two time
intervals of 3 to 4 hours.

19. • In addition to the use of
enrichment in nutrients,
live food mediated delivery
of therapeutic drugs has
emerged as a new tool for
disease treatment in larval
culture. This is normally
done through liposomes.

20. • The overall nutritional Quality of the live food organisms depends on the
content and nutritional balance of carbohydrate, protein and lipids. The
content of essential amino acids and protein in live food must meet the
requirement for growth and cell maintenance of fISh larvae. The enriched live
food might be starved in the culture tank before they are consumed by the
larvae and in this period, the protein, lipid and fatty acids decreases. The loss
of all these components increase with increasing water temperature. The
total lipid content loss is at higher rate than the protein content. Highest loss
rate is for DHA and considerably lower for EPA.
• It is concluded that survivability of marine larvae in hatcheries depends on
the larval nutrition, which in turn is due to the Quality loss (potency loss of the
stock on continuous use, mutation etc) of live food organisms.
Bioencapsulation can be effectively and successfully employed to cut down
the larval mortalities in hatcheries

21. • WIKIPEDIA
• FAO MANUAL 361 ON LIVE FEED
• CMFRI JOURNAL,LINK:-
http://eprints.cmfri.org.in/7869/1/B140-
14_imelda_joseph.pdf