Livefeed culture, Priyanka Chatterjee

LIVE FEED CULTURE FOR HATCHERY

Submitted By - Priyanka Chatterjee
Roll No - 14, CUSAT,
School of Industrial Fisheries, 2nd Semester

Submitted to- Dr. M. Harikrishnan

Introduction:
Types of feed are an important constituent in ornamental fish
farming and high expenditures are generally incurred on feed. Hence
the right type of feed in appropriate quantity would ensure a higher
survival and growth and also a higher economic return. The feed
requirement of different species varies and hence a thorough
knowledge of the food and feeding habits of the culturable species is a
pre-requisite. Nowadays different types of feed are available in the
hobby shops; however most of these feeds are expensive. Hence the
culturists can prepare the feed using locally available materials
which would be more economical and cost-effective. Feed are of two
types viz. formulated dry feed and live feeds.

Important Live Feed:
Among the different live feeds micro algae and micro zooplanktons
together plays an important role in larval production system. Among
the organisms that have been used as live feed, the most important
are nauplii of the brine shrimp, Artemia spp., the rotifer Brachionus
plicatilis, calanoids and copepods, cladoceran crustaceans (Daphnia
spp. and Moina spp. ), and many species of microalgae, diatoms
Skeletonema costatum, Thalassiosira sp. Chaetoceros gracilis,
Chaetoceros calcitrans, the flagellates Isochrysis galbana,
Tetraselmis sp. Pavlova lutheri, and the chlorococcales Chlorella spp
are the most important. The selection of these live feed organisms are
based on many factors such as nutritional requirements of the
cultured larvae, size of the mouth gape and development of the
digestive tract of the cultured larvae, nutritional value, and
availability of the live feed and suitability for mass scale production.
Though much effort have been taken worldwide to supplement live
feed totally or partially with artificial feeds, various study pointed
out that supply of suitable live feed organisms fortified with vitamins
and fat are essential for the successful completion of the larval
stages.

1. Micro Algal Culture:
Suitable micro algae can be isolated through various techniques viz.
Pipette method; centrifuge or washing method, photo tactic
movements, agar plating method and serial dilution and the isolated
species can be cultured in mass quantity using suitable culture
media. Although most algae are photoautotrophic and can grow in
purely inorganic media, many other required organic compounds, the
requirements of which may be either absolute or stimulatory. While
most of the microalgae can be successfully cultured on synthetic
inorganic media, a few genera require organic compounds for their
rapid growth and therefore the culture are supplemented with soil
extracts, yeast extracts or organic salts. Since, the microalgae in any
water body require the nutrients such as nitrates and phosphates
roughly in a ratio of 10:1(N:P) for its normal growth and
reproduction, the culture media used in the laboratory should have
sufficient quantities of these elements besides other growth
promoting agents would definitely reflect on the growth of micro
algae especially in a culture system. The most widely used culture
media are „Conway‟ or Walne‟s medium, Suto, PM, SEAFDEC,
Gulliard f, f/2,f/4, MN, ASPW etc. And fresh water microalgal
medium are Bold basal, BG 11, PHM 1, Botoryococus and Zarrouk
media.

Mass culture of microalgae
Mass production of phytoplankton for rotifers and “green water” in
most Mediterranean hatcheries is limited to a few species such as:

Chlorella sp, Isochrysis galbana, Pavlova lutheri, Nannochloropsis
oculata and N. gaditana, Dunaliella tertiolecta and Tetraselmis
suecica. These species have been selected on the basis of their size,
nutritional value, culture easiness and absence of negative side
effects, such as toxicity. Their nutritional value shows a great
variability not only among different species, but also in genetically
different populations of the same species (strains). For hatchery
purposes, the species to be cultured should both fit well the local
rearing conditions and have a high nutritional value for rotifers. The

increasing availability of nutritional boosters as enrichment diets for
both rotifers and brine shrimps, has made this choice easier.

Mass culture of microalgae

Population dynamics
Microalgae population dynamics can be described by different
phases:
the lag-phase, where, just after the inoculum, the cells increase
in size, but not in number, and begin to absorb the nutriens
supplied with the culture medium;
the log-phase (or esponential phase), where cells reproduce very
fast and population growth is exponential;
the transitional phase (or declining growth phase), where
growth rate slows down;
the stationary phase, where cells remains constant in number
and reproduction is balanced by death;
the decline phase, where cell number decreases since death rate
exceeds growth.
It is advisable to harvest phytoplanktonic organisms during their log
phase, since in the new culture they will grow more rapidly and will
yield a more viable population.

Mass production systems
For aquaculture purposes, microalgae are mass produced in three
main ways: (i) batch (or discontinuous or multistep back-up system)
culture, (ii) semi-continuous culture, and (iii) continuous culture.
In the batch culture a small axenic stock culture produces a series of
cultures of increasing volume where the algal population of each
culture vessel is entirely harvested at or near its peak density, i.e.
while still conserving a good growth potential, to be used either as
inoculum for other culture vessels, or to feed rotifers or be used in
fish larval tanks. It typically makes use of small (few litres) to
medium size (500 liters) containers, and it is kept indoors and under
strictly controlled, if not properly axenic, conditions. It is considered
by many authors the easiest and most reliable method of algal
production, provided that the working protocol is strictly enforced.
Algal quality is less erratic than in the semi-continuous method, even
if the latter is more productive for any given volume.
In the semi-continuous system the algal population, when mature, is
partially harvested at intervals. The harvested culture volume is
replaced by fresh medium to keep growth going on. This culture is
adopted to produce large amounts of algae and frequently uses large
outdoor tanks. Their main drawbacks are: (i) the unpredictable
duration, (ii) the risk of contamination by other organisms as
competitors (other microalgal species), contaminants (bacteria) and
predators (ciliate protozoa feeding on the algae), as well as (iii) the
building up of metabolites, which can affect quality.
The continuous system is a steady-state continuous flow culture in
which the rate of growth is governed by the rate of supply of the
limiting factor. It is a balanced axenic system where the algal
population is harvested and fertilised continuously. This method,
though the most efficient over extended periods, produces limited
amounts of high quality cells and requires complex equipment as well
as advanced management. A relatively recent development of this
system is represented by the photo-bioreactor, a continuous culture
device that increases the density of cultured microalgae to very high
levels under predictable environmental and microbiological
conditions.

The microalgae produced can be concentrated to a dense liquid
suspension by centrifugation, and can then be stored for more than
one month in the refrigerator, still giving excellent viability when
used. A new industry is now appearing, whose concentrated algal
products can also fulfil the hatchery needs, saving the timeconsuming and expensive production of microalgae in the hatchery.
The system described below is the batch culture, by far the most
widely adopted method by Mediterranean hatcheries. Before its
description, additional instructions are given concerning facilities,
the preparation of the culture medium, and the equipment required.

Old fashioned unit using artificial light for algae mass culture

Mass culture facilities for microalgae
Algae are cultured in a dedicated sector of the live feeds production
section, which is made of three working areas inside the hatchery
building: a lab for duplicating small cultures, a conditioned room to
maintain small culture vessels and pure strains and finally a large
area for the mass cultures in PE bags or, less frequently, tanks. In
the warmest Mediterranean areas, a light greenhouse can replace the
latter.
Small volume cultures are kept in vessels ranging from 20-ml test
tubes up to 18 l carboys. They can be made of borosilicate glass,
polycarbonate, PET or any other material able to stand a sterilization
process. These vessels are placed on glass shelves lightened by

fluorescent tubes and equipped with a CO2 enriched air distribution
system.
Hot-extruded tubular PE film is utilised for larger volumes bags. The
film is usually 0.25 mm thick and its stretched width ranges from 45
to 95 cm. Two bag designs are widely adopted in Mediterranean
hatcheries: the smaller suspended bag and the larger one placed
within a steel wire cylindrical frame. The first type has a capacity of
60 I (single) to 150 I (double or U-shaped), whereas the latter, that
stands on a saddle-like GRP base to improve circulation, can contain
up to 450l. Their top is closed by a plastic cover to prevent
contamination.

A typical scheme of a batch type production
All units are equipped with artificial lights, usually fluorescent
tubes, an aeration system, often with an additional source of carbon
dioxide, and stands for the culture vessels, i.e. light shelves for small
volumes and metal racks or wired frames for PE bags.
The unit also stores the special equipment to process pre-treated
seawater, such as fine filters and sterilizers, as well as a laboratory
where nutrients and glassware are prepared and stored, and where
the necessary monitoring operations are performed. Standard
cleaning procedures have to be strictly followed to maintain proper
hygienic conditions.

Preparation of the culture medium
In planktonic mass production, seawater is the medium of all culture
vessels. The use of other mediums such as agar is limited to the
preservation of pure algal strains. Ideally, seawater should be free
from pathogens and pollutants. With this aim in mind, seawater is
treated to remove suspended solids, contaminants and organisms and
to improve its original parameters to fit the quality standards set for
proper growth of microalgae. These methods are outlined below,
while the description of related technical equipment can be found in
the Engineering part of the manual.

Mechanical filtration
The most common systems of seawater mechanical treatment
(applicable to all culture volumes: strains, small volumes and mass
cultures) for microalgae production (which also apply to rotifers and
brine shrimps cultures), are described below.
Raw seawater is first pre-treated to remove the bulk of suspended
solids and contaminant organisms. Different methods are followed,
but the most commonly used is a combination of settling and sand
filtration. If properly dimensioned, a settling tank is a useful device.
Not only it improves the water quality at no cost by decanting its
suspended matter, but also provides a reservoir of seawater to tackle
unpredictable problems in seawater supply, such as a damaged main
pump station or a temporary contamination of seawater (oil spill,
river plume, exceptional storm). Settled water is then filtrated
through a sand or bag filter that retains particles as small as 50,10 or
5 mm depending from the sector of destination. At this point filtered
water can be used for most purposes in the hatchery, provided for
some sectors like phytozooplankton it undergoes UV sterilisation
before use. In the live feeds sector a higher degree of filtration is
required and pre-treated water is further micro-filtered to a size of 5
mm for large culture volumes and down to 1 mm for small volumes
and strain cultures for this finer filtering polyethylene wire
cartridges, bag filters or diatomaceous earth filters are used. Such a
fine filtration should even remove bacteria and other microorganisms, but in reality the filtering capacity is not absolute and
cannot totally guarantee such results, in particular under hatchery

working conditions. It is therefore recommended to proceed with the
final step, sterilization of filtered seawater.

