Faculty of Bioscience Engineering. Academic year

Transcription

1 Faculty of Bioscience Engineering Academic year Creating added value to consumption mussels (Mytilus edulis), using a formulated feed in combination with micro-algae. Kresensia Mtweve Promoter: Dr. Ir. Nancy Nevejan Supervisor: Mieke Eggermort Thesis submitted in partial fulfillment of the requirements for the academic degree of Master of Science in Aquaculture

2 COPYRIGHT The author and the promoter give the permission to use this thesis to for consultation and to copy parts of it for personal use. However, any other use falls under the limitations of copyrights, in particular the obligation to explicitly mention the source when citing parts out of this thesis Gent University, 26 August 2011 Promoter. Dr. Ir. Nancy Nevejan Author Kresensia Mtweve i

3 ACKNOWLEDGEMENTS First and foremost, I would like to thanks the Almighty God for giving me the ability to undertake this research work to completion. He kept me effective and strong all throughout my entire journey. This work was made possible through moral support and kind assistance received from the following people whom I owe a particular debt of gratitude. I would like to express my sincere and profound gratitude to my promoter Dr. Ir. Nancy Nevejan of the University of Gent, Laboratory of Aquaculture and Artemia Reference Center (ARC), for guidance, kind support, advice, encouragement, corrections, useful suggestions and overall supervision throughout the research period. She really made this work successful. I also register my sincere thanks to my tutor Mieke Eggermort for supervision, encouragement and marvelous advice for every possible solution for the problems I faced during my experiments. Another vote of thanks goes to laboratory members of the ARC especially Geert Vandewiele and Anita De Haese for their technical support and cooperation. I sincerely thank the collaboration of Le petit pecheur company (Dutch company) and ARC management to allow student to do thesis in their company. I am further greatly indebted to my mother Romana, brothers and other relatives for their love, support and encouragement throughout my entire life. Mother thanks you for always being there for me. I lastly owe my sincere thanks to my fellow Master of Aquaculture students at Ghent University for their cooperation during my study period. You really made my life meaningful and left me with good memories. ii

4 ABSTRACT The mussel industry is faced with seasonal variation in production due to a cycling of energy storage and utilization which are mainly attributed to the reproductive activity. This cycle results to the reduction of quantity and quality of mussel meat which lead to seasonal fluctuation of the performance of mussel industry. This study evaluates the effects of the experimental artificial diet 101Mer (INVE Technology) and micro-algae on the quality of the meat of mussels (Mytilus edulis). Mussels were fed with artificial diet and micro-algae (Isochrysis galbana and Tetraselmis suecica) at 0.05% of live biomass. The result showed a decrease in body weight and condition index due to uncontrolled spawning and the insufficient feeding level. When the feeding level was increased to 0.2% of live biomass, and a Nannochloris atomus was compared to the artificial diet, the dry weight and condition index decreased in the first week by 20% and 15% respectively, but there was a noticeable improvement in the week that followed for the mussels that received the artificial diet at 100% and 75%. The biochemical analysis showed that, the mussels fed with the artificial diet had 13 to 20% higher of lipid and carbohydrate content than the non-fed mussels and mussels fed with N. atomus. The result of the fatty acid analysis demonstrated poor nutritional value of N. atomus since the PUFA s EPA and DHA were virtually lacking. This study suggests that similar feeding trials with artificial diets should not be carried out during the spawning season and the duration of the experiments should at least be 4 weeks since mussels needs two to twelve days for the digestive enzymes to respond the changes in diet. iii

5 TABLE OF CONTENTS COPYRIGHT...i ACKNOWLEDGEMENTS... ii ABSTRACT... iii TABLE OF CONTENTS...iv LIST OF FIGURES... viii ACRONYMS AND ABBREVIATIONS... x CHAPTER ONE INTRODUCTION General introduction Main objective Specific objectives... 4 CHAPTER TWO LITERATURE REVIEW Classification of blue mussel Morphological description of blue mussel Habitat... 9 iv

6 2.5 Reproduction Culture Techniques Cultivation on Suspended culture Cultivation on the Sea bed Cultivation on Poles Ecological function Nutritional requirement of bivalves Carbohydrates Proteins Lipids Vitamins and minerals Diets Live micro-algae Artificial diets Cereals Spray-dried algae Micro-encapsulated particles v

