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GROWTH PERFORMANCE OF ROTIFER (Brachionus rotundiformis)

FED WITH GREEN ALGAE (Nannochloropsis oculata)
AT DIFFERENT SALINITIES








EDEMIE ANTONIO NUEVA








A THESIS PROPOSAL SUBMITTED TO THE FACULTY OF THE
FISHERIES DEPARTMENT
COLLEGE OF AQUATIC SCIENCES AND APPLIED TECHNOLOGY
MARIANO MARCOS STATE UNIVERSITY
CURRIMAO, ILOCOS NORTE








IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DECREE BACHELOR OF SCIENCES IN FISHERIES








JULY 2014


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GROWTH PERFORMANCE OF ROTIFER (Brachionus rotundiformis) FED
WITH GREEN ALGAE (Nannochloropsis oculata) AT DIFFERENT
SALINITIES


INTRODUCTION


Rotifers of the genus Brachionus constitute an important group of organisms
in aquatic ecosystems but also play a notable role in technological research for
exploiting aquatic living resources (Yfera 2001). They are also used worldwide,
alone or in conjunction with other types of food, to rear early developmental stages of
marine finfish and crustaceans (Suchar et al. 2006). Brachionus spp. are ideal as a
first exogenous food source for aquaculture species due to their small size, slow
swimming speed, ability to stay suspended in the water column and ease of culture
(Fielder et al. 2000).
Food and salinity are among the most important factors that determine
community typology in aquatic ecosystems (Miracle et al. 1989).They also influence
the life history characteristics and population dynamics of rotifers (Bosque et al.
2001). Food has an important role in maintaining rotifer culture stability (De Arauju
et al. 2001).Several food items including various algal species have been used to
produce rotifer mass cultures and to improve their nutritional quality (Srivastava et al.
2006).
The use of rotifers as live feed has demonstrated to improve aquaculture
yields, in particular Brachionus spp. Herein, N. oculatais one of the most important
feed used to produce Brachionus spp. (Spolaore et al. 2006a); however, scarce


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information was found regarding the effect of culture media on microalgae response
as well as the effect of such microalgae on the rotifer productive performance.
It is important to take a daily assessment of rotifer mass cultures, as many
external (environmental) factors can stress the rotifer population and contribute to
sudden culture crashes. Accumulation of dissolved organic substances in the culture
water increases the turbidity of the water and eventually leads to a decline in rotifer
numbers. Even under optimal temperature, salinity and diet conditions, a culture may
collapse due to the increase of unionized ammonia, and the presence of certain
bacteria (Yu et al., 1990) and protozoans (Maeda and Hinto, 1991).

Objectives of the Study
This study will be conducted to determine the growth of rotifer B.
rotundiformis fed with N. oculata at different salinities cultured under laboratory
conditions. Specifically, it aims to:
1. To determine theeffect of salinity on the growth of rotifer B.rotundiformis
in terms of density
2. To determine the effect of the amount of green algae N. oculata given as
food for rotifer; and
3. To correlate relationships between salinity and amount of food given to
the growth of B. rotundiformis.



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Scope and Limitations of the Study
This study will only determine the growth in terms of cell density (cell/ml) of
B. rotundiformisfed with N. oculata at different salinities cultured under laboratory
conditions. The study will be done in the month of February 2015. Actual culture of
rotifer will conducted for 4 days.






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REVIEW OF LITERATURE

Rotifers are valuable live food for the culture of the larvae of most fish
species. Several characteristics of rotifers including their very small size, relatively
slow motility have contributed to their usefulness as good prey for active larvae
(Lubzens et al. 1989).Rotifers are a common first live feed used in Marine
Aquaculture Centre (MAC) and globally in larviculture. Common in tropical waters,
the rotifer species B. rotundiformis also displays within it, a smaller strain,
comprising the ultra-small rotifers (with 90-150m length), based on assays of male
mate recognition (Hagiwara et al. 1995).
Rotifers can be nutritionally improved when fed with good quality
microalgae, another live feed that is extremely good in boosting the fatty acid content
in rotifers. In MAC, rotifers are fed a combined microalgae-and-yeast diet. It had
been shown that microalgae are rich in essential fatty acids like eicosapentanoic acid
(EPA 20:5n-3) and docosahexanoic acid (DHA 22:6n-3). For example, the
microalgae Nannochloropsisoculata is rich in 20:5n-3 (Kovan et al., 1990).