Mechanical sand filter for the water inlet

Sterilization
Different methods of water sterilization have been developed. The
following description refers to the most common methods adopted for
hatcheries. The choice is based on local availability of equipment and
service and depends also on the amounts of water to sterilize, which
are related to the size of the hatchery.

UV light sterilisation (applicable to all culture volumes)
UV light with a wave length of 265 nm (short wave UV or UV-C) has
a strong germicidal effect based on its capacity to break the DNA
helix. It is produced by special high or low pressure mercury vapour
lamps whose germicidal capacity depends on several factors such as
their power, seawater transmittance (transparency to UV), type and
quantity of microorganisms to be destroyed, degree of purification
required, water flow (contact time) and temperature. Seawater to be
sterilized flows through one or more sealed chambers where it is
irradiated by one or more lamps placed inside quartz tubes
(transparent to UV light). The thickness of the water film inside the
irradiation chamber should be such as to allow the maximum
sterilization effect. If the power of UV lamps and chamber design are
properly dimensioned, the contact time between water and quartz
tubes is only of few seconds.

Compact water treatment unit for zooplankton
For practical purposes, and under normal hatchery conditions, an
intensity of at least 40 mJ/cm2, provided at the end of the life span of
the lamps, removes 99% of most unwanted micro-organisms for fish
farming from the treated water volume. With its auxiliary equipment
(manual or automatic wipers, UV sensors and stabilizers, computeraided control), this method is very effective and manageable and fully
justifies its cost.

Chlorine sterilization (applicable to all culture volumes)
Active chlorine is a strong oxidizing agent, commercially available as
liquid bleach (sodium hypochlorite or NaOCl) and as bleaching
powder (CaOCl2). The percentage of active chlorine in these
chemicals should always be checked in advance as it changes widely
according to the producer: commercial grade NaOCl usually contains
5-15% active chlorine, while CaOCl2 contains 60-70%. Annex 7
describes how to prepare hypochlorite solutions, to assess their active

chlorine content and the residual chlorine in treated water, as well as
the methods to sterilize seawater and deactivate residual chlorine.
Independently from the method employed, a final dosage of 5 to 10
ppm of active chlorine is used to sterilize seawater. The contact time
between water and chlorine should be at least one hour, after which
any residual chlorine must be neutralised with sodium thiosulphate,
Na2S2O3, (see Annex 7 for details). This technique is now widely used
as final sterilisation step of water in larger vessel and of the culture
equipment (air and oxygen tubing and diffusers, detritus traps,
submersible water heaters)

Chlorine container for laboratory use

Use of an autoclave, or wet vapour sterilization (applicable
to small volume cultures)
With this method, applicable only up to 5-6 I volumes depending on
the autoclave size, seawater is sterilised together with the culture
vessels, usually made of Pyrex® glass, due to their resistance to heat.
The autoclave should work at 120°C under a 2 atm. pressure.
Sterilization time ranges from 10 min (100-ml flasks) to 20 min (200-

ml flasks) and 30 min (up to 5-6 I vessels). The neck of each container
has to be covered with a loosened aluminium foil stopper to let
vapour out during the sterilization.

Dry vapour sterilization (applicable to small volume
cultures)
This method replicates the previous one, but the autoclave is
replaced by an oven. As it works in dry vapour at ambient pressure,
glass vessels filled with seawater are heated at 160-170°C for 2 to 3
hours. Dimensions and stoppers of vessels to be sterilized are the
same ones used for wet vapour sterilization.

Enrichment
The exponential growth of the micro algal populations is regulated by
four most important parameters: light, pH, turbulence and nutrients.
Whereas the first three can be easily adjusted specific nutrients have
to be added to the culture medium in proper quantities.
The main nutrients required are nitrogen (N) and phosphorus (P),
followed by trace minerals, vitamins and chelating agents. Nutrient
solutions are prepared in advance according the type of microalgae
cultivated. With the exception of N and P solution, the other
nutrients are stored as primary stock solutions, which are used to
prepare the working solutions according to the day-by-day production
schedule. Aseptic conditions have to be maintained in the
preparation of the enrichment solutions. The vitamin solution cannot
be sterilised because heating will deactivate the vitamins. The
microalgae selected for the reproduction of sea bass and gilthead sea
bream require the following fertilizers that refer to the enrichment
medium Guillard f/2.

Primary stock solutions
Trace elements and vitamins are first prepared as concentrated
primary stock solutions: in this way, if properly stored, they may last
several months. Trace elements are prepared as four different
solutions, each stored, like vitamins, in a separate container. To
prepare one litre of each stock solution, the following quantities (in
grams) are required.

Trace element stock solutions
Solution A: ZnSO4 · H2O (30g) + CuSO4 · 5H2O (25g) + CoSO4 · 7H2O
(30g) + MnSO4 · H2O (20g)
Solution B: FeCl3 · 6H2O (50g)
Solution C: Na2MoO4 · 2H2O (25g)
Solution D: EDTA · 2H2O (50g)
To prepare the solution put the components, according to the
proportions indicated above, into one 1 -I graduated Pyrex® bottle
and fill with distilled water (DW) to the mark. Deionised water can
also be employed if distilled water is not available. When the
components are fully dissolved, store at ambient temperature,
avoiding exposure to direct light.
Vitamins stock solutions
B12 Cyanocobalamin (0.1 g)
B1 Thiamin (10g)
H Biotin (0.1g)
Place the indicated quantity of each vitamin into a sterilized 1 -I
graduated Pyrex® bottle filled to the required volume with sterilized
DW. When fully dissolved, store in refrigerator and keep away from
light. The B12 solution should preferably be stored in a dark or
aluminium wrapped bottle.
Warning: do not sterilize any vitamin solution.

Working solutions
The working solutions represent the way to add the nutrients, trace
elements and vitamins directly to the seawater medium. Two
working solutions are prepared by diluting the above mentioned
stock solutions, whereas the third solution is prepared directly from
industrial grade chemical salts of N, P and K.

Working solutions ready to use

Mineral salts working solution
Mineral salts solution: NaNO3 (300g) + KH2PO4 (30g) +NH4Cl (20g)
Put the salts into one 1-I screw-capped oven-resistant glass bottle
and fill with DW to the mark. If not available, deionised water can
also be employed. When fully dissolved, sterilize either in autoclave
or oven. Store at ambient temperature, avoiding direct light. Due to
the comparatively higher requirements for the mineral salts stocking
solution, quantities in excess of one litre are usually prepared at one
time. Use 5 to 10-l glass autoclavable vessels, then store in 5- or 10-I
plastic carboys with bottom tap.
Use: 1 ml per litre of seawater medium.

Trace elements working solution
One litre of trace element working solution is prepared according to
the sequence and quantities oulined below:
Solution D (100 ml) + Solution A (10 ml) + Solution B (10 ml) +
Solution C (10 ml).
Use only sterile glassware: a 100-ml cylinder and three 10-ml
pipettes (one per solution). Mix the four solutions into a 1-I screwcapped oven-resistant glass bottle and fill with DW or deionised
water and sterilise either in autoclave or oven. Store at ambient
temperature, avoiding direct light.


Vitamins working solution
Mix the following amounts of vitamins stock solutions: solution B12
(10 ml) + solution B1 (10 ml) + solution H (10 ml).
Use only sterile pipettes, one per vitamin. Dilute the mix to one litre
volume with sterilized distilled water. Pour in a dark sterilized bottle
(or wrapped in aluminium foil) and store in the refrigerator just
before use.
Warning: do not sterilize any vitamin solution

Culture equipment sterilization
As for water medium and nutrients, also the equipment should be
kept clean and disinfected to prevent contamination.
Small glassware such as volumetric pipettes, Petri dishes, Pasteur
pipettes and beakers are sterilized either in an autoclave or in an
oven. Bigger glass containers as Erlenmeyer flask, balloons and
carboys are sterilized after being refilled with seawater. Pipettes are
divided by volume capacity and stored into capped metal containers
or wrapped in aluminium foil. Polyethylene (PE) bags are considered
sterile as they are obtained by hot extrusion and do not need special
treatments. Tanks, plastic jugs for nutrients and piping for pump
transfer are disinfected with hypochlorite.

Pipette container used with oven sterilization

All up-scaling tools and consumables that may be in contact with
algae (aluminium foil, platinum needles, necks of tubes, flasks and
balloons) are sterilized by flame using a Bunsen burner. On the
glassware neck, flaming should lasts until all water drops have
evaporated.
Sterilized hydrophobic cotton is commonly used as disposable
stoppers for all glass containers. For its sterilization, it is packed in
aluminium foil and sterilized in autoclave or oven. Cotton stoppers
are disposed of after use. Hygiene and cleaning procedures in the live
feed production sector are outlined in Annex 6.

Enrichment of culture vessels
This section describes how to proceed with the fertilization of the
different culture vessels. All working nutrient solutions are diluted at
1 ml per litre of seawater medium. Pure strain cultures in test tubes,
in marine agar and their initial small volumes are fertilized with half
dosage (0.5 ml/l).

Phytoplankton strain cultures
With small vessels, this operation should be carried out in a
dedicated room kept clean and equipped with all the necessary tools,
glassware and consumables. To reduce the risk of contamination, a
UV-light ceiling lamp can be installed to provide germicidal
irradiation when the room is not in use.
Enrichment procedure for small vessels (up to 20l capacity):
1 prepare on the bench the required vessels containing sterilized
water, nutrient working solution and the necessary tools;

2 carefully loosen their aluminium foil caps;
3 heat each flask neck and the inner side of the aluminium foil caps
with a burner; close again;
4 heat opening and screw cap of each bottle containing stock solution
in the same way;
5 take a sterile pipette from its sealed container (if in a metal case,
open and close it at the flame);
6 with the pipette place one nutrient solution at a time in each flask,
using a new pipette for each solution;
7 repeat the flaming of necks and aluminium caps on the enriched
flasks.
Enrichment procedure for polyethylene bags (up to 500l capacity):
1 The bags are filled with the same treated and UV-light sterilized
seawater utilised for small V vessels, the only difference being that
the bags are not sterilized. Due to the larger amount of nutrients
involved, use clean and sterilized graduated cylinders to fertilize
bags.
2 Cut a 20 cm-long slit on the upper rim (empty part) of the bag;
3 add 1 ml per litre of each solution.
When cutting the bag, bear in mind the final culture level, i.e. after
the addition of the inoculum.

Batch culture of microalgae
Mass culture of phytoplankton starts from pure strains of selected
species and proceeds by upscaling from small (0.5 I) up to large
volumes in PE bags (450I) or tanks (1 000 l and up). In finfish
breeding, where large amounts of microalgae are not required, the
last upscaling step is the PE bags. See annex 8 for a typical example
of microalgae upscaling protocol, and annex 9 for the daily work plan
and culture file for microalgae.