7 Algae paste Single cell protein CHAPTER THREE MATERIAL AND METHODS Experimental setup Sampling Fatty acid Dry weight Biological analysis Laboratory analysis Lipid Protein Ash Carbohydrate Fatty acids Statistical analysis CHAPTER FOUR vi

8 4.0 RESULTS Survival rate Increase in dry weight Condition index Biochemical composition Lipid Ash Protein Carbohydrate Fatty acid Dry weight of algae CHAPTER FIVE DISCUSSION CHAPTER SIX CONCLUSION AND RECOMMENDATION REFERENCES vii

9 LIST OF FIGURES Fig. 1: Major internal anatomical features of a mussel... 8 Fig. 2: Developmental stages of the mussel Fig. 3: Cultivation of mussels on suspended culture Fig. 4: Mussel cultivation on the Seabed Fig 5: Mussel cultivation on Pole Fig. 6: Experimental set-up Fig. 7: Survival rate of adult mussels fed different diets in experiment Fig. 8: Survival rate of adult mussels fed different diet in the experiment Fig. 9: Increase in dry weight of adult mussel fed different diet in the experiment Fig. 10: Increase in dry weight of adult mussels fed different diet in the experiment Fig. 11: Condition index of adult mussel fed different diet in the experiment Fig. 12: Condition index of adult mussel fed different diet in the experiment Fig. 13: Percentage lipid in adult mussel Fig. 14: Percentage ash of adult mussel Fig 15: Percentage protein of adult mussel Fig. 16: Percentage carbohydrate of adult mussel viii

10 LIST OF TABLES Table 1: Production (in thousand tonnes) of bivalves worldwide Table 2: Classification of mussel... 5 Table 3: Treatments of experiment Table 4: Treatments of experiment Table 5: Selected fatty acid composition of the formulated feed and micro-algae Table 6: Selected fatty acid composition of adult mussel experiment Table 7: Selected fatty acid composition of adult mussel experiment ix

11 ACRONYMS AND ABBREVIATIONS µg Microgram µm Micromole ARC CI Cm CP DHA DW EPA FAME Fig. G H 2 SO 4 HCl KCl Kg Artemia and Reference Center Condition Index Centimeter Chrompack Docosahexaenoic acid Dry weight Eicosapentaenoic acid Fatty acid methyl esters Figure Gram Sulphuric acid Hydrochloric acid Potassium hydroxide Kilogram x

12 M M mg/g DW Min Ml Mm N Meter Molarity Milligram per gram dry weight Minutes Millilitre Millimetre Nomality N 2 Nitrogen gas NaOH Pg Pp PUFA rpm SPSS Stdev Vol Sodium hydroxide Picogram Page Polyunsaturated fatty acids Revolutions per minute Statistical Package for the Social Science Standard deviation Volume xi

13 CHAPTER ONE 1.0 INTRODUCTION 1.1 General introduction Aquaculture has been expanding all over the world during the last couple of decades due to increased demand for fisheries products and serious decline of world s wild fisheries. The total global production from aquaculture was about 65 million tons in 2007 (2009) with total value of about $95 million, US (FAO, 2008). Bivalves (mussels, clams, scallops, and oysters) make up a significant proportion of the world s fisheries production. Annual landings of bivalves from capture fisheries and aquaculture operations increased from 11 million tonnes in 2001 to 13.5 million tonnes in 2007 (Table 1) (FAO, 2007). Mussels have become a popular consumption product in the last 10 years. During the period 2001 to 2007, mussel landing from capture fisheries was tonnes while landing from aquaculture recorded a total of tonnes (FAO, 2007). About 90% of the world s mussel production between 2001 and 2007 was obtained from aquaculture operation (FAO, 2007). In 2008 aquaculture of mussels has continuously increased to almost 1.6 million tons (FAO, 2008). Mussel culture is one of the largest aquaculture industries in many countries of the world including Europe. Mussel culture in Europe produces approximately 50% of the annual world-wide harvest of mussels (Smaal, 2002). In Europe, mussel production has increased dramatically since 1950 (Kristensen, 1997, Smaal and Lucas, 2000). But in the last decades it has stabilized, and European production decreased relative to total world production. The decrease of production was because the carrying capacity has been fully exploited, as lower growth rates have been recorded in years with lower primary production (Van Stralen and Dijkema, 1994, Smaal et al., 2001). The 1

