Name/s: Araneta, Allen Patrick Class and Section: Fish 125 - Sec 2

Daquioag, Ariadne Marie Date Submitted: February 15, 2020

Dimaano, Antoniette Lorain
Hilaus, Edrose Janne
Serag, Divine Angela
Date Performed: January 18, 2020

Assessment on Growth Dynamics of Artemia sp. and Brachionus rotundiformis

Abstract
Growth of aquaculture species in their larval stage is ensured by nutrients coming
from natural food such as zooplankton. This experiment assessed the growth rates of Artemia
sp. (brine shrimp) and Brachionus rotundiformis (rotifer). Furthermore, it aimed to know the
morphology, growth and hatching rate, and ecology of brine shrimp and rotifer; and
understand their culture. The rotifer was cultured from a starter population and was grown
with different feed types. Their population density was determined every after 24 hours for
five days to determine its growth curve. The artemia, on the other hand, was disinfected and
hatched. After 24 hours, the hatching rate was determined. It was found out that rotifer has
grown best when fed with Tetraselmis sp. Moreover, the rotifer population growth peaked on
day 4 and plummeted the next day. This is explained by the life cycle of rotifer which only
lasts for 3.4-4.4 days. They are opportunistic zooplankton that can undergo parthenogenesis
when the need arises. The hatching rate of artemia, on the other hand, was 31.24%. The
hatching rate of artemia cysts was greatly affected by several factors. Higher hatching rate
can be achieved with good quality cysts, careful handling, and proper storage.

Introduction
Natural food sources play a vital role in larval stages in culturing fish species. These
are zooplankton, which are often herbivores and grazers that usually feed on phytoplankton.
They are able to swim in water columns and are constantly available to fish and shellfish
larvae are likely to stimulate larval feeding response (Das et al. 2012). In an aquaculture
system, these live fish food organisms contribute the most to the cultured fish species’
nutrition.
The success in the hatchery production of fish fingerlings for stocking in the grow-out
production system is largely dependent on the availability of suitable live food for feeding
fish larvae, fry and fingerlings. The most common live fish food organism used in
aquaculture are rotifers. Rotifers are commonly known as wheel animalcules. Its rate of
reproduction larval cultures depend on food quality and quantity, salinity, temperature and
pH of the medium since they rely on mass cultures. Rotifers are ideal for fish feed in
aquaculture for they are generally considered to have low sensitivity to pesticides and have
frequently shown increased abundance in temperate model ecosystems. ( Dahms et al. 2010)
Another fish food organism used in aquaculture is the nauplii of brine shrimp Artemia
sp.. Artemia forms dormant embryos, known as cysts, which may account for a convenient,
suitable and excellent larval food source. It is also linked to the evolution of aquaculture
practices and as years go by, was later produced in cans and bottles for easier access. These
cysts are available in large amounts all year round which makes them high on demand
especially in the aquaculture ventures. This type of fish feed organism is said to be an
excellent food for shrimp larvae and newly hatched fish cultures for its high nutritional
content. (Lavens & Sorgeloos 1996)

Objectives
Specifically, the study aimed to compare the growth rate of rotifers fed with four
different phytoplankton diets namely; Tetraselmis, Isochrysis, Nannochloropsis, and a mix of
Tetraselmis and Isochrysis. Additionally, it wanted to determine the hatching percentage of
Artemia and the factors that might have affected it. Furthermore, it also wanted to identify the
morphology, growth rate, reproduction, nutritional value, and hatching process (for Artemia
only) on both zooplanktons to compare their significance on aquaculture.