Flask sterilization and enrichment

Pure strain culture
The culture of pure strains of the selected micro algal species is the
starting point of the mass production process. Strain quality is
therefore essential for any successful production process. A good
selection of pure strains of different algae should always be kept in a
dedicated facility in the hatchery. This practice also allows the
selection of the most suitable strains under local conditions and at a
given time.

Pure guaranteed strains of algae, as well as of rotifers, are normally
available from a few laboratories and institutions in the
Mediterranean region and Europe. It is strongly recommended to
regularly renew their lines, not only in case of culture crashes, but
also to control the often unavoidable contamination or decline in
quality. Strains from other hatcheries should better be avoided
because of their possible decline in quality and the associated risk of
introducing contaminated strains.
The algal pure strains are kept under standard controlled
environment in conditioned rooms or in especially designed
incubators, in which routine work can be performed under strict
hygienic control. The pure strain cultures are usually kept in small
glass containers, such as 10 to 25 ml test tubes or 100-ml glass
Erlenmeyer flasks, closed by a sterile stopper (screw cap or a folded
aluminium foil).
Pure-strains cultures should be maintained at a steady or resting
stage, i.e. under environmental conditions which allow them to
reproduce, but not to increase exponentially in number. In this way,
their sexual reproduction is fostered, thus increasing their genetic
variability, and the growth of unwanted organisms such as other
algal species, bacteria and ciliate protozoa is prevented. Culture
parameters are therefore kept below the values adopted for mass
production. In particular, only half dose of nutrients is used, water
temperature is kept at around 14-16°C, light intensity ranges from
300 (test tubes) to 1000 flux (flasks) and no aeration and carbon
dioxide are provided.
Under routine conditions, strain cultures are usually renewed every
month. In the replication process, an inoculum of 0.1-0.2 ml (from
test tubes) or 0.5 to 1 ml (from Erlenmeyer flasks) is taken from the
best old culture which is free of contamination, to inoculate three
new vessels of the same size to start a new strain. The old culture is
then either utilised for upscaling, or is discarded. Strain culture
vessels should be stirred at least once a day by hand, paying
attention not to stir bottom debris up.
Warning: in the management of pure algal strains quality is
essential: get rid of any tube or flask which is found to be
contaminated by bacteria, fungi, ciliates, nematodes or different algal
species.

Protocol for test tubes replication
The steps to duplicate algal strains at test tube level are described in
the following paragraphs. All required equipment and consumables
have to be well cleaned and sterilised in advance.

Pure strains kept under controlled conditions
Choose the test tubes that show the best algal populations at naked
eye and check under the microscope a sample taken from each of
them for contaminant organisms (use a new sterile pipette for each
sampling). Then keep only the uncontaminated cultures and put
them on a stand avoiding any stirring.
For each selected test tube:
1 prepare four sterilized test tubes with cap on a stand;
2 prepare at least 50 ml of sterilized seawater medium, enriched with
half dose of the standard nutrients mix (see above);
3 heat necks and caps of all test tubes (new and old vessels) by means
of a manually operated burner and let them cool;
4 fill each new test tube with 10 ml of seawater medium taken with a
graduated pipette;
5 with a sterile 1 ml-pipette take 0.5 ml of mature culture from near
the surface of the selected test tube;

6 Inoculate 0.1 ml of the old culture into each new test tube; be
careful to make the drops fall freely into the culture medium without
touching the tube walls; with one hand open and close the tube cap;
with the other hand handle the pipette; do not mix or agitate the
tubes;
7 place the used pipettes into the rinser cylinder;
8 after inoculation, heat the upper part of each new test tube
thoroughly and cover with its cap, previously flamed; as an
alternative, use sterilized hydrophobic cotton or flamed aluminium
foil as stopper;
9 discard the old culture, if hot needed for other replicas, and clean
the empty tube following the usual cleaning routine for glassware
(annex 6);
10 write date and algal species on all new test tubes with a
waterproof marker;
11 place the newly inoculated test tubes on a rack in a shelf reserved
to pure strain cultures.

Protocol for purification of algal strains
Even under axenic conditions, pure strains can be contaminated by
other algal species or micro-organisms: in such cases before upscaling the cultures the micro algal populations have to be purified
before being used as inoculum.
Two methods to purify contaminated algal cultures are of common
use: successive dilutions of the original contaminated culture, and
picking up of single cells from the original culture. Both techniques
are also applied when new algal species are isolated from the wild.

Dilution method
A simple technique to purify a contaminated strain is to proceed with
repeated sub-cultures obtained by progressive dilutions of the
original sample. Dilution rates can be very high, depending on the
number of the sub-cultures. In the procedure described below a

dilution rate of 10-10 is reached. The same nutrient quality and
environmental condition of the initial culture are used.
Example of dilution:
1. Prepare 10 sterile test tubes with caps on a tube rack;
2. Sterilize 500 m I of seawater in an Erlenmeyer flask, then fertilize
it with half dose of usual mix of nutrients when cool;
3. Using a sterile 10-ml pipette add 9 ml of enriched seawater to each
tube, close loosely with flamed caps and number them 1 to 10;
4. Put the test tube containing the strain to be diluted in the rack,
remove the cap and flame its neck;
5. Using a 1-ml sterile pipette take 1 ml and add it to the tube No. 1,
then stir gently;
6. Using a new sterile 1 ml-pipette repeat the previous step by taking
1-ml inoculum from tube No. 1 and inoculate it into tube No. 2;
7. Repeat the same procedure with the remaining tubes, each time
pipetting 1 ml from the previous tube (gently stirred) into the next
one; flame necks and caps and let them coo);
8. Keep under controlled environmental conditions.
When cell growth reappears, check samples of the tubes under the
microscope and get rid of the tubes that are still contaminated,
typically the initial ones, and keep only the purified cultures, usually
in the more diluted tubes. Repeat the process using the last dilutions
if necessary, and in any case at least every three months to always
have a safe amount of purified cultures ready at hand.

Picking up method
Two systems which produce single cells are described below: the agar
plate method and the capillary method:

Agar plate method: A solid medium can grant more stable
conditions for the growth of the desired species. This technique needs
some sterile equipment like Petri dish and platinum hooks.

Example:
1. Prepare the solid medium by adding 1.5 g of agar powder to 100 ml
of sterile fertilised seawater;
2. Heat the medium using a Bunsen burner, stirring with a sterile
glass rod to dissolve the agar, and pour it on 3 to 5 sterilised Petri
dishes;
3. Allow to cool and solidify;
4. Using a sterilized 1 ml-pipette take 0.1 ml from the initial
contaminated strain culture and drop it into each Petri dish;
5. Spread the sample over the agar with a sterile platinum hook and
incubate at desired environmental conditions placing the dish upside
down, so that water drops will not form on the lid and then fall on the
culture;
6. Once algal colonies are observed, take a sample by means of a
sterile platinum hook and check under the microscope
7. Monospecific colonies should be kept in agar for successive
replication or might be up-scaled by replication (i.e. a small portion of
the colony together with some agar, using a sterile hook) on a 50-ml
or 500-ml Erlenmeyer flask to continue in the liquid medium.

Capillary method:
This technique follows the dilution method, but the inoculum is
obtained by selecting single cells of the desired species by means of a
capillary pipette handled under a microscope.
The isolation or purification of cultured strains should be repeated as
many times as required to produce contaminant-free cultures.

Upscaling culture conditions
As indicated above the upscaling of microalgae production starts
from small containers (0.5 ml) and proceeds through various steps up
to the mass production in PE bags. Each step involves an increase of
the culture volume: when mature (i.e.: in log phase), the algal

population of a smaller volume is sacrificed to replicate the same
vessel and to inoculate larger vessels.
Small-medium size (0.5 to 10l) cultures of microalgae are usually
kept in borosilicate-glass containers with large necks, such as
Erlenmeyer flasks or other flat bottom round flasks (balloons) and
carboys. The flasks are usually closed with a stopper made of
sterilised cotton or of plastic which can stand sterilization in
autoclave.

Petri dish with culture solid medium
All these cultures are provided with a proper medium to support
algal growth (treated and fertilized seawater), good aeration
supplemented with carbon dioxide (CO2) as an additional carbon
source, and a strong light. In this way it is possible to reach quickly
the log-phase growth. Aseptic conditions should be maintained to
avoid contamination and culture crashes.
For practical purposes the main parameters for the cultures are
usually the same for the different species of algae mentioned in the
sections above:
temperature 20 ± 2°C
salinity
25 to 30 ppt
light intensity 4000 to 8 000 flux

aeration
50 to 100% of the culture volume per minute
carbon dioxide 2 % of air volume
Algae flask replication

Temperature
For mass culturing purposes, the optimal temperature for the above
mentioned algal species ranges between 20 and 24°C. Generally
speaking, temperatures lower than 16°C and above 27°C will slow
down growth rates, whereas those above 30°C are normally lethal.
That makes room conditioning necessary as artificial lights and
insufflate air from the aeration system can raise temperature
dangerously.

Low temperatures are used for pure strains only, where growth
should be kept as slow as possible. As low temperatures also affect
bacterial growth, non-axenic cultures should be maintained at the
lowest possible temperature consistent with a good growth, to
prevent bacterial growth.
Salinity
In the selected species salinity does not represent a limiting factor
within a range of 15 to 40 ppt.
Light
Light is the source of energy for photosynthesis and therefore in
mass cultures algae are usually kept in continuous light. Fluorescent
tubes are the commonest choice for providing light due to their low
power consumption, low installation cost and limited heat production
compared to bulb lamps. Spectral quality suitable for algal growth is
provided by “Cool white” and “Daylight” models, often installed in
equal numbers. The commonest tube size is 42” with waterproof
contacts. See Part 4 Engineering for their installation in the
hatchery‟s algal unit.
Even if each algae species has its own preferred light intensity for
best growth, for practical purposes intensity in mass cultures is kept
in the range of 2 500-8 000 flux, while higher intensity is used on the
small volumes shelves (5 000-8 000 flux) because of their higher cell
density. Large volumes (bag cultures) can also be illuminated by
natural daylight, entering from sufficiently large windows or from a
continuous glass wall, provided that the cultures are not directly
exposed to external contamination.