14 commercial mussel industry is determined mainly by reproductive activity of the mussel, primary productivity of the environment and the water temperature (Smaal et al., 2001), which considerably alter the meat content. Table 1: Production (in thousand tonnes) of bivalves worldwide from capture fisheries and aquaculture operations from 2001 to Species Oyster Fisheries Aquaculture Mussel Fisheries Aquaculture Scallop Fisheries Aquaculture Clams, cockels,arkfish Fisheries Aquaculture Source: (FAO, 2007) The mussel industry is faced with seasonal variation in production due to a cycling of energy storage and utilization which are mainly attributed to the reproductive activity (Thompson, 1977, Mulvey and Feng, 1981, Fisher and Newell, 1986, Gabbot, 1975). This cycle results to the reduction of quantity and quality of mussel meat which lead to seasonal fluctuation of the performance of mussel industry. In Western Europe the blue mussel are very managed from February to March till and June to July. During winter season, mussels accumulate glycogen ready for spawning in spring time. March and April is the period which there is massive spawning. May 2

15 to July is the periods which mussels regain their body, at this period the meat quality is seriously depleted since there use most energy to rebuild their body after spawning. During this period mussels are unsuitable for commercial markets (Lubet, 1959, Lubet, 1969). Mussels also show adaptive responses to variable food quantity and quality (Hawkins and Bayne, 1992, Hawkins et al., 1996). Fluctuations in food supply and demand are reflected in changes in body weight and biochemical composition (Dare and Edwards, 1975, Bayne et al., 1978, Pieters et al., 1979, Rodhouse et al., 1984). Water temperature has proven to be a determining factor in regulating reproduction in bivalves (Robinson, 1992, Rodrıguez-Jaramillo et al., 2001) with seasonal temperature changes frequently correlating with gonad growth and gametogenesis (Heasman et al., 1996, Ojea et al., 2008). Relative increase in temperature commonly favors the process of vitellogenesis (Robinson, 1992). Indeed, increase in temperature leads to an increase in metabolism thereby stimulating the use of energy reserves for vitellogenesis (Griffond et al., 1992). Few studies have been done on fattening of bivalves using reared algae in order to compensate the seasonal variations in the occurrence of natural feed (De Pauw, 1984, Watanabe and Ackman, 1974, Robert, 1990). The cost of micro-algae production in commercial hatcheries has been estimated to be US$160 to more than US$200 per kg dry weight (Coutteau et al., 1993). Commercially fattening mussel using the micro-algae needs huge amount of algae which will be so expensive and notoriously difficult to cultivate on a commercial scale. Artificial feeding of mussel has recently become important in conjunction with the effort to solve the mussel industry problems. Most studies have been done on the partial or complete replacement of live algae with artificial diet in larvae stage bivalves (Langdon and Waldock, 1981, Knauer and Southgate, 1997a, Knauer and Southgate, 1997b, Perez-Camacho et al., 1998, Pernet et al., 2004, Fernandez-Reiriz et al., 2006, Nevejan et al., 2007) some worked on juvenile bivalves (Langdon 3

16 and Onal, 1999, Coutteau et al., 1994, Onal et al., 2006) and broodstock bivalves (Laing and Lopez-Alvarado, 1994, Heras et al., 1994, Nevejan et al., 2008). However, to the best of my knowledge, no information has been documented on the use of artificial diet on the fattening of adult mussel in order to increase the meat content and fetch higher market price. This thesis aims to fill a gap in the fattening aspects of adult mussel, particularly on comparisons between the artificial diets and micro-algae on the meat quality of mussel. 1.2 Main objective The objective of this thesis is to evaluate the quality of the meat mussels using a formulated feed in combination with different micro-algae Specific objectives To determine the effect of partial or total replacement of algae with artificial diet on the mussel weight increase. To assess the effects of feeding artificial diet, micro-algae and starvation on protein composition, lipid and glycogen utilization. 4

17 CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 Classification of blue mussel Linnaeus (1958) classified the blue mussel from phylum to species level (Table 2). Classification was based on shell form, hinge structure or gill structure. Gill structure differences provide the basis for naming various orders such as order Lamellibranches and Filibranchia to which mussels belong. These two orders consist of bivalve with gills filaments attached by ciliary junctions rather than by tissue. Table 2: Classification of mussel Phylum Mollusca Class Bivalvia Subclass Lamellibranchia Super order Pteriomorpha Order Mytilida Family Mytilidae Genus Mytilus Species Edulis Scientific name: Mytilus edulis Common name: Blue mussel Common name: Blue mussel Source: Linnaeus (1958) The mussel belongs to the family Mytilidae. Members of this family are characterised by equalsized valves with dorsal external ligament. They also have a hinged structure with very small or no teeth and their gills are separated by filaments. The two adductor muscles draw the body part 5