Methods
A. Rotifer Culture
The carboy and plastic tubings were disinfected using 10 ppm chlorine soaked for 24
hours and were then neutralized using freshwater that was filtered under UV treatment.
Rotifer starter, about 10-20 individuals/ml, was inoculated to dense Tetraselmis sp. The algae
was prepared by the earlier batch of class 4 days ahead of the rotifer being stocked. The algal
density and the rotifer count of inoculum was determined while the volume of starter,
dependent on the rotifer starter population, and volume on the culture tank was counted and
calculated.
Rotifer was counted by placing samples into vial from carboy using pipet. Few drops
of Lugol’s iodine were placed into the vial and the rotifer was immobilized. The sample was
placed properly in the Sedgewich-Rafter Counting Chamber. The rotifer was counted starting
from left to right area of the chamber using the counter for definite count.
B. Artemia Culture
Artemia hatching tank was disinfected using 20 ppm chlorine and soaked for 24-48
hours. The tank was cleansed with a mixture of chlorine and detergent then was washed
thoroughly until the remaining chlorine was eliminated. Airstones and plastic tubings were
disinfected using 10 ppm chlorine and were then neutralized with freshwater filtered using
UV treatment.
2 g of Artemia cysts were hydrated by being placed in 10 L freshwater for 1 hour
aided with aeration at 28 °C. A 200 ppm hypochlorite solution was prepared joining 28 ml of
liquid bleach in 1 liter of freshwater. The cysts were soaked for 30 minutes in the solution at
a density of 50 g cysts/liter. These were efficiently washed on a 125 μm screen using
freshwater in which the cysts were ready for the hatching incubation stage.
The disinfected transparent cylindroconical-shaped tank was used. The valve at the tip
of the tank was managed to aid in the harvest. Aeration supplied air down to the end of the
funnel-shaped portion of the tank in which strong aeration was provided. Seawater that was
disinfected and filtered was placed within the tank. A low amount of illumination of about
2999 lux was applied at the surface area of water for 24 hours. The cysts were incubated at a
density of 1 g/liter for 24-48 hours. Amount if cysts that was required was dependent on
hatching efficiency of the cyst batch and the amount of nauplii. The Artemia was harvested
by switching off aeration. Once the cyst shells floated, it was extracted from the surface while
the remaining unhatched cysts and nauplii sunken and was concentrated at the bottom of the
tank. These concentrations of unhatched cysts were siphoned for the removal of such
accumulation. Nauplii were collected with the use of fine mesh screen, less than 150 μm, and
was washed thoroughly for the removal of potential contamination. Nauplii was cleansed and
disinfected then after was stored at 4 °C. After 24 hours of incubation, a sample pipetted and
was placed into vial. Lugol’s iodine solution was dropped for immobilization. The sample
was placed in Sedgewich-Rafter Counting Chamber and its cells were counted.

Results
A. Rotifer Culture
Figure 1 shows the growth curve of rotifer on Tetraselmis sp. diet as determined by its
cell density for five consecutive days. The initial cell count after its culture preparation was
13.3 cells/ml, 53 cells/ml after two days, 118 cells/ml after three days with addition of 2 L
live feed, 168 cells/ml after four days added with 2 L feed, and 102 cells/ml after five days
with addition of 4 L feed. There was an increase in the cell count from the first day until the
fourth; however, a decrease in count was observed on the fifth day.

Figure 1. Growth rate of the Rotifer population on Tetraselmis sp. diet is determined by its
cell density for five days.
Figure 2 shows the growth curve of rotifer on Isochrysis sp. diet as determined by its
cell density for six consecutive days. The initial cell count after its culture preparation was 14
cells/ml, 44 cells/ml after the first day, 61.8 cells/ml after two days, 73.5 cells/ml after three
days with additional 1 L live feed, 60.5 cells/ml after four days with additional 1 L feed, and
62 cells/ml after 5 days with addition of 3 L feed. There was an increase in the cell count
from the first day until the third; however, a decrease in count was observed on the fourth day
and a slow increase after the fifth day.

Figure 2. Growth rate of the Rotifer population on Isochrysis sp. diet is determined by its cell
density for 6 days.
Figure 3 shows the growth curve of rotifer on Nannochloropsis sp. diet as determined
by its cell density for six consecutive days. The initial cell count after its culture preparation
was 14 cells/ml, 39.3 cells/ml after one day, 66.3 cells/ml after two days, 47.5 cells/ml after
three days with addition of 2 L live feed, 74.5 cells/ml after four days with addition of 1 L
feed, and 96 cells/ml after five days with addition of 3 L live feed. There was an increase in
the cell count from the first day until the second; however, a decrease in count was observed
on the third day and an increase after the fourth day and until the fifth.

Figure 3. Growth rate of the Rotifer population on Nannochloropsis sp. diet is determined by
its cell density for 6 days.
 Figure 4 shows the growth curve of rotifer on combination of Isochrysis sp. and
Tetraselmis sp. diet as determined by its cell density for six consecutive days. The initial cell
count after its culture preparation was 12.7 cells/ml, 35.8 cells/ml after one day, 66.5 cells/ml
after two days, 79.5 cells/ml after three days with addition of 2 L live feed, 104 cells/ml after
four days with addition of 1 L feed, and 97.8 cells/ml after five days with addition of 2 L
feed. There was an increase in the cell count from the first day until the fourth; however, a
decrease in count was observed on the fifth day.