Lighted table used for mid size culture up scaling

Aeration
Aeration is used to maintain the culture in turbulent state,
preventing settling of cells and exposing all cells to light. It also
supplies carbon dioxide, which is fixed by the algae during
photosynthesis, and provides essential pH stabilization.
Transparent PVC tubing, of 6 mm internal diameter, are commonly
utilized to deliver air to the culture vessels. In small vessels this
tubing is connected to glass pipes which fit the stopper and reach the
bottom of the vessel, while in PE bags they are simply forced into a
small hole near their bottom (the water pressure keep the holes
sealed), whereas in tanks a weight keep them submersed.
Aeration should be moderate in the first two days of culture, then
should be increased adjusting it according to culture growth. For this
purpose screw clamps or cheaper plastic needle aquarium valves are
required to adjust the air flow. If aeration produces foam, it is an
early warning of culture troubles.

Carbon dioxide
As its normal content in air is low (approx. 0.03%), carbon dioxide is
often supplied at 2% by volume to optimize culture growth.
Commercial grade CO2 bottles are utilised, and the gas is injected
into the main air pipe via a dispensing vessel in which gas bubbling
reveals the gas flow. Since carbon dioxide is heavier than air, to
prevent stratification the pipe makes some ups and downs after the
point where it is injected.
Depending on the algal species, the CO2 supply, and the volume of
inoculum, working cultures normally reach their log-phase in 5 to 7
days. At this point, cultures can be utilized either to start new
cultures, to be fed to rotifers or to be used as “green water” in fish
tanks..
The volume of the algal inoculum is usually 15 to 20% of the new
volume. Smaller or larger inocula could be used to decrease or
increase growth rate. In culture up-scaling, the new vessels have to
be inoculated with a sufficiently high microalgae density in order to
ensure a rapid growth and to limit the risk of contamination with

different algae or other micro-organisms (protozoa, nematodes, fungi,
etc.).

Scaling up protocol
The following section describes in detail the operation to replicate
different volumes.

Inoculation of flasks from test tube
Follow the same procedure as previously described for pure strains.
Each 0.1-1 flask will receive 50 ml of enriched medium and 0.5 ml of
inoculum. At this stage, no aeration is required. When mature, each
small flask will inoculate a new 2-l flask.

Alternative use of olives containers for algae culture

Very small PE bags of
phytoplankton
at
Ittica

Ugento

Medium and large size bags

Inoculation of 2 and 6-l flasks from another 2 or 6-l flask:
1. Prepare the necessary amount of new flasks filled with sterilised
seawater, as well as all equipment required for the operation
(pipettes, nutrients, cotton stoppers, aluminium foil, glass tubing,
etc.);
2. Select the mature culture that will be used as inoculum, checking a
sample under the microscope for contaminants;
3. Remove its cap and flame its neck; then close with a flamed
aluminium foil stopper and let it cool;
4. In the meanwhile, add the fertilizing working solutions to each
new flask, at a rate of 1 ml/l; using a new sterile pipette for each
solution;
5. Flame their necks and aluminium caps thoroughly;
6. When cool, remove the stopper and pour some algal culture of the
old flask into the new vessels at a rate of about 10% of the receiving
volume, avoiding to wet their neck with the inoculum, then gently
shake flasks to mix the new culture;
7. Flame thoroughly their necks, introduce the sterilized glass tubing
for aeration and close tightly with sterilised cotton stopper (or any
other type of sterile cap);

8. Write date and algal species on the new flask;
9. Place the flasks on the lighted shelf and connect to the air delivery
system, adjusting its flow to a gentle bubbling;
10. After one hour, check all new vessels for a proper air bubbling.
Use only the upper layer of the old culture, leaving dead cells and
debris in the flask used to inoculate the new ones. The size of the
inoculum for small volumes is only 10% of the new volume because of
the high cell density. Remember to get rid of every contaminated
flask. The above mentioned procedure applies to the upscaling of the
other medium size vessels.
PE bags inoculation with the help of a self-priming pump

Inoculation of a PE bag from a flask or from another bag
1. Prepare the bag, either suspended to a rack or placing it inside a
cylindrical frame made of wire;
2. Fill the bag with sterilized water; leaving enough free space for the
volume to be inoculated; wait a couple of hours to check for possible
leaks; if found, seal them or replace the bag;
3. Introduce nutrients and inoculum;
4. Fit two PVC tubes for aeration to the bottom of the bag, connect
them to an air line and adjust the air flow to a gentle bubbling;
5. Add the nutrients using a graduated cylinder at the usual rate of 1
ml/l of each working solution;
6. Select a mature culture that will be used as inoculum, either from
a large flask or from another bag, and check a sample under the
microscope for contaminants;
7. In case a flask is used as inoculum, remove its cap and flame its
neck; then close with a flamed aluminium foil stopper and let it cool;
8. Then, remove the stopper and pour the algal culture from the flask
into the bag. The inoculum should be about 10% of the receiving
volume;
9. If the inoculum comes from another bag, with a self-priming
plastic pump transfer the inoculum from the old bag into the new
one. The volume to be transferred should be about 15-20% of the
receiving volume (a bigger inoculum is necessary to compensate for a
less dense culture);
10. on each bag mark date, algal species, origin of the inoculum (bag
or flask) and the bag serial number.
To reduce the risk of contamination, smaller bags are usually
inoculated from flasks, whereas larger bags are inoculated from
smaller bags.

Large volume cultures

Monitoring algal populations
A regular check of microalgae cultures is essential to prevent crashes
and to keep high quality standards. The main parameters to be
monitored are: colour, density, pH and contaminant levels. As an

example, a change in colour to opaque grey and a pH level lower than
7.5 may indicate a high degree of bacterial contamination. A lighter
colour than normal may reveal insufficient nutrients or poor lighting.
However, during the peak production season in the hatcheries a close
monitoring of algal cultures can hardly be assured, due to the chronic
lack of staff and time. Mass cultures are normally checked at naked
eye by experienced staff and strict controls are usually restricted to
pure strains and small vessels.
To partly by-pass this problem, the staff can do a good preliminary
job outside the peak production season, when they are not so
absorbed by production and there is some more time. Then test
culture cycles can be closely monitored, the algal culture growth can
be followed daily by counting the number of cells per ml with a
haemocytometer (see below), and their average growth curves can be
plotted against values obtained with a colorimeter. During more busy
days, a colorimetric monitoring (comparing values against the test
curves) gives a fast and reliable indication of algal cultures growth.
These test cultures are useful for a number of other reasons as it is
possible:
to determine the growth curve for each species of algae under
local conditions;
to devise criteria for quick identification of possible troubles
(eg.; presence of foam, sedimentation pattern, changes in
colour, etc.);
to determine optimal utilization time, i.e. the age at which the
algal population reaches the peak of the log-phase;
to adjust environmental conditions to maximise production;
to control possible contaminants and try countermeasures.

Counting microalgae
A Neubauer haemocytometer can be used for counting microalgae
cells with diameters ranging from 2 to 20 mm and at densities up to
500 million cells/ml.
This device consists of a thick rectangular slide with an H-shaped
trough delimiting two counting areas. With its special cover slip in
place, each area forms a chamber 0.1 mm deep. The total area of each
chamber is 9 mm2. Each chamber is divided into 1 mm2 squares: the

four corner squares are subdivided into 16 smaller squares, whereas
the central one is subdivided into 25 smaller squares, 0.2 x 0.2 mm,
each with an area of 0.04 mm2.
To count cells in a sample, proceed as follows:
1. Take a 5-ml sample from each culture and place each sample in a
separate test tube. Add one drop of Lugol solution and mix well;
2. Prepare clean Neubauer slides and covers;
3. Put one drop of well mixed algal suspension on each Neubauer
chamber and cover with its cover slip;
4. Check under low magnification that the algal cells are evenly
distributed: avoid the presence of air bubbles, over flowing, under
filling and uneven distribution of cells. Allow the cells to settle for 5
minutes before counting;
5. Start counting at the top left square and count only those cells
which lie within or touch the boundary lines chosen according one of
the two possibilities: either left and bottom boundary lines or right
and top boundary lines;
6. For cells larger than 6 mm and not too dense, make a total count in
each of the four corner squares and in the central square; then repeat
this count in the second chamber;
7. For minute cells and denser populations, count the cells in 5
smaller squares in the larger central square, then repeat this count
in the second chamber;
8. Calculate average cell density as follows: if all cells are counted in
individual blocks each with an area of 1 mm2 and a volume of 0.1
mm3, the average cell density expressed as cells/ml is given by the
total count divided by the number of blocks and multiplied by 10 000;
9. If all cells in ten (five in upper chamber + five in lower chamber)
smaller squares (volume = 0.004 mm3)in the central block are
counted, the average cell density is the total count multiplied by
4 000000 and divided by 10, i.e. the total count multiplied by 400 000;

10. Record the count for each sample and introduce it in the algae
population growth curve prepared for each culture container.

2. Micro Zooplankton Culture:
Micro zooplanktons, the rotifer Brachionus plicatilis, calanoids and
copepods, cladoceron crustaceans (Daphnia spp. and Moina spp.) can
be isolated from their natural environment, and after isolation and
purification the required organisms can be subjected to mass scale
production through feeding algae and rice bran. Artemia nauplii can
be produced through decapsulation of Artemia cysts. The tiny size
and enormous reproductive potential of rotifers make them a popular
choice as a food item among breeders of marine fishes and
invertebrates.

Mass culture of rotifers
For the breeding of many marine finfish species the rotifer
Brachionus plicatilis is, up to now, the only live feed that can be used
in their very early larval stages. While not a compulsory choice in
seabass feeding, its mass production is required for the successful
breeding of gilthead Seabream (and of other Sparids, grouper and
grey mullet), whose small mouth cannot accept larger preys at the
onset of larval feeding. Among other valuable characteristics as live
feed for fish, B. plicatilis was also chosen due to the relative easiness
to culture it in large scale.
Two main strains are used. The so-called small strain (S-type) and a
large strain (L-type), 50% bigger in dry weight than the S-type. The
average length of the lorica in the adult S-type rotifers is 130 µm,
whereas it is 240 µm in the L-type. The two strains also show
different temperature and salinity tolerances. In the last years
mainly S-type rotifers are reared in the hatcheries.