Growth Dynamics of Rotifer
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


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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:
Lag-phase (just after inoculation): 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;
Log-phase (exponential growth): rapid growth stage, showing a significant
increase in numbers of rotifers and good fertility rate;
Transitional phase (declining growth): where growth rate slows and number
of egg bearers decreases;
Decline phase (negative growth):where almost only old rotifers without eggs
are found and their number decreases rapidly as death rate exceeds growth rate.

Microbial conditions
Bacteria are always associated with mass production of rotifers and may cause
unexpected mortality or suppressed growth to rotifers. In some other cases, no harm
is caused to the rotifers but infected rotifers cause a detrimental effect on fish larvae,
resulting in poor survival and growth. Although most bacteria are not pathogenic for
rotifers, their proliferation must be avoided since a real risk of accumulation and
transfer via the food chain can cause detrimental effects on the predator (Dhert 1996).


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Rotifers as Vector of bacteria:It is assumed that rotifers, the first food
administered to fish larvae, are the major carriers of bacteria (Muroga et al.1987).
Although in most hatcheries, special efforts are made to keep the rotifer cultures as
clean as possible (Minkoff 1994) the billions of rotifers and their accompanying food
inevitably create a high load of organic material which is rapidly colonised by
bacteria.
Effect of associated microbiota on rotifer cultures: Although little is known
about the importance of the microbial biomass in live feed production systems, the
microbiota associated with it seems to play a major role in the instability and
variability of the live feed cultures themselves (Hirayama 1987) and of the cultures of
the marine predator-larvae (Muroga et al. 1987).

Culture Method
Batch method:A method of harvesting all rotifers being cultured before
population of the rotifer reaches the stationary growth phase. Actively growing
rotifers are continuously being used to maintain stocks. Initial density of the rotifer
cultures is 50 individual/ml (BFAR-NIFTDC). Stocks are harvested after 3-4 days
when densities reach 100 individual/ml or more.
Semi-continuous cultures:The semi-continuous culture is also known as
thinning culture since the rotifer density is kept constant by periodic harvesting
(Coves et al. 1990;Girin 1974). Contrary to the batch culture, this long-term culture is
maintained at low densities for a period of 714 days without water renewal (Lubzens


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1987). The size of the culture tank is usually larger than that used in the batch
cultures.
Continuous culture:Increasingly, recirculating aquaculture technology has
come into favor with aquaculturists wishing to maximize rotifer production per unit
volume while decreasing labor inputs. By incorporating biological filters and foam
fractionators for fine solids control, it is possible to maintain higher levels of water
quality, thus increasing the stability of a culture.

Water Quality Parameters Required for B. rotundiformis Culture
Temperature: The optimal temperature for rotifer culture is generally between
20 and 28C, although this may change with the composition of the culture medium,
the species and strain cultured. Most commonly cultured species of rotifer tolerate
temperature between 25 and 27C (BFAR-NIFTDC, 2014).
Rapid changes in temperature affected the availability of rotifers in the water
column and both species recovered slowly over time. The B. rotundiformis were more
tolerant of transfer to increased temperature which follows their preference for higher
production temperatures (Komis 1992).
Nutrients:Algae, bakers yeast, ground shrimp meal, flour, rice bran, frozen
algae, formulated diets are some of the food sources that have been exploited for the
cultivation of rotifers (Arimoroet al. 2004).
pH:Water quality parameters such as pH may have influenced availability of
rotifers; however, it is most likely that the changes in availability were caused by the