Figure 4. Growth rate of the Rotifer population on combination of Isochrysis sp. and
Tetraselmis sp. diet is determined by its cell density for 6 days.
B. Artemia Culture

Graph 1: Cell count of the growth of Artemia cyst and nauplii during incubation period.
 Graph 1 shows the cell count of the growth of Artemia cyst and nauplii during
incubation period. The total count of the Artemia cyst is 885,500 cells while the amount of
Artemia that underwent its next larval stage as nauplii is 276,666.67 cells. Both larval stages
of Artemia can be summed with the total of 1162166.67 cells. The hatching rate percent of
the Artemia cyst is computed to be 31.24%.

Discussion
A. Rotifer Culture
Rotifers are mostly freshwater metazoans which do not grow greater than 2 mm in
size. They are labelled as opportunistic organisms, with an abundance of females, since they
often reproduce by parthenogenesis. They are able to reproduce up to ten generations before
they die. Generally, they live up to 3.4-4.4 days, which agrees with the results shown above.
Females have the complete set of body parts namely: the head, trunk, and the foot. The head
contains the locomotory organ called the corona which mobilizes them through annular
ciliation. Thus, the name Rotatoria which means “bearing wheels”. In addition, this circular
motion also helps them feed on small food particles. This food is effectively digested with the
mastax which is found on its trunk. Both the corona and mastax determine the preferred food
of rotifers. The foot is their appendage and is important in taxonomic classification. Males on
the other hand, are less developed than females in terms of size, growth rate, and body
structure. Since they develop 2-3 hours longer than females, they grow only a quarter in size
of females with no digestive tract and bladder, only a relatively big testis full of sperm.
Rotifers only reproduce sexually under favorable conditions (Lavens & Sorgeloos 1996;
Kakkassery 2003; Yin et. al. 2015).
The biology of rotifers heavily influences their suitability as food for larvae. Since
these larvae are very small after hatch; they also require small prey as food. Given this, it is
important to provide the larvae with small live food, yet can easily be mass cultivated in large
quantities. Both are notable characteristics found in rotifers. These organisms also swim
slowly; allowing them to be easily caught, specifically for small fish larvae. Additionally,
these organisms are filter feeders; they can be fed on by various and small-sized food such as
algal and yeast cells. More importantly, their nutritional composition can be easily and
quickly adjusted according to the requirements of their predators (Dhont et al. 2013; Lubzens
et al. 1989)
The specimen cultured for this study was Brachionus rotundiformis. This species is
often used in aquaculture along with Brachionus plicatilis. The main difference between the
two is their size. B. plicatilis is the large (L-type) rotifer while, B. rotundiformis is the small
(S-type) rotifer. B. rotundiformis is more preferable when culturing tropical fish (e.g.
rabbitfish, grouper) with small mouth openings. However, this species is quite laborious to
culture given that they require higher temperatures for growth (28-35℃). When this
parameter is reached, metabolic rates are heightened along with greater reproduction leading
to the addition of more food. (Lavens & Sorgeloos 1996)
Nevertheless, when it is fed with high-quality feed, it provides great nutritional value
to the fish larvae. They are not naturally known to have high nutritional value since they act
as nutrient carriers that deliver nutrients from phytoplankton to bigger target fish species.
Hence, they need food like algae which are the ideal feed for growing and enriching rotifers.
The commonly used phytoplankton as live feed for rotifers are Nannochloropsis spp.,
Chlorella spp., Tetraselmis spp., and Isochrysis galbana. However, Nannochloropsis strain
has been found to have significant effects on the rotifer reproductive rate. As mentioned
above, rotifers are filter feeders, which means that they feed on minute organisms by
randomly straining the water. Nannochloropsis is one of the smallest alga normally reaches 2
µm so rotifers can easily digest these cells. It also has very high protein, lipid, and calorie
content. Eicosapentaenoic acid commonly known as EPA, a type of omega-3 fatty acid, is a
highly-valued nutrient that this phytoplankton species can provide in good amounts.
Furthermore, its cell wall can resist bacterial breakdown; hence, it will have no contribution
to fouling, excessive bacterial proliferation, or stickiness. On the other hand, Isochrysis sp. is
also small in size and a good source of the essential fatty acid DHA (docosahexaenoic acid);
hence, it can be used as natural feed if the target species for culture should require this
nutrient. (Feeding . . . c1995-2019; Lavens & Sorgeloos 1996)
Cheng et al (2011) conducted a study on the population dynamics of rotifer B.
rotundiformis by assessing the availability of food level. The results of the study states that
food availability has a direct relationship with the population of the zooplankton. B.
rotundiformis is at its maximum growth at high food level. In comparison of the different