18 toward the median line. The foot is elongated while a byssus, which is a beard like structure helps the mussel for attachment to substrate (Newell, 1989a). Generic and specific designations are mainly characterised based on shell characteristics. Mytilus edulis, the "blue mussel", is usually described as having thin shells, bluish black in colour with a maximum length of 10 cm. This length is attained after a period of about 18 month (Korringa, 1976). 2.2 Morphological description of blue mussel The shell of blue mussels is triangle- like in shape with elongation in the anterior posterior axis and broader at the posterior shell edge. Main functions of shell are to protect the inner part of the body from predators and against desiccation (Quayle and Newkirk, 1989). The shell is made up of three layers; the outer pigment layer is called periostracum. This layer is mainly composed of the protein conchin, and its function is to protect the prismatic layer from abrasion and dissolution by acids. The middle layer called ostracum mainly composed of calcium carbonate and less protein. Inner layer is called nacre, this is a thin layer made of calcium carbonate minerals and pigments which is continuously secreted by mantle (Newell, 1989b). The internal anatomy has distinctively characteristics; the posterior adductor muscle is much larger than the anterior adductor muscle. In empty shells, the scars of the posterior adductor muscle and retractor muscles both posterior pedal and byssal are clearly visible (Fig.1) (Yonge and Thompson, 1976). In the center of the visceral mass is the darkly pigmented foot which can be extended to secrete a new byssus thread that secure the mussel to the inhabitant. The mantle which extends from either side of the visceral mass is attached to the entire periphery of both valves of shell. New shell growth is initiated from the mantle margin (Wilbur and Saleuddin, 1983). In two places of posterior ends, the mantle is modified to form an inhalant and exhalent siphon this is tube likestructure on which the water flows (Quayle and Newkirk, 1989). 6

19 The alimentary tract of M. edulis consists of an anterior mouth, esophagus and stomach joined to a long complicated intestinal tract that ends toward posterior anus. Mouth has two pairs of accessory structures; labial palps the outer palps which are collecting the incoming particles and the inner palps which transfer the acceptable food particles to the mouth, where the digestion begins (Jorgensen, 1949, Jorgensen and Riisgard, 1988). The ejected materials are expelled without passing through the digestive tract as pseudo faeces (Healy, 1998). Ingested particles enter the stomach, where they are subjected to a combination of enzymatic and mechanical attack from the continuously revolving crystalline style. Particles are also sorted in the stomach, with some particles being channelled by ciliary currents into the digestive diverticula (Newell, 1989a). Other particles of possibly lower nutritional value or given in excess of the capacity of digestive diverticula are removed by ciliary tracts from the stomach into the mid-gut. Particles directed into the ducts of the digestive diverticula are taken into digestive cells by endocytosis and are subject to intracellular digestion for periods of many hours (Seed, 1976). Assimilated material diffuse into the blood system and the undigested fragments (glandular faeces) are ejected into the duct and thence into the stomach and mid-gut. At this point, intestinal and glandular faeces are joined into a single mucus-coated faecal ribbon that passes through the anus and is carried away in the exhalent water current (Quayle and Newkirk, 1989). 7

20 Fig. 1: Major internal anatomical features of a mussel (source: BIODIDAC). 2.3 Distribution Mytilus edulis are extensively spread in European waters and along the Atlantic coast of North America, in temperate areas and polar waters (Quayle and Newkirk, 1989). Blue mussels are very successful species due to its ability to resist to a wide range of environmental conditions. It has being reported that, mussel can acclimatize temperature of about C with the upper limit of 29 0 C for adults and larvae grew well in temperature of about C with salinity ranging from 15 to 35 ppt. (Resgalla et al., 2007). Also it can stand firm in desiccation, and oxygen stress (Karayucel and Karayucel, 1999). This species occupies a wide range of microhabitats and are able to reproduce in intertidal and sub-tidal zones (Bohle, 1972). 8

