Culture of Rotifer with Comments.docx

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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 UNIVERSITYCURRIMAO, ILOCOS NORTE

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

JULY 2014GROWTH 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 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 StudyThis 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; and3. To correlate relationships between salinity and amount of food given to the growth of B. rotundiformis.

Scope and Limitations of the StudyThis 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.

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 RotiferRotifers 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: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 conditionsBacteria 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).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 MethodBatch 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 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 CultureTemperature: 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 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.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 RotifersWhen 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. oculataMicroalgae 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 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. OculataComment by casatLIB01: indicate the culture method for N. oculata, what is the ideal stocking density, the ideal salinity and other water quality parameters, also, indicate the culture practices if there is any.

METHODOLOGY

Locale of the studyThe 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 organismRotifer (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 designThe 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).

Table 1.Treatment I to be used in the studySalinityAmount of foods (Green Algae)Density of RotiferReplicates

102x 106 cells/ml50 ind/ml3




Table 2.Treatment II to be used in the study

SalinityAmount of foods (Green Algae)Density of RotiferReplicates





Table 3.Treatmen III to be used in the studySalinityAmount of foods (Green Algae)Density of RotiferReplicates





Table 4. Treatment IV to be used in the studySalinityAmount of foods (Green Algae)Density of RotiferReplicates





Source: BFAR-NIFTDC

Feeding TechniquesEach 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 RotiferB. 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: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. Comment by casatLIB01: Please indicate the procedure in culturing N. oculata

The growth of rotiferB. rotundiformisand egg ratio is computed using the formula:Specific Growth Rate: = (ln Nt ln No) /tWhere: = specific growth rate (SGR) N0 = initial densityNt = 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 ParametersWater 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 AnalysisOne-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.