Brachionus rotundiformis and Brachionus plicatilis

Population dynamics
Rotifers can reproduce both sexually (mictic reproduction) and
asexually (amictic reproduction), according to the environmental
conditions. Usually amictic, rotifers may turn to sexual reproduction
when sudden changes in salinity or temperature take place. Then,
they produce large resting eggs, similar to brine shrimp cysts.
However, in hatcheries is the asexual reproduction that provides the
large amounts of animals required for the early feeding of fish larvae.
Rotifer population dynamics under mass rearing conditions follow
different phases, mimicking those of microalgae:
the lag-phase, when, just after the inoculum, rotifers begin to
consume the phytoplankton of their culture medium and the
number of both egg-bearing individuals as well as the quantity
of amictic eggs increases;
the log-phase (or exponential phase), where rotifers reproduce
very fast and population growth is exponential;
the transitional phase (or declining growth), where growth rate
slows down and egg-bearing rotifers become rarer;
the decline phase, where almost only old rotifers without eggs
are found and their number decreases rapidly as death rate
exceeds growth rate.
The quality of the rotifer population to start new cultures is even
more important than in the case of microalgae. To be used as
inoculum, the rotifer population must still be in the middle of its logphase with at least 20% fertility rate (measured as percentage of eggs
over total rotifers. Populations in their last declining phase,
characterised by limited motility, scarce repletion and absence of eggbearing animals, should always be discarded. With a proper

inoculum and under optimal rearing conditions, a rotifer population
should reach its harvesting density within 4 to 5 days.
Under hatchery conditions, rotifer populations can reach the
following densities:
in flasks and bags, after 5 to 7 days:
- S-type rotifers, 500 to 700 in d/ml
- L-type rotifers, 150 to 250 in d/ml
in tanks, after 4 to 6 days:
- S-type rotifers, 1000 and more in d/ml
- L-type rotifers, 400 in d/ml

Mass production systems
As this microscopic animal is a filter feeder, its nutritional value
strictly depends on its food. In hatcheries, the species is first cultured
on microalgae, following the same scale-up protocol described for
microalgae, then its final mass production is achieved in large tanks
where artificial diets are fed to rapidly increasing numbers,
improving at the same time their nutritional value.
The rotifer B. plicatilis is a rather sturdy species able to tolerate a
wide range of salinity, temperature and ammonia levels. It can also
use several food sources, provided that particle size remains within a
2-20 µm range. Obviously, the highest growth rate is achieved under
more restricted environmental parameters, and is closely related to
the selected rotifer strain and feeding provided,. In particular, good
yields are obtained with high dissolved oxygen levels, temperature at
25°C, pH at 7.5-8.5, salinity in a 20-30 ppt range, less than 1 mg/l
free ammonia (NH3), and moderate turbulence. Light is required only
when rotifers are fed microalgae.
Dissolved oxygen levels
During the upscaling phase in algal vessels, the oxygen requirements
of rotifers are fulfilled by microalgae photosynthetic activity. In mass
culture, artificial diets, enrichment boosters, high rotifer densities

and metabolic products, contribute to deplete oxygen in the culture
medium. To keep its levels within safe margins, i.e. above 80%
saturation, a strong aeration is supplied, linked to an emergency
oxygen delivery system. See section below for technical details.
Temperature
In mass culture, a temperature as high as 30°C is acceptable, but as
bacterial and other contaminants would also increase at these levels,
a more manageable 20-25°C range is commonly adopted.

Medium and large volumes cultivated in the same room
pH
An acceptable range is pH 5-9, but the buffer capacity of seawater
keeps it within a narrower range. A low pH also influences the
balance between the toxic un-ionized ammonia and the ionized form
(NH4+). The lower pH also influences the balance between the toxic

un-ionized ammonia and the ionized form (NH4 reduces the fraction
of toxic NH3.
Salinity
The acceptable salinity range for this rotifer species is quite broad: 160 ppt. However there are differences between the two strains, the
optimal salinity for the S-type strain being 18-20 ppt, whereas L-type
grows better at 30 ppt.
Turbulence
A moderate to strong turbulence is required to keep food particles
and rotifers in suspension. The aeration system should provide
enough water circulation, but the position of the air diffusers should
be adjusted in such a way to avoid stirring and re-suspension of
bottom sediments: air stones are hung 15 cm above the bottom, both
along the periphery and in the centre of the tanks.

Mass culture facilities for rotifers
Inside the hatchery building, phytoplankton shares the same culture
facilities with rotifers, which have a specific section with large tanks
for their final mass production. In Mediterranean hatcheries, these
tanks have a volume ranging from 1 to 10 m3. These tanks are made
of FRP, PE or PVC-lined concrete. As water circulation and particles
sedimentation are important in rotifer culture (see below), a widely
adopted design is the round tank with conical bottom. This section for
mass production of rotifers is equipped with an air distribution
system, air conditioning and seawater heaters to keep high water
temperatures. Special filtering devices to harvest and rinse rotifers
are also available.
As mass culture of rotifers does not require light (except for the
phases in which algae are used as food), a normal lighting system is
installed. Treated seawater (filtered and sterilized) is provided by the
same source that feeds the algal section.
As in microalgae production, hygiene is important and requires the
strict implementation of standard cleaning procedures

Preparation of the culture medium
The same procedures and precautions described in the algal
production section apply to rotifer culture. The only enrichment
added to the rotifer culture medium, be it a log-phase algal culture or
treated seawater plus artificial diet, is represented (cyanocobalamin)
as fertility by the addition of vitamin B12 booster for the rotifers. Its
dosage is usually 100 ml of B12 stock solution per m3 of rotifer culture
in tanks, whereas small vessels and bags are fertilized at the rate of
1 ml/litre. In both cases the vitamin is added together with the
inoculum.
To prepare the stock solution put 0.1 g of vitamin B12 into a sterilised
1-l graduated Pyrex bottle and fill with sterilised DW to the mark.
When fully dissolved, store in the refrigerator. Warning: never
sterilise vitamin solutions.
Pure strain culture
The first phases of rotifer cultures (strains and small volume
cultures) are performed in the same conditioned room where
microalgae are kept, but on separate stands to avoid the risk of
possible contamination. The same propagation techniques described
for microalgae strains are used for rotifer strains. As for microalgae,
culture conditions discourage exponential growth. Rotifer strain
inoculums are kept at 1 or 2 individuals per ml in a low-density algal
culture and no vitamin is added. Both test tubes and small flasks are
used to maintain rotifer strain cultures.
Upscaling rotifer cultures
A clean culture of egg-rich rotifers from a 0.5-l flask is usually
inoculated directly into a 5 to 10-l flask, bypassing the 2-l flask stage.
The inoculum should provide an initial density of 10 to 20 rotifers per
ml. A larger inoculum results in a faster pace of population growth.
Rotifers should always be inoculated in algae cultures which have not
yet reached their peak growth (that is their log-phase).
Environmental conditions should be the same ones at which algae
are kept. However, aeration can be reduced to diminish the amount
of foam and bottom sediments produced by the metabolic activity of
rotifers.

Vitamin B12 is routinely added to all new vessels, at 1 ml of stock
solution per litre of algal culture. Mature rotifer cultures from small
vessels (5 litres or larger) are poured into bags with an algal
population that has not yet entered its full log phase. Five to ten %
inoculum is used at this stage, i.e. one 5-liter vessel is used to
inoculate one 100-l bag, following the rule of the new culture starting
at 30-50 rotifers/ml. Using a sterile volumetric cylinder, add 1 ml of
the Vitamin B12 per litre of culture; mark the date on the bag surface,
together with the origin of the inoculum and the serial number to
facilitate its handling and record keeping.
If the population is growing normally and remains free from
contaminants, the horizontal culture method is also frequently used
for rotifer culture upscaling. In this case, a good bag makes it
possible to inoculate a group of new bags with a initial density of at
least 30-50 rotifers/ml.
In the same way as it happens in microalgae cultures, also rotifer
cultures are subject to occasional failures (culture crashes). A culture
crashes either when rotifers do not multiply as foreseen, or when the
entire culture dies abruptly. The possible causes are described below
in the Monitoring section. As a precaution against these problems,
always maintain a certain number of culture bags in excess of those
required by the production schedule. They could be useful to replace
quickly a crashed culture at any stage.

Mass culture
Rotifer mass culture is carried out in large tanks as mentioned above.
Because of the very high density achieved (up to 1.000 individuals
per ml or more), rearing procedures and protocols to maintain strict
hygienic conditions (see Annex 6) have to be rigorously applied. Such
routine procedures are described below.
There are basically two main mass rearing methods for rotifers: the
older technique that is based on algae and baker‟s yeast as food for
the rotifers, and the more recent one that uses an artificial diet, the
Culture Selco® produced by INVE SA of Belgium (or similar). Both
are described below.

Tank preparation
Before starting a new production cycle, normally after the harvest of
the previous rotifer culture, prepare the tank as follows:
1. Rinse with tap water to eliminate the bulk of organic debris;
2. Wash it thoroughly with brush and detergent and rinse it again;
3. Wash or spray the tank walls with 500 ppm active chlorine
solution (Annex 6); after a couple of hours, drain the tank and rinse it
well until the chlorine smell is gone;
4. Let the tank dry and fill it with sterilized heated water only when
needed; as an alternative possibility, fill the tank with seawater and
sterilize with hypochlorite, then neutralize the residual chlorine with
sodium thiosulphate.
Repeat the procedure for the equipment to be used in the tanks:
aeration tubing, drain valves and suspended traps. A practical
procedure is to assemble all small equipment in the new tank, fill
with SW and sterilize with hypochlorite: the equipment will be
disinfected as a consequence.

Inoculation
To be used as inoculum, the rotifer population must still be in the
middle of its log-phase with at least 20% fertility rate (measured as
percentage of eggs over total rotifers). Never use rotifers which have
already reached the last phase characterised by limited motility,
scarce repletion and no egg presence.
In algae/yeast fed tanks, the initial density of inoculum, one of the
most important factors in rotifer culture, should be kept at least at
100 animals/ml, with an optimal density of 150-200 animals/ml. High
density cultures with artificial feeding need up to 500 rotifers per ml
(see below). With an initial density of 200 animals/ml, rotifer density
should reach its peak within four to six days at 25°C.
Tank inoculum may come from large rotifer bags (vertical upscaling),
or from other tanks (horizontal upscaling). One day before being
harvested to be used as inoculum, rotifers in the tanks should be fed

a fertility booster such as Protein Selco® by INVE SA. In order to
reduce contamination and culture crash risks, horizontal upscaling
should be limited to 7-8 cycles, after which the inoculum should be
again taken from large bags.

Feeding
As previously indicated two feeding methods are most widely
adopted: 1) a combination of algae and baker‟s yeast and 2) a totally
artificial diet. For its reliability and higher output, the latter is
progressively replacing the first method.