21 2.4 Habitat Blue mussels occur naturally in marine environments on shallow sandy bottoms being in large communities attached to one another, known as beds. They may be found in habitats ranging from slightly brackish shallow estuaries to highly saline deep offshore environments, but tend to occur in bays and estuaries that have elevated levels of nutrients from land runoff, causing an increase in phytoplankton (Quayle and Newkirk, 1989). In many areas mussels play an important role in benthic community structure. In some areas mussels can also form dense reefs on hard bottom or soft sediments in the sub tidal and intertidal zones (Coen and Grizzle, 2007). 2.5 Reproduction Blue mussel is dioeciously (separate sexes) though rare cases have been reported to be hermaphrodites (Bayne, 1976). Mussels produce gametes and release them into the water where fertilization takes place. The process of releasing gametes into the water is called spawning and mussels are ready to spawn when they are sexually matured. Sexual maturity is reached when are about six to twelve months old. When unfavorable environmental conditions occur, like prolonged periods of exposure to air, sexual maturity is delayed until the second year (Saurel et al., 2004). After fertilization, normally after 24hours at temperature of 18 C the ciliated swimming trochophore stage begins, they are also called straight hinge (Bayne, 1976). They have a digestive tube which helps larvae to start feeding on algae. The veliger stage follows at about 40 to 72 hours after fertilization, (Bayne, 1964). This stage is characterized by formation of one transparent shell with mean shell length of about µm and a velum giving the larva a "D"-shape (fig. 2) (Sprung, 1984). The veliconch stage occurs next with shell length of about µm, this stage differs from straight-hinged stage. At this stage umbo can be distinguished and velums are well develop which help in swimming and collect food (Aypa, 1990). 9

22 When the larvae are about 210 to 250 µm they develop foot and a pair of pigmented eye spots they are called pediveliger larvae which signifies that metamorphosis is about to occur (Fig. 2). The pediveliger will sink, alternately swim and crawl until it finds a surface favourable to attachment (Sprung, 1984). According to Bayne (1976), fertilization to pediveliger stage takes about 15 to 35 days depending on temperatures, salinity and feed ration. The larvae tend to concentrate near the surface until metamorphosis approaches at that time when they migrate towards the bottom (Seed, 1976). Fig. 2: Developmental stages of the mussel. Image credited to the United States Army Corp of Engineers. 2.6 Culture Techniques Aypa (1990) identifies three main categories of culture methods for mussel cultivation. Bottom culture method, suspended culture and Bouchots culture method. These are then divided into a variety of culture methods as practiced in many countries, based on the prevailing hydrographical, social and economic conditions. 10

23 2.6.1 Cultivation on Suspended culture The suspended mussel culture techniques first developed by using rafts as a floating platform. Mussel seed from coastal rocks or collectors hung from the raft are tied to the ropes using a fine mesh net, which decomposes a few days later (Hatcher et al., 1994). After 5-6 months when mussels have reached 4-5 cm, the ropes are taken up to reallocate the mussels onto longer pieces of rope. Each rope produces enough seed for about three new ropes (or thinning out ropes) where mussels remain until they reach marketable size 7-10 cm (Perez- Camacho, 1987). In the long line system, the hung rope is firmly attached horizontally near the water surface and maintained at 1-2 m below the surface with buoys (Fig. 3). Mussels are grown on vertical ropes known as droppers' which hang from the horizontal rope for a length of 4m. The droppers are placed a minimum of 0.5 m and have free space from the bottom to avoid predation. Mussels grown on long lines can become smothered by naturally settling juvenile mussels and other fouling organisms ( (Aypa, 1990). 11

24 Fig. 3: Cultivation of mussels on suspended culture. (Source: excursion to Ireland) Cultivation on the Sea bed (bottom culture) Bottom culture as the name implies is growing mussels directly on the bottom (Fig.4). The culture plots have a depth ranging from 3 6 meters. In this culture system a firm bottom is required with adequate tidal flow to prevent silt deposition, removal of excreta, and to provide sufficient oxygen for the cultured animals (Aypa, 1990). Bottom culture or seabed culture is largely practiced in Europe especially in the Netherlands, Germany, Ireland and the United Kingdom (Aypa, 1990). Bottom culture is based on transferring wild mussels to a sheltered culture plot where the density is reduced to improve growth and fattening (Baylon, 1990). This method is said to be less labour intensive than suspended culture, 12