LITERATURE CITED

Arimoro, F.O. and P.C. Ofojekwu. 2004. Some aspects of the culture, population dynamics and reproductive rates of the freshwater rotifer, B. calyciflorus fed selected diets. J. Aquatic Sci. 19(2): 95-98.Bosque, T. R. Hernadez. R. Prez. R. Todol. And R. Oltra. 2001.Effects of salinity, temperature and food level on the demographic characteristics of the seawater rotifer SynchaetalittoralisRousselet. Journal of Experimental Marine BiologyBureau of Fisheries and Aquatic Resources-National Integrated Fisheries Technology Development Center(BFAR-NIFTDC). 2014. Mass production of rotifer B. rotundiformis. handout.Coves, D. P. Audineau. And J.L. Nicolas. 1990. Rotifer-rearing technology. In: Barnabe, G. Ed. Aquaculture, vol. I, Ellis Harwood, West Sussex, England, pp. 232245. and Ecology, 258, 55-64.De Arauju, A.B. and A. Hagiwara. 2001.Application of enzyme activity test for the diagnosis of rotifer mass culture.Bulletin of Faculty of Fisheries of Nagasaki University, 82, 85-91.Dhert, P. 1996. Rotifers. In: Sorgeloos, P. Lavens, P. Eds. , Manual on the production and use of live food for aquacultre. Fisheries technical paper no. 361. Food and Agriculture Organization of the United Nations, Rome, pp. 4978.Epp, R.W. and P.W. Winston. 1978. The effects of salinity and pH on the activity and oxygen consumption of Brachionusplicatilis (Rotatoria). Comp. Biochem. Physiol. 59A, 9-12.Fielder, D.S. J. Purser. and S.C. Battaglene. 2000.Effect of rapid changes in temperature and salinity on availability of the rotifers Brachionusrotundiformis and Brachionusplicatilis. Aquaculture, 189, 85-99.Fulks, W. and K.L. Main. 1991. Rotifer (Brachionusplicatilis) production systems. In: Fulks, W., Main, K.L. (Eds.), Rotifer and Microalgae Culture Systems. Proceedings of a US-Asia Workshop, Honolulu, HA, January 28-31. pp. 3-52.Girin, M. and B. Devauchele. 1974. Production du rotifereBrachionusplicatilis.O.F.Muller en elevagemixte.avecle copepodetisbefarcata Baird. 3rd Meeting I.C.E.C., Work Group Maricult. Act. Coll. CNEXO 1, pp. 8799.Hagiwara, A. T. Kotani, TW. Snell. M. Assavaaree. and K. Hirayama. 1995. Morphology, reproduction, genetics and mating behaviour of small, tropical marine rotifer Brachionus strains (Rotifera). J. Exptl. Marine Biol.Ecology. 194: 25-37.Hirayama, K., 1987. A consideration of why mass culture of the rotifer Brachionusplicatilis with bakers yeast is unstable. Hydrobiologia 147, 269270.Komis, A., 1992. Improved production and utilization of the rotifer Brachionusplicatilis Mller, in European sea bream (Sparusaurata Linnaeus) and sea bass ( Dicentrarchuslabrax Linnaeus) larviculture. Doctor in Agricultural Sciences Thesis, University of Gent, 277 pp.Kovan, W M, A. Tandler, GW. Kissil, D. Sklan, O. Friezlander, and M. Harel. 1990. The effect of dietary (n-3) polyunsaturated fatty acids on growth, survival and swimbladder development in Sparusaurata larvae. Aquaculture. 91: 131-141. Lubzens E, A. Tandler, G. Minloff. 1989. Rotifers as food in Aquaculture.Hydrobiologia 186/187, 387 400.Lubzens, E. 1987. Raising rotifers for use in aquaculture.Hydrobiologia 147, 245255.Ludwig GM, and S. Lochmann. 2000. Culture of the sunshine bass, Moronechrysops X M. saxatilis fry in tanks with zooplankton cropped from ponds with a drum filter. J. Appl. Aquacult. 10(2): 11-26.Minkoff, G. 1994. Control of Vibrio type bacteria in rotifer mass cultures.Tinamenor S.A, pp. 18.Miracle, M. and M. Serra, 1989. Salinity and temperature influence in rotifer life history characteristics.Hydrobiologia, 186/187, 81-102. Muroga, K. and H. Yasunobu, 1987. Uptake of bacteria by rotifer.Bull. Jpn. Soc. Sci. Fish 53, 2091.Oie, G. and Y. Olsen. 1993. Influence of rapid changes in salinity and temperature on the mobility of the rotifer Brachionusplicatilis.Hydrobiologia 255/256, 81-86.Spolaore, P.C. E. Joannis-Cassan. Duran and A. Isambert 2006a.Commercial applications of microalgae. Journal of Bioscience and Bioengineering 101: 87-96.Srivastava, A. K. Hamre. J. Stoss. R. Chakrabarti. And S.K. Tonheim. 2006. Protein content and amino acid composition of the live feed rotifer (Brachionusplicatilis).With emphasis on the water soluble fraction.Aquaculture, 254, 534-543. Suchar, V. A. and P. Chigbu. 2006.The effects of algae species and densities on the population growth of the marine rotifer, Colurelladicentra.Experimental Marine Biology and Ecology, 337, 96-102.Tanasomwang, V. and K. Muroga. 1989.Effects of sodium nifustyrenate and tetracycline on the bacterial flora of rotifers Brachionusplicatilis.Fish Pathol. 24, 2935Yfera, M. 2001. Studies on Brachionus (Rotifera): an example of interaction between fundamental and applied research.Hydrobiologia, 446/447, 383-392.

Internet Sources:Anonymous.Untitled. 2014. Retrieved on July 24, 2014at www.reed mari culture. com/support_rotifers_feeding.phpAnonymous.Untitled. 2014. Retrieved on July 28, 2014 at www.fao. org/ docrep/ 005/x3980e/x3980e07.htm

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Culture of Rotifer with Comments.docx