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different salinities. Epp and Winston (1978) found that changes in pH from 6.5 to 8.5
had no effect on rotifer activity or metabolism.
The pH range for most culture algal species is from 7-9 with the optimum
range being 8.2-8.7. Complete culture collapse due to due to the disruption of many
cellular processes can result from a failure to maintain an acceptable pH (
Aeration: Aeration (which determines the DO) for rotifers is a critical factor
(in yeast diets, but especially if given emulsified compound feeds). Therefore the DO
must be monitored to ensure adequate DO level (at least 3.0ppm), and sufficient
turbulence to allow feed and rotifers to be kept in suspension (but not excessive
turbulence, which would stress the rotifers and re-suspend any solid waste).
Salinity:The effect of salinity on the availability of rotifers in the water
column was greater than the effect of temperature. Generally, availability of rotifers
decreased as salinity was reduced. The B. plicatilis were slightly more tolerant at
lower salinity than the B. rotundiformis and availability was not affected by rapid
transfer to salinities that were 10 lower than rotifer cultures.
Optimal reproduction at the usual culture temperature of 25C occurs at any
salinity within the range 4-35 (Komis 1992); however, rotifers are generally
cultured at salinities between 10 and 20 (Fulks et al., 1991). Availability of B.
rotundiformis, however, decreased when they were rapidly transferred to salinities
5 lower than the rotifer culture salinity. The availability of rotifers increased over
time indicating that the rotifers had begun to acclimate to the conditions.


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Oie and Olsen (1993) found that sudden transfer of rotifers from ambient
salinity of 20 to 5 resulted in their immediate immobilization.The effect of
salinity was also found to be more pronounced than temperature. Recovery from
immobilization was slower, but about 80% of the rotifers had regained mobility after
30 min. The effect of immobilization was even more pronounced when the
temperature was increased to 28C in conjunction with a reduction in salinity.

Harvesting of Rotifers
When rotifers reach their peak in the plastic vessels, ponds or small-scale
culture; it is advisable to harvest them to avoid sudden crash. A hand net of mesh size
(50 m) is recommended for this experiment. For small scale cultures, the entire
culture volume is filtered through the net and the rotifers collected in the plankton net
bucket, is emptied into a plastic or any suitable container for onward transfer to the
fry holding tanks(Ludwig et al. 2000).

Biology of N. oculata
Microalgae belong to highly diverse group of photoautotropic organisms,
which are important for aquatic animal feeding. They play important roles of primary
producers in mariculture as food for consumers such as rotifers, copepod, daphnia,
and brine shrimp etc., which are feed to late larval and juvenile fishes.
N. oculata can be found in the marine environment but also occur in fresh and
brackish water. They are known to be abundant in waters with high-nutrient loading


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such as coastal waters and estuaries. All of the species are small, non-motile spheres
which do not express any distinct morphological features.N. oculata is a 2-4 micron
(m) green flagellate. This fast growing species is easy to maintain. This
phytoplankton is the most commonly thought of when the term green-water is used.
This is a dark green alga with a thick tough cell wall that interestingly is readily
consumed by rotifers.
Marine microalgae are widely recognized as the best feed for growing and
enriching rotifers. Microalgae are what rotifers naturally feed on in the wild, and
provide the complete chemical composition that larval fish need for proper neural
development. Microalgae are also the easiest feed to work with. Yeast and emulsion
products rapidly foul a rotifer cultures, creating high levels of bacteria and ammonia,
and causing the rotifers to stick together. Microalgae such Nannochloropsis have a
cell wall that resists bacterial breakdown so there is no fouling, excessive bacterial
proliferation, or stickiness.(www.reedmariculture.com/support_rotifers_feeding.php)

Culture Method of N. Oculata







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METHODOLOGY

Locale of the study
The study will conduct indoors at Mariano Marcos State University-College
of Aquatic Sciences and Applied Technology (MMSU-CASAT) at the Algal Room
from November 2014 to January 2015.

Source of the experimental organism
Rotifer (Brachionus rotundiformis) and the green algae (Nannochloropsis
oculata) will be obtained from the Bureau of Fisheries and Aquatic Resources-
National Integrated Fisheries Training Center (BFAR-NIFTDC).

Experimental Set Up and design
The experiment will follow a Completely Randomized Design (CRD)
consisting of five treatments, each treatments will replicated 3 times. The treatments
and replicates will randomly distributed in a total of fifteen containers with a capacity
of six liters(Table 1).