Mass culture with algae/yeast as food
The initial method of mass culturing rotifers in Mediterranean
hatcheries makes use of a common and easily available food staple,
the bakers‟ yeast Saccaromyces cerevisiae. It is a labour and cost
sparing food, which has no nutritional value for rotifers that feed on
bacteria associated with the yeast.
Compared to the artificial diets, this method has a lower yield and
requires more time, typically one extra day. Density at harvest rarely
exceeds 450 rotifers/ml with an average daily increase ranging from
19 to 33%. In addition, rotifers should be enriched with high levels of
(n-3) HUFA and vitamins. A major constraint of this method is the
absolute necessity to improve the otherwise very poor nutritional
quality of yeast-fed rotifers before their distribution to fish larvae.
The enrichment procedure, which takes place the day before
harvesting the rotifers, is explained below.

Protocol:
1. Fill the tank with sterilized sea water diluted with tap water to
obtain a 20 ppt salinity; check the chlorine content of tap water and,
if present, neutralize it with an excess of sodium thiosulphate (see
Annex 7). Take care to leave enough space for the algal cultures to be
supplied as food (about 30% of the tank volume).
2. Place the air diffusers and switch on the aeration;
3. Place the traps for ciliates and impurities;

4. Select the most suitable rotifer bags to be used for inoculum,
checking for contaminants and using only clean batches;
5. Filter the selected batch or batches (see below for procedure);
6. Inoculate the tank to achieve an initial density of 150-200
rotifers/ml. This is considered day 0;
7. Add algal culture as 20% of tank volume to provide rotifers with
their initial food; as usual, the algae should be in their log-phase and
from non contaminated cultures, even if of different species.
8. The next day (day 1) fill the remaining 10% volume with algal
culture;
9. On the tank file, record all information on the culture growth, food
distributed and environmental parameters monitored (see below for
details and Annex 9 for a file sample);
10. From day 1 on, feed with bakers‟ yeast at the following rates,
according to the recorded rotifer density.

The daily amount of fresh yeast is divided into 4 equal rations fed at
2 and 8 am, 2 and 10 pm (the last two rations are given by the night
watchman). Each yeast ration is taken from weighed out of the yeast
cake kept in the refrigerator and placed in a plastic bucket filled with
tap water at a concentration of 100 g/l. A kitchen or better an
industrial blender helps to separate yeast cells and keep them
suspended in water. Feed immediately and discharge any leftover.
Prepare fresh for every meal.

Mass culture with Culture Selco® as food
A different technique based on a compound feed has been developed a
few years ago by the Belgian Company INVE SA. The product,
named Culture Selco® (CS) is a dry and complete rotifer diet that
does not require algae and is also effective as enrichment medium.
Particle size (5 to 7 µm) and physical characteristics ensure an
optimal uptake by rotifers. The feed composition includes proteins
(>35%), lipids (>15%, of which 23% are PUFA), carbohydrates (30%),

carotenoids and other micronutrients as minerals and vitamins A,
D3, F and C.
The average daily production of rotifers fed on CS ranges consistently
from 45 to 60% of the initial rotifer density. In addition rotifers are
enriched with high levels of the essential (n-3) PUFA and vitamins
This diet has made rotifer mass culturing reliable and
predictable, and has consistently reduced the need for algal cultures
and their associated labour and facilities requirements. New rotifer
cultures can be easily started from old ones, thanks to their enhanced
fertility.

Feeding
The following schedule gives the CS daily feeding rate (DFR),
expressed as grams of CS per million rotifers. The day before harvest
rotifers are enriched with other artificial diets made by INVE,
Protein Selco® or DHA Protein Selco®.
To prepare CS suspend the amount required for a single meal in tap
water, up to 50 g CS/l, and mix vigorously for 3 minutes (use a
kitchen or better an industrial blender). Mixing or shaking by hand
or using a magnetic stirrer is not sufficient to separate the CS cells.
Remember that cell agglomerates left in the feed suspension cannot
be ingested by rotifers because of their large size. Take care not to
overfeed as uneaten feed can also quickly spoil water quality. Feed
the daily amount in four to six meals evenly distributed over the 24-h
period. In case it would be necessary, the feed suspension can be
stored at temperatures below 8°C, and the amount needed for the
whole day can be prepared at one time. The feeding ration can thus
be distributed from the stored suspension at each meal.
Before transferring rotifers to a new tank, place about 25% of the
first day‟s food ration in the tank, so that the feed would be already
evenly spread when the rotifers enter the medium. Add a few drops
of silicon based antifoam agent, such as Rhodorsil Antimousse AM
70414 by Caldic Belgium NV.

Aeration
Aerating the rotifer culture is essential to provide oxygen, to keep
rotifers and food cells suspended and to optimise tank cleanliness.
The aeration rate and the air diffusers positioning should be carefully
adjusted. A reasonably strong aeration is detected by an evenly
spread water turbulence at water surface without large air bubbles.
Number and positioning of air diffusers depend on tank shape and
volume. In all cases, however, they should be suspended at about 15
cm above the tank bottom to prevent re-suspension of sediments. As
to the number of diffusers, a reliable rule of thumb is to keep a
distance of approximately 60 cm among them.

Cleaning cultures
In round tanks with conical bottom, bottom sediments are removed
by letting them first settle for 10 15 minutes in absence of aeration
(take care to check DO levels during the operation). Then the bottom
valve is opened for a few seconds and it is closed when the out
flowing water is again clean. Repeat the operation twice a day, in the
morning and evening.
Complete the cleaning procedure by removing any greasy layer that
may be formed at the water meniscus with a sponge or a paper cloth.
Never dip your hands in water.
The rotifer tank water can be rapidly polluted by particulate matter,
faeces and flocks of uneaten food. Their removal may prevent an
excessive bacterial development and will increase the oxygen
available for rotifers. As the continuous aeration of the water volume
prevents in part the settlement of particles, their removal is achieved
by means of “particle traps”. These devices consist of floating mats of
coarse sponge-like material, such as the housing polishing mat
Scotch Briteä. In the case of a 3,000-l tank, three pieces measuring
15 x 100 cm are hung vertically in the water (by means for example
of wooden stick laid across the tank top rim), and are kept vertical by
a terminal weight. The water circulation pushes particulate matter
against the trap, where they adhere and clog the mat. These mats
are also an ideal substrate for Vorticella, a sessile ciliate that
competes with rotifers for food. To be effective, the traps must be
cleaned at least twice a day.

To clean particle traps proceed as follows:
1. Take them out of the tank carefully, avoiding dripping of trapped
material back into the water;
2. Remove all particles with a high pressure jet of tap water;
3. Dip the trap in a 500 ppm hypochlorite solution for one hour to
disinfect;
4. Rinse well with tap water, dip in a thiosulphate solution to
neutralize residual chlorine and place again in the tank.

Harvest
At harvest, rotifers are filtered and rinsed before being fed to fish or
utilized as inoculum for new tanks. For this purpose, a double
submerged filter is used. The inner filter has a mesh size of 250-300
µm to retain larger particles, flocks of agglomerated food particles
and ciliates which would rapidly clog the finer filter. The outer filter
has a 50 µm filter mesh. Its capacity should be large enough to keep
safely the whole rotifer population for the time needed to complete
harvest and rinsing. Both filters are placed inside a large wheeled
container full of water to avoid pressure build-up from the outgoing
water that would smear rotifers against the net. A gentle air
bubbling along the inner side of the filter helps to keep the filter free
from clogging.
Harvesting and rinsing protocol for round-conical tanks with central
drain:
1. Prepare the harvesting device, always clean and disinfected with
hypochlorite;
2. Inject pure oxygen into the tank to be harvested for 10 to 15
minutes to have a supersaturated medium (at 10 ppm DO) in which
rotifers could safely stand filtering operation;
3. Switch off aeration for 10 - 15 minutes and purge the bottom
sediments;
4. Into the central drain fit a PVC pipe with holes at 15 cm from the
drain; wait 5 minutes;

5. Fix a flex hose to the bottom drainage valve and place the other
end into the harvesting device;
6. Open the valve and start filtering;
7. Regulate the water flow so that the filter does not clog and the
culture water does not overflow; do not exceed 100 l/minute;
8. Stop water flow and clean the clogged pre-filter whenever
necessary to avoid overflow of rotifer concentrated water;
9. While harvesting, check for possible loss of rotifers through the net
by sampling some filtered water with a beaker or a Petri dish;
10. At the end of filtration, close the valve and rinse for 10 to 15
minutes the rotifers with filtered sterilized seawater at the same
temperature as the tank of origin.

High density rotifer culture
Recent advances in mass culture technique yield higher rotifer
densities. As an example, the procedure described below shows how
to produce one billion rotifers daily with a battery of five 1-m3 round
tanks with conical bottom.
Protocol:
Inoculum: 500 rotifers/ml, harvesting at 1 000 rotifers/ml in four
days. Prepare five tanks following the above mentioned procedure.
Environmental parameters
oxygen: keep 7-9 ppm with pure oxygen integration, preferably
controlled by an automatic monitoring device
pH:7.5-8.5 add 10% hydrochloric acid when needed
TAN Total Ammonia Nitrogen:< 6 mg/l
NH3:< 1 mg/l
Aeration: reasonably strong. Hang three air stones placed at 15
cm above the bottom along the tank wall for air injection, and
place one in the centre at 20 cm from the cone tip for oxygen
injection
Particle traps: place four mats along the tank wall.

Cleaning operations (three times a day):
1. Remove traps, air tubing and diffusers and wash carefully with tap
water;
2. Stop aeration for 10 minutes and purge the tank cone through its
bottom valve;
3. Put back in place clean and disinfected traps, air tubing and
diffusers.