25 but the mussels are more subject to predation, siltation, poor growth and the harvest is less predictable (Beaumont et al., 2007). Fig. 4: Mussel cultivation on the Seabed (bottom culture). (Source: Mark A. Wilson (Department of Geology, the College of Wooster) Cultivation on Poles (Bouchots culture). In this system, ropes with spat attached, are wound around large vertical poles (bouchots) in the intertidal zone (Fig. 5). It is considered to be the original method for farming mussels (Gosling, 1992). Poles of about 3 m long and 20 cm in diameter are driven into the sea bed with m exposed above the ground. The poles are spaced 1 meter apart and arranged in rows with 3 m distance between rows (Baylon, 1990). Mesh netting is used to cover the mussels to prevent them from being detached and lost. A barrier is placed at the bottom of the pole to prevent predators such as crabs from reaching the mussels. This method of culture requires large tidal ranges in order to supply the densely packed mussels 13

26 with food. Bouchots culture was first developed in French; the word bout means enclosure (Aypa, 1990). Fig 5: Mussel cultivation on Pole (Bouchot culture) (source: Ruud van der Lubben) Mussels are a nutrient-dense seafood choice. They are low in fat ; mussel and oyster contain about 20% to 28% calories from fat when compared with beef and pork which have over 40% of calories from fat (King et al., 1990). Shellfish also provide high quality protein and high amount micronutrients or vitamins and minerals. Mussels also are rich in omega -3 fatty acids (Baylon, 1990). In blue mussel meat, the concentration of EPA and DHA are found to be 0.84g/100 g ration of mussel meats while salmon has high amount EPA/ DHA of 1.17g/100 edible portions. Sea scallop and northern lobster have a lower EPA/ DHA content of about 0.23 to 0.2g/100 edible portions respectively (King et al., 1990). Omega -3 fatty acids are important because they help to promote 14

27 good growth, good functioning of the brain, retina and sperm. They also play a role against inflammation, blood pressure maintenance and blood clotting enhancement (King et al., 1990, Raper et al., 1992). In 100g of edible portion of mussel, the concentration of iron was found to be 4.0g and that of zinc and copper was found to be 1.6 to 0.1g respectively. Iron is a dietary essential mineral in heme molecules of haemoglobin, the component of the red blood cell that carries oxygen in the bloodstream (Centres for Disease control and prevention, 1998). Zinc is needed for the body's defensive (immune) system to work properly. It plays a role in cell division, cell growth, wound healing, and the breakdown of carbohydrates (Whitney and Rolfer, 1999). Copper helps in the formation of red blood cells. It also helps in keeping the blood vessels, nerves, immune system, and bones healthy (Whitney and Rolfer, 1999). Although mussels are good source of many essential vitamins such as the vitamins B and vitamin C, they do better than other foods when it comes to vitamin B-12, selenium and manganese. The quantity of vitamin B 12 in 100g of mussel meat is believed to be 12.0µg (Raper et al., 1992). Vitamin B- 12 is essential for a healthy nervous system, the formation of red blood cells and proper growth and development (Whitney and Rolfer, 1999). 2.7 Ecological function Blue mussels, being filter feeders, have a lot of functions within the ecological system. They can readily absorb water-borne toxic chemicals and substances which can harm the health of consumers and other organisms along the food chain. They play a significant role in improving the water quality and clarity (Moal et al., 2005). Mussels help to reduce the effects of wave action and floods due to their ability to form mussel banks that stabilize the bottom stream (Baylon, 1990). 15

28 Mussels are a food source for several different kinds of terrestrial and aquatic animals, including ducks, raccoons as well several species of fish. Mussel beds also create microhabitats to species that live on the bottom in estuaries (Ryan and Davis, 2004). 2.8 Nutritional requirement of bivalves Mussels, like any other animal, require energy to carry out the metabolic processes necessary for their life. They obtain energy from the food they ingest by the process of digestion, in which the food is broken down into its basic components. Several authors noted that lipids, carbohydrates and proteins are the major components into which food is broken (De Zwaan and Zandee, 1972). Lipids, carbohydrates and proteins are stored in the liver. These compounds are sources of energy for the bivalves. However, they are all not used at the same time and rate (Epifanio, 1979). Many studies have been carried out to compare the food value of several diets to consistently support rapid growth of bivalves cultured under hatchery conditions (Coutteau et al., 1993, Utting and Millican, 1998). These comparisons allow characterization of better potential diets for growth. Identification of such characteristics facilitates the development of optimal diets for bivalves (Whyte et al., 1990). The list of micro-algae species used in marine research institutions and commercial hatcheries is still short and there is a continuous attempt to find new and better microalgae feeds or the substitutes (Coutteau and Sorgeloos, 1992) Carbohydrates Carbohydrates comprise of carbon, oxygen, and hydrogen, are the primary source of energy for bivalves. This energy is obtained from the monosaccharide glucose (C 6 H 12 O 6 ), a simple sugar that is the building block of complex carbohydrates, such as starch and cellulose (Whyte et al., 1989). In mussel carbohydrates have two major biological functions; as a long-term energy storage in terms of glycogen and as structural elements (Robledo et al., 1995). Seasonal cycles of energy 16