phytoplankton diet fed on B. rotundiformis, the culture with Tetraselmis sp. diet reached the
highest peak (168 cells/ml) among the four growth curves. This can be linked to the volume
of feed added to the rotifer culture on each succeeding day, to compensate for the growing
population of B. rotundiformis. Tetraselmis sp. was constantly fed to the culture in a greater
amount than the other algae. Therefore, as suggested by this study, Tetraselmis sp. is the
recommended live feed for B. rotundiformis culture, with addition of 2 L feed per day when
observable exponential growth of the zooplankton occurs. Additionally, Tetraselmis is a
motile food species along with Isochrysis. Due to this, they are able to meet with their
predators more frequently (Cheng et al. 2011; Rahman et. al 2018)
B. Artemia Culture
One of the most widely used live feed in aquaculture, specifically for newly-hatched
fish larvae, is the nauplii of the brine shrimp Artemia. It is a primitive arthropod with a
segmented body and leaf-like appendages. It thrives in areas of high salinity, a physiological
adaptation to avoid its competitors and predators. However, brine shrimp cannot migrate
through the seas from one saline biotope to another; hence, they are found in different parts
of the world. (Cultured Aquatic . . . c2010-2020; Lavens & Sorgeloos 1996)
A notable characteristic of this organism is that it forms biconcave-shaped cysts.
Cysts are dormant embryos which are metabolically inactive. In dry environmental
conditions, they do not experience further development. These cysts take form to avoid early
predation. However, when submerged in seawater, the cysts become spherical as they
become hydrated and resume their metabolism. This is because of the innate highly
hygroscopic characteristic of dry cysts. During the first hours, the volume of the cysts
increases to a maximum of 140% water content. At 60% water content and onwards, active
metabolism starts, provided that the environmental conditions are favourable for growth. For
about twenty hours of constant immersion in seawater, the breaking stage occurs wherein the
outer membrane of the cysts burst. The embryo in the umbrella stage comes out but is still
surrounded by a hatching membrane. After this hatching occurs, the membrane is ruptured
freeing the nauplius inside. (Cultured Aquatic . . . c2010-2020; Lavens & Sorgeloos 1996)
Artemia is a less labor intensive food source compared to rotifers. They can be readily
bought in cans and hatched into swimming nauplii after a 24-hour incubation period in
seawater. Afterwhich, they can be directly fed to the larval culture. They also have some
similar nutritional components with rotifers. EPA is present in Artemia which was proven by
Millamena (1988) to have improved the postlarval stage of Penaeus monodon, in terms of
survival and growth in later stages. Additionally, its protein composition can be easily
digested by larvae yielding high values for protein utilization. They are also palatable and
easy to capture as they lack that effective escape response from predators. Moreover, since
they are non-selective filter feeders they can be enriched with additional nutrients in order to
improve their nutritional composition. Enrichment materials may include lipophilic
compounds, prebiotics, and probiotics that raises their resistance against pathogens. In
relation to this, possible contamination of such pathogens actually depends on geographical
location. For instance, surface runoffs may carry toxic contaminants like chlorinated
hydrocarbons which were found on the strains produced in China and Italy. Hence, it is
essential to administer proper disinfection before hatching (Hamsa 2017; Lavens & Sorgeloos
1996; Léger et al. 1987; Millamena et al. 1988).
Proper disinfection of Artemia cysts should be conducted before it can be fed to fish
larvae. This is due to the possibility that they can be a source of pathogenic bacteria which
can cause development of diseases when introduced to a culture system. Specifically, marine
fish and shrimp larvae are very susceptible to microbial infections. Artemia cyst hatching
solutions may account for Vibrio sp. as their main bacterial flora. Most Vibrio are lethal to
fish that are under stress and non-optimal environmental conditions. In these situations, these
bacteria can cause disease or even mortality outbreaks. Furthermore, the microbial load of its
outer covering may reach more than 10 7 CFU/ml, composed of bacteria, fungi and other
organic impurities. High cyst densities and increased temperature activates the metabolism of
bacteria therefore high cellular production takes place. Moreover, if hatching solutions are
not disinfected, it may become turbid and result in reduced hatching yields. In this study,
sodium hypochlorite (bleach) was used for disinfection. Gomez-Gil (1994) claimed that
solutions of sodium hypochlorite, formaldehyde, and hydrogen peroxide are effective agents
in bacterial load reduction. Sodium hypochlorite and formaldehyde gave the best results by
killing more than 90% of heterotrophic bacteria. However, only sodium hypochlorite fully
eliminated the presence of Vibrio species. (Lavens & Sorgeloos 1996; Gomez-Gil 1994)
However, for complete sterilization, cyst decapsulation should be conducted.
Decapsulation is the process of removal of the hard shell that covers the dormant Artemia