Enrichment
Rotifers have a limited nutritional value for marine finfish larvae. In
the past their nutritional value was upgraded by an enriching
process before their harvest through feeding them with microalgae
rich in PUFA and vitamins such as Chlorella, Nannochloropsis and
Isochrysis.
At present, the enrichment is provided by specially formulated
artificial diets like the above mentioned Selcoâ products. This oil
emulsion gives excellent results in terms of high levels of EPA, DHA
and vitamin C, which was not possible with the only use of algae.
Moreover, labour, time, investment and running costs are spared.
Rotifers can be enriched either in their mass culture tanks or after
harvesting by placing them in dedicated enrichment tanks.
The first method produces an enrichment of the tissues, as it is
continuous along the entire culture period. The acquired fatty acids
reserves are more stable and are less exposed to a rapid decrease in
nutritional value during starvation. This method also saves time and
reduces handling losses.
The second system is short term enrichment or, rather, a gut
enrichment. It implies the harvesting and rinsing of the rotifers and
the preparation of a separate enrichment tank.
Enrichment with algae:
use selected algae as specified above;
enrich for at least 8 h;
maximum rotifer density: 500/ml;

microalgae density: Isochrysis 5 million cells/ml; Chlorella sp. o
Nannochloropsis spp. 12 million cells/ml;
resulting average total PUFA content of enriched rotifers: ± 7
mg/g dry weight
Enrichment with Selco® products or other similar products:
follow the instructions provided by the manufacturer;
enrichment should take between 6 to 12 hours;
maintain the oxygen level at or above 4 ppm throughout the
entire procedure;
dry products can be used directly in the production tank,
whereas oily products are only fed in enrichment tanks;
use an antifoam product during enrichment to prevent rotifers
losses by foam aggregation;
during the enrichment process check frequently rotifer
mortality and dissolved oxygen content; the latter should be
kept above 80% saturation, with addition of pure oxygen if
necessary.
The content of nutrients decreases rapidly in rotifers that are not
immediately consumed by fish larvae. In starving rotifers the total
PUFA loss reaches 60% after 6h at 18°C. Even in green water, i.e.
with microalgae, this loss remains important (about 40% after 6 h).
To prevent this degradation in nutritional quality, enriched rotifers
not immediately fed to fish should be stored in containers at low
temperature as follows:
Storage time should not exceed 14 hours;
Temperature should be kept between 5 and 10°C by means of
insulated tanks and blue ice or ice bags.
Rotifer density should not exceed 2 500 to 3 000 in d/ml;
Oxygen level should be kept at or above 4 ppm.

Monitoring rotifer populations
Check all rotifer cultures daily for both quantitative and qualitative
evaluations. From each vessel, flask, bag and tank, take a 1 ml
sample and observe under the stereoscopic microscope.

Measure the following parameters: quantitative parameters:
total number of rotifers per ml
total number of eggs per ml
fertility as percent of total eggs over the total rotifers
qualitative parameters:
average number of eggs per individual (estimate)
repletion (presence of food in the stomach, note 0 for empty, +
for medium full, ++ when full)
motility (++ active, + slow, 0 absent)
filtration (activity of the ciliated corona)
In addition the following qualitative parameters of the culture should
be checked:
presence of foam at the culture surface or sediment on the wall
and bottom of the container
presence of other micro-organisms, such as protozoa, fungi,
bacterial flocks, etc. (identification and frequency, note 0 when
clean, + for medium contamination, ++ large contamination)
All these information must be recorded in the individual file of each
culture. All daily monitoring procedures for the rotifers sector are
given in Annex 10.

3. Production of the brine shrimp Artemia
Artemia is a tiny saltwater crustacean brine shrimp and the nauplii
of which are excellent diet for the aquarium fishes, both for the
adults as well as for their young‟s ones. The artemia cysts which are
generally collected from the salt pans are available in markets in
tins. The Artemia cysts can be hatched in glass jars at room
temperature of 27-300c.
Having a larger size than rotifers, the larval stages of a small
crustacean, the brine shrimp Artemia salina are used as the second
(after rotifers), and last live food fed to fish larvae before their
weaning on artificial feed. Strictly speaking, Artemia is not
cultivated in the hatchery as is the case for algae and rotifers, but
their larval stages are obtained by incubating and hatching their
resting eggs, available as dry storable cysts, which can be purchased
from specialised suppliers. The first Artemia larval forms, the

nauplii, which are also the smallest and richest in yolk, are followed
by a larger metanauplius, whose nutritional value has to be boosted
by feeding them special enrichment diets 12 to 24 hours before being
offered to fish.
Many types of brine shrimp cysts from different locations in the
world are marketed: they differ in naupliar size, important in
gilthead Sea bream first feeding, in nutritional value and in hatching
characteristics. For practical purposes they can be categorized as
follows.
Cysts giving small Instar I-nauplii (with a length of around 430
µm at yolk- sac stage) with high levels of the essential highly
unsaturated fatty acids (n-3 HUFA). An example is the AF
strain of the Belgian producer INVE. Such nauplii allow an
early switch from rotifers to Artemia in larval feeding.
Cysts producing medium-sized Instar I-nauplii (around 480 µm)
with high levels of n-3 HUFA, such as the AF 480 strain of
INVE. These nauplii are useful to switch from the smaller
nauplii to the bigger metanauplii.
Cysts hatching large Instar I-nauplii (around 520 µm) with low
levels of n-3 HUFA, such as the EG strain of INVE. These cysts,
which are the commonest and cheaper, are widely used to
produce metanauplii which represent the bulk of live feeds in
larval fish rearing. Their enrichment is mandatory to increase
their essential fatty acids content to meet the nutritional
requirements of young fish.

Two additional parameters characterize the Artemia batches: the
number of cyst per gram and their hatching rate (the number of
nauplii produced per gram of cysts). The best strains can give about
290 000 - 300 000 nauplii per gram of cyst hatched, with a hatching

rate close to 95%. In a hatchery the use of good quality cysts allows a
synchronization of the production cycle on a 24-h period, with the
harvest of freshly hatched nauplii coinciding with the start of the
incubation of new batches.

Disinfection and decapsulation of brine shrimp cysts
Artemia cyst shells are usually contaminated with bacteria spores of
fungi and other microorganisms. Fish larvae can be infected when
untreated empty shells, unhatched cysts or cyst hatching medium
residues are introduced into the larval rearing tank.
Before incubation cysts should therefore be disinfected. This process
also improves hatching by reducing the bacterial load of the hatching
medium. Disinfection is done by keeping the cysts for a few minutes
in a hypochlorite solution at a maximum density of 50 g/litre. This
product is easily available as commercial grade bleach. The duration
of the treatment varies according to the active chlorine concentration
of the disinfecting solution. Typical duration is:
1 minute in a 10 000 ppm solution,
20 minutes in a 200 ppm solution.
As in commercial bleach the chlorine content may range from 5 to
15%, it is mandatory to check the actual chlorine concentration in the
bleach that is going to be used. This can be done either by titration or
by determination of the refractive index. The titration method is
explained in Annex 7.
The following example shows how to disinfect one kg of cysts in a 200
ppm hypochlorite solution obtained from household bleach with 5%
active chlorine:
1 one kg of cysts needs 20 l of fresh water for the disinfecting
solution;
2 if this solution is going to be used for a 20 minutes bath you will
need 20 l x 200 mg/l = 4 000 mg = 4 g active chlorine;
3 the quantity of 5% bleach required to give 4 g active chlorine is: (1
000/50) x 4 = 80 ml

4 pour 80 ml of 5% bleach in 20 l of fresh water;
5 add one kg of cysts; place an air stone for continuous aeration to
keep cysts in suspension, and keep the cyst in the solution for 20
minutes;
6 harvest cysts on a sieve (125 µm mesh size) and rinse thoroughly
with plenty of tap water;
7 transfer the rinsed cysts to the incubation tank.
A more effective way to obtain completely contaminant-free cysts is
decapsulation, which implies the elimination of the cysts thick
external layer, the chorion, by chemical oxidation. This process,
which requires greater attention, has additional advantages. As they
spend less energy to hatch after the removal of the chorion, the
hatching nauplii have better nutritional value. Moreover, fish do not
risk to suffocate by gulping empty or unhatched cysts offered
together with the nauplii, as it may happen when using disinfected
cysts.
The decapsulation process consists in four steps: hydration,
treatment in a chlorine solution, washing and deactivation of the
residual chlorine. The example described below refers to the
decapsulation procedure of one kg of cysts.
The hydration, a necessary step as the complete removal of the
chorion may only happen when cysts are spherical, proceeds as
follows:
1. Water volume required: around 6 l per kg (maximum amount: 200
g/l); both fresh and sea water can be used; water temperature should
be between 20 and 25°C;
2. Duration: 45 minutes;
3. Aeration: sufficiently strong to keep cysts in constant suspension;
use an open end pipe in a 10 l bucket;
4. Collect the hydrated cysts on a sieve and treat them immediately
with the decapsulation solution.

The decapsulation solution requires a source of hypochlorite, usually
liquid bleach (NaOCl), and an alkaline product to increase pH level of
the decapsulation solution above pH10. Usually technical grade
caustic soda (sodium hydroxide NaOH) is utilized. The first product
is added at 0.5 g active chlorine per gram of cysts, and the second as
0.15 g of sodium hydroxide per gram of cysts. For hydrated cysts the
procedure is as follows (figures refers to one Kg of cysts):
1. Prepare 0.5 g Cl x 1 000 g cysts = 500 g of active chlorine, equal to
3 33 l of a 15% bleach;
2. Prepare 0.15 g NaOH x 1 000 g cysts = 150 g of NaOH, equal to
0.375 l of a 40% NaOH solution;
3. Put the bleach and NaOH in a suitable container (e.g.: a 20 l
plastic bucket) and fill with seawater to 14 litres (14 - 3.33 - 0.375 =
about 10.3 l of seawater)
4. Provide a strong aeration and eventually if available add antifoam;
5. Place the hydrated cysts in the bucket;
6. Control the temperature: it should remain within 25°-30°C. In case
of higher temperatures, add ice to prevent that it reaches 40°C which
are lethal for the cysts;
7. Verify cyst colour changes. The change in cyst colour confirms that
decapsulation is in progress: the cyst colour shifts from dark brown to
grey and finally to orange, which is the colour of the nauplius body
seen by transparency through its outer cuticular membrane, left
exposed by the dissolution of the chorion. The process usually lasts 5
to 15 minutes.
8. Using a pipette or a graduated cylinder, check floatability: non
decapsulated cysts will float and decapsulated cysts will sink; as soon
as all cysts have turned orange, stop the process by harvesting them
on a sieve and rinse thoroughly with plenty of tap water and rinse
well until no more chlorine smell is noticed;
9. The residual hypochlorite adsorbed by the decapsulated cysts has
to be neutralised by dipping them in a 0.1% solution of sodium
thiosulfate (Na2S2O3. 5H2O) for 5 minutes;

10. Then, after a final rinsing, they are transferred to the incubation
tank.
Warnings
Decapsulation is an exothermic reaction: during the process the
temperature will raise by approximately 12°C, from 20 to about 32°C.
The chemicals used for this process are toxic to humans and must be
handled properly. Wear gloves and protective eyeglasses.
Check if the local legislation requires any special permission for (or
bans) the use of any of these products.
Always check the chlorine content of the bleach used, to optimize the
entire procedure and give constant results. See Annex 7 for detailed
description of procedure and reagents

Besides incubation, the decapsulated cysts can be either stored or,
unless not commonly, be fed directly to fish. For short-term storage,
up to one week, keep in the refrigerator at a temperature of 0 - 4°C.
Decapsulated cysts that have to be stored for a longer period have to
be dehydrated in a saturated brine solution as follows:
Per each kg of decapsulated cysts prepare a brine solution by
dissolving 1.7 kg kitchen salt (fine crystals) in fresh water to
reach a final volume of 6 l (solubility after 1 h is ± 270 ppt),
Aerate with an open pipe for 12 h,
Place the decapsulated cysts in the brine solution overnight or
for minimum 3 hours at room temperature,

Drain cysts on a 125 µm sieve, transfer them to a plastic
container topped with fresh brine and store in a refrigerator or
a freezer.
As the water content of cysts should be about 16-20% after this
process, maximum hatchability is guaranteed for a storage period of
only a few months.
In an emergency, they can be directly offered to fish larvae, provided
that water circulation and aeration can keep them in suspension for
a sufficient time to be ingested by fish. As the decapsulated cysts are
50% smaller than newly hatched nauplii, they may be used as initial
food for small fish larvae.