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Table 1.Treatment I to be used in the study

Salinity Amount of foods
(Green Algae)
Density of Rotifer Replicates
10 2x 10
6
cells/ml 50 ind/ml 3
10 3x 10
6
cells/ml 50 ind/ml 3
10 4x 10
6
cells/ml 50 ind/ml 3
10 5x 10
6
cells/ml 50 ind/ml 3

Table 2.Treatment II to be used in the study

Salinity Amount of foods
(Green Algae)
Density of Rotifer Replicates
20 2x 10
6
cells/ml 50 ind/ml 3
20 3x 10
6
cells/ml 50 ind/ml 3
20 4x 10
6
cells/ml 50 ind/ml 3
20 5x 10
6
cells/ml 50 ind/ml 3


Table 3.Treatmen III to be used in the study
Salinity Amount of foods
(Green Algae)
Density of Rotifer Replicates
30 2x 10
6
cells/ml 50 ind/ml 3


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30 3x 10
6
cells/ml 50 ind/ml 3
30 4x 10
6
cells/ml 50 ind/ml 3
30 5x 10
6
cells/ml 50 ind/ml 3
Table 4. Treatment IV to be used in the study
Salinity Amount of foods
(Green Algae)
Density of Rotifer Replicates
40 2x 10
6
cells/ml 50 ind/ml 3
40 3x 10
6
cells/ml 50 ind/ml 3
40 4x 10
6
cells/ml 50 ind/ml 3
40 5x 10
6
cells/ml 50 ind/ml 3
Source: BFAR-NIFTDC

Feeding Techniques
Each container will be fed three times a day. At an intervals ( 9am, 12am,
3pm). Rotiferswere fed different amount of food of N. oculata at different salinities in
all treatments.

Counting and Monitoring of Rotifer
B. rotundiformis cell densities will be measured daily by counting with the use
of Sedgwick-Rafter slide viewed under the high power objective of a compound
microscope. A tally counter will be used to count the cell number. Water sample from
each replicate bottle is taken daily and cell density counting is made three times per
replicate. One way to count rotifers is as follows:


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1. Mix the culture medium using stirring rod to avoid sedimentation of the
rotifer.
2. Collect a 1 ml sub-sample with a pipette and load a Sedgwick-Rafter slide.
3. Then add one to two drops of formalin or Lugols solution to immobilize
the rotifers.
4. Place the Sedgwick-Rafter slide onto a microscope under 40x
magnifications.
5. Count the total number of rotifers and the number of rotifers with eggs.
Samples may need to be diluted prior to loading the Sedgwick-Rafter slide if
rotifer densities approach 1000/ml.

Culture of N. oculata
Marine microalgae are the best diet for rotifers and very high yields can be
obtained if sufficient algae are available and an appropriate management is followed.
However, the culture of microalgae as a sole diet for rotifer feeding is costly due to
the labour intensive character of microalgae production. In most places, however,
pure algae are only given for starting up rotifer cultures or to enrich rotifers. Usually,
large amounts of cultured microalgae, such as the marine alga Nannochloropsis, are
usually inoculated in the cultivation tanks together with a starter population
containing 50 to 150 rotifers.ml-1.




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The growth of rotiferB. rotundiformisand egg ratio is computed using the formula:
Specific Growth Rate:
= (ln Nt ln No) /t
Where: = specific growth rate (SGR)
N0 = initial density
Nt = final density at day-t of culture period
t = culture period (day)
Egg Ratio:
RT = Nt / N x 100%
Where: RT = Egg ratio (%)
N = B. rotundiformis total number
Nt= Amount of eggs

Water Quality Parameters
Water quality parameters such as pH, salinity, and temperature of the water in
the experimental containerwill be monitored and recorded daily using pH paper,
refractometer and thermometer.

Statistical Analysis


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One-way analysis of variance (ANOVA) will be used to check for the
differences of the salinities of the treatments. Mean differences will be measured at
5% level of significance. A post-test using the Duncans Multiple Range Test will
also be used to further test significant findings.




















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