embryo. The cyst shell consists of three layers, namely: alveolar layer, outer cuticular
membrane, and embryonic cuticle. The outermost hard layer is the alveolar, consists of
lipoproteins filled with chitin and haematin. It protects the embryo against mechanical
disruption and UV radiation. In the mid-portion, the outer cuticular membrane acts as a
permeability barrier that protects the embryo from penetration of molecules larger than CO2.
Lastly, the transparent and highly elastic embryonic cuticle that develops into the hatching
membrane. This proves that the embryo has a strong encystment; however, the procedure can
be done through short-term exposure to a hypochlorite solution. A decapsulated cyst offers a
number of advantages to their predators. Unhatched cysts and left over shells can be ingested
by the predators, but not digested resulting in problems in the gut. Moreover, decapsulated
nauplii has higher energy content and individual weight since they did not spend energy
struggling out of their shells. It also lowers illumination requirements for hatching. Finally,
decapsulated cysts are easily digestible energy-rich food for the fish. (Lavens & Sorgeloos
1996)
The hatching percentage of Artemia cysts determine the amount of viable cysts that
can be fed to the larval culture. Prior to processing in cans, the cysts are harvested in
hypersaline environments since they get more energy reserves when there is extreme salinity.
When storing, water content and temperatures must be kept low. Dry cysts increase the
tolerance for high temperatures compared to those that are moist. However, it requires only in
short periods of time since longer exposure to such conditions kill the organism. During
hatching, favorable environmental conditions must be achieved. It should have a pH range of
8-8.5, for it induces the activation of the hatching enzyme. Temperature must be kept within
the range of 25-28°C with low salinities of 5-35 g.l-1. Additionally, the technology used must
suit the ideal conditions needed to improve hatching percentage. The container used must be
tailored in a way that adequate DO levels are still maintained when harvesting due to their
tendencies to aggregate at the bottom. Additionally, it must be transparent for light to reach
the organism. Since they are positively phototactic, they will accumulate at the direction of
the light source making them easier to harvest. (Lavens & Sorgeloos 1996)
In this study, the Artemia shrimp from Snowville, Utah yielded a very low hatching
percentage (31.24%) compared to the hatching percentage of cysts from the Great salt lake
(>90%). During hatching, proper protocol was followed however, prior to it, correct handling
of the product was not administered. Hence, it was inferred that the major factors that may
have influenced such results include improper storage of cysts, moisture and temperature. In
the storage phase, the can has been opened for more than a month which led to the infiltration
of air moisture through the opening of the container. Within that period, water particles must
have adsorbed on the surfaces of the cysts therefore increasing mortality rate. For
temperature, this referred to the transportation of the product into the country. It could be that
there was high surrounding temperature in the storage area of the vehicle that could have
brought stress on the cysts. (Lavens & Sorgeloos 1996)
C. Comparison between Artemia and Rotifer Culture
Both zooplanktons, Artemia and Rotifer, are widely used as live diets in larviculture
along with copepods. The advantage of using Artemia nauplii is that it provides convenience
by being stored as dry powder (cysts); hence, it is portable and easy to store. However, the
nutritional value of Artemia is varied among strains and within batches in each strain, causing
unreliable outputs in marine larviculture. On the other hand, rotifers work well in mass
production. Under optimum environmental conditions, the female exhibit asexual
reproduction by parthenogenesis in which reproduction does not require fertilization. Rotifers
also exist in large and small sizes which provide options depending on the mouth size of the
target species to culture. Furthermore, yeast-based products can be used for a cost-effective
diet. Similarly, its nutritional value is not always predictable. (Dhert & Sorgeloos 1995)

Conclusion
In general, Tetraselmis sp. was the most preferred food of rotifers among the
phytoplankton diets in this study due to its motility. Although the biology of rotifers (mastax
and corona) mostly prefer smaller planktons like Nannochloropsis, the availability of food
source had a greater effect. For Artemia, the low hatching percentage was gravely affected by
improper storage of cysts, high temperatures, and moisture.
The effectiveness of both organisms as larval food source depended on storage
convenience, nutritional value and production density. Furthermore, it extends to the choice
of fish species given that different feeding adaptations heavily influences their food choice.
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Appendix

Table 1: Data of the Artemia Cyst and Nauplii after Incubation Period and its Hatching
Percent.
Artemia Cyst Artemia Nauplii Hatching Percent (H%)

885,500 276,666.67 31.24 %