Incubation
As soon as the dry cysts are placed in water to be hydrated, the
embryos inside the cyst start their metabolic activity. The free
swimming nauplius, called instar I, hatches after about 20 to 24
hours if incubated under optimal conditions as follows:
- Tank design: round with conical bottom, white gel-coated inside
with a semi-transparent window near the cone tip for harvesting; a
drain with a valve is installed in the cone tip;
- Filtered and sterilized seawater, 35 ppt salinity;
- Water temperature 28-30°C;
- strong aeration to provide a vigorous water agitation, to keep cysts
in suspension: air is provided through the open end of a 1/2 inch PVC
pipe placed close to the tank bottom;

- dissolved oxygen level above 4 ppm;
- pH over 8; if needed, add sodium bicarbonate (NaHCO3) at the rate
of about one gram (previously dissolved) per litre;
- a strong illumination of 2 000 flux at the water surface during the
first incubation hours; light can be provided by two neon m3 (photo
STM Aqua trade) tubes (2 x 58 W) placed just above the tank rim;
-cyst density for incubation: 2.5 g per litre.

Harvesting of nauplii
Artemia should be harvested when at the energy-rich instar I larval
stage, just after hatching. This occurs in about 22 h at 28°C. To
assess the proper time, sample the incubation medium with a 5-ml
glass pipette and check for nauplii and umbrella stages (embryos still
attached to their cyst) under the stereomicroscope.
To harvest nauplii, proceed in the following way:
1. Purge the bottom tip of unhatched cysts that has sunk by opening
the drain valve for a few seconds;
2. Fit a flexible hose at the drain valve and place its open end into a
container full of seawater equipped with a central filter.
3. Stop aeration, switch off the light above the tank, cover its top
with a light-proof lid and place a light source at the window on the
tank bottom: empty cyst shells will float; unhatched cysts will sink,
whereas freshly hatched nauplii will concentrate at the tip of the
conical bottom, attracted by light because of their positive
phototactism. To attract the nauplii use a 150 W bulb lamp placed in
a waterproof holder;
4. Wait 10 minutes, the time the nauplii will separate from their
empty shells and start to harvest the tank, either fully or partially,
by draining it into the filter. After another 10 minutes, a second
harvest may be carried out if all nauplii were not harvested earlier.
Do not drain the tank completely to keep floating empty shells in it.
Adjust the water flow to avoid clogging the screen/water overflowing.
Suggested maximum water flow is 100 l/min. If the water level in the

filter stands higher than the water level in the tank, it would also be
possible to harvest nauplii using a small submersible pump. When
filtering, check with a glass beaker for possible losses of nauplii that
may escape the filter.
5. During the settling time monitor the oxygen level, which should
not drop below 2 ppm, as well as excessive crowding nauplii above
the tip of the conical bottom. If necessary prior to harvest inject pure
oxygen into the culture tank for 10 to 15 minutes to raise the
dissolved oxygen content up to 10 ppm.
6. Rinse the nauplii thoroughly (for about 15 minutes), preferably
with fresh water, to wash out the hatching debris.
7. Put the rinsed nauplii in a temporary container filled with a
known volume of filtered and sterilised seawater and count them (see
the procedure below).
8 .Transfer nauplii to the storage tank filled with sterilised sea water
and adjust the water volume to give a maximum density of 4 million
nauplii per litre; provide a moderate aeration from the cone tip and
add blue ice (or sealed ice bags ice) to keep water temperature at 5 to
10°C or better use a cold storage refrigerated tank or a freezer when
available. At higher temperatures their nutritional value decreases
rapidly.
9. If the nauplii have to be enriched, transfer them to a previously
prepared enrichment tank.

Counting and evaluating Artemia nauplii
To assess the hatching results and to feed the larval rearing tanks at
the established densities you have to count the Artemia nauplii.
Three methods are described below, one for high nauplii densities,
such as after harvesting and in a cold storage tank, one more
common for counting the nauplii when they are in the incubation
tank and the other for low nauplii densities, similar to those which
can be found in fish tanks.
Counting high density nauplii samples:
take a 10-ml sample of the population to be estimated;
dilute the nauplii concentration by adding 90 ml of sea water to
obtain a total sample volume of 100 ml;
take three sub-samples with a 1-ml pipette, avoiding to suck air
bubbles;
transfer each sub-sample to a Petri dish;
add a few drops of the fixative staining solution Lugol (see
Annex 7) to each of the Petri dishes and wait until all nauplii
are immobile and deeply stained;
add water so as to distribute the nauplii over the whole surface
of the three Petri dishes;
put each Petri dish on a grid and count the nauplii present in
each 1 ml sub-sample;
calculate the average number of nauplii per ml by dividing the
sum of the three counts by three;
The total nauplii density per litre is given by the average
number of nauplii per ml multiplied by 10 to give the number
per undiluted ml and finally by 1 000 to get the final density
per litre.
Counting nauplii samples from the incubation tank:
take with a test tube a 50-ml sample of the population to be
estimated;
take three sub-samples with an automatic 0.1-ml pipette;
transfer each sub-sample to a 3 cm-wide Petri dish, whose
bottom has been subdivided in a 5 mm-grid;

add a few drops of the fixative staining solution Lugol (see
Annex 7) to each of the Petri dishes and wait until all nauplii
are immobile and deeply stained;
add water so as to evenly distribute the nauplii over the whole
surface of the three Petri dishes;
count the nauplii present in each 0.1 ml sample;
calculate the average number of nauplii per ml by dividing the
sum of the three counts by three;
The total nauplii density per litre is given by the average
number of nauplii per ml multiplied by 10 to give the number
per ml and finally by 1 000 to get the final density per litre.

Counting low densities nauplii samples:
Take three 50 or 100 ml samples, using a graduated cylinder
which has been cut at the 50 or 100 ml mark;
Transfer each sample to a large Petri dish;
Add several drops of Lugol (see box below) to each of them and
wait until all nauplii are immobile and deeply stained;
Put each Petri dish on a grid and count the nauplii present in
each sample;
Calculate the average number of nauplii present in each
sample;
Calculate the nauplii density per litre by multiplying this count
either by 20 (50 ml sample) or by 10 (100 ml sample).
The two main criteria to evaluate hatching results are:
Hatching rate: the number of nauplii hatched per 100 cysts;
good batches have a hatching rate around 90-95%;
Hatching efficiency: the number of nauplii produced per gram of
cysts; top quality cysts yield about 300 000 nauplii/g.

Enrichment and storage
Older larval stages of brine shrimp, the metanauplii, are used
typically as feed for growing fish post-larvae. However, their poor
nutritional value has to be boosted by dedicated enrichment diets
that are rich in essential n-3 HUFA. Such diets can only be given
when Artemia feeds actively, which is during its larval stages of

instar II and instar III. First feeding in brine shrimps actually
coincides with their moult into the second Instar stage.
The best results are obtained when the hatching time and the
moulting pace are exactly known. The beginning of the enrichment
can easily be determined by observation. After the first 18 hours
small samples are taken every hour and checked under the
microscope. The appearance of second instar stage is easily detected
since it is larger than the first instar and presents a gastrointestinal
tract. Enrichment will begin as soon as the first instar II stages
appear.
The duration of the enrichment process as well as the type of product
to be used depend on the HUFA content that is desired. Full
enrichment takes 24 hours and two doses of enrichment emulsion, at
time zero and 12 h later. Short-term enrichment takes 12 h and only
the initial dose.
Optimal enrichment conditions
For water quality and container design, see previous section;
initial nauplii density: between 150,000 and 300,000 nauplii/l;
Vigorous water agitation to keep the nauplii in suspension and
pure oxygen to keep dissolved oxygen above 4 ppm throughout
the enrichment period: use one open PVC pipe for air and a
micro-bubbles diffuser for oxygen;
Lighting: not required.
Prepare the enrichment meal as specified by the producer, and make
sure to prepare a new enrichment emulsion for each meal. At the end
of the enrichment time harvest the metanauplii as usual, rinsing
them thoroughly with seawater until no oily emulsion is noticed in
the out flowing water.

Cold storage of enriched Artemia nauplii
Enriched metanauplii rapidly lose their nutritional value at room
temperature, as it happens to rotifers, unless they are stored in cold
seawater (4 to 10°C). Keep density below 4 million per litre.
Equipment and procedure are the same described for cold storage of
nauplii.

Other Live feed for Fish:
Infusoria:
The infusorians are a tiny single celled protozoan which exits in
almost all water bodies. They form an excellent starter food for the
newly hatched spawn after the yolk sac absorption is complete.
Among the many species of infusoria, paramecium and stylonychia
are the commonly cultured species for feeding the larvae. They are
mostly seen in ditches contain rotten kitchen waste having foul
smell.

Tubificid worm:
Tubifex are small, reddish worm up to 2cm long, and they occur in
large number in flowing sewage drains. When disturbed, they enter
into the mud.

CONCLUSION
The pivotal part of the fish seed production is production of live feeds
in which the nutritive quality of live feeds are further enhanced
through bio encapsulation with EPA, DHA and micro algae. High
survivability and healthy larval production are the baseline factors
for the seed production of ornamental fishes.

Reference
1. Ornamental Fish Breeding, farming and Trade
2. CIFE Manuel
3. www.fao.org

Livefeed culture, Priyanka Chatterjee

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Livefeed culture, Priyanka Chatterjee