29 storage and utilization are generally attributed to reproductive activity (Gabbot, 1975, Thompson, 1977, Mulvey and Feng, 1981, Fisher and Newell, 1986). Glycogen is used as an energy source during the quiescent and early stages of gametogenesis, but in later stages it is conserved, possibly to be used in the lipid storage cycle, and the proteins become the major energy source (Pronker et al., 2007) Proteins Proteins are the major organic materials involved in every process that occurs in cells and tissues of animal. It s composed of amino acids, the key group of essential nutrients required by all animals for growth (Glencross, 2006). In the lifecycle of mussels, proteins are important in the formation of oocytes because protein is the main constituent of eggs, followed by lipids and glycogen (Holland, 1978). Proteins also provide energy during gonad maturation (Barber and Blake, 1985) and maintain the energy level in periods of inadequate food supply and carbohydrate levels depletion (Whyte et al., 1990) Lipids Lipids are large biological molecules that are diverse in their chemical composition. They are essential structural components of living cells (Enright et al., 1986). Lipids are divided broadly into two categories: namely, neutral lipids (NL), which are the stored fat and are mainly composed of triglycerides, and phospholipids (PL) and cholesterol, which are building blocks of membranes. Identification of lipid composition is important for physiological studies (Uno et al., 2009). Lipids constitute an important nutrient store in bivalve molluscs. They are usually used as an energy source during gametogenesis (Walne, 1970a, Beukema and De Bruin, 1979, Holland, 1978) and constitute the principal nutritional reserve in eggs and larvae as well as conditioning their viability (Helm et al., 1973). 17

30 Phospholipids are notable components of cellular membranes by forming lipid biolayers, essential for growth and to a less are extent as energetic reserve (Holland, 1978, Beninger and Lucas, 1984, Soudant et al., 1996b) Vitamins and minerals Vitamins are essential organic compounds required in small quantities to ensure proper metabolism and thus, good health. Unlike carbohydrates and proteins, vitamins have little caloric intake and are not macromolecules made up of building blocks. Their chemical composition varies and they have unique structures of their own (Hilton, 1989). Vitamins and minerals are required for the regulation of the body's metabolic functions and growth of the shells. These nutrients can be found naturally in the feeds consumed by bivalves while minerals such as calcium can be taken directly from the water. Concerning artificial diets, many feeds are prepared in order to provide additional nutrients necessary for growth of the bivalves (Conklin, 1997). Bevelander (1952) observed that the growth of the shell of the mussel does not depend on the feed availability. According to Sick et al. (1979) the shell weight of unfed Crassostrea virginica increased linearly with increase of calcium concentration. In comparison with fish and crustaceans, there is very little information about the nutritional requirement of bivalves. This is due to the lack of suitable artificial diet for bivalves. 2.9 Diets Live micro-algae Micro-algae play a crucial nutritional role for marine animals in open ocean and consequently in marine aquaculture. Most marine invertebrates depend on micro-algae for their whole life cycle. 18

31 The main consumers of micro-algae in aquaculture are filter feeders (mainly larvae, juveniles and broodstock of mollusk) (Muller-Feuga et al., 2003). The biochemical components of the micro-algae depend on the algal species, culture condition and harvesting growth stage. According to Brown et al. (1997), micro-algae contain 6-52% protein, 5-23%, carbohydrate, 7-23% lipid and 6-39% minerals. Almost all algae species have similar amino acid composition and they are rich in the essential amino acids such as arginine, histidine, isoleucine, leucine, and lysine (Brown et al., 1997). Fernandez-Reiriz et al. (1989) reported that the biochemical composition of algae can vary with culture age. Carbohydrate and lipid contents were observed to be increased with the exponential phase of the culture, while protein levels increased in the stationary phases of the culture in the case of diatoms and Rhodomonas sp. and decreased in Isochrysis galbana, Pavlova lutheri, and Tetraselmis suecica. Many studies have been conducted to determine the best micro-algae species that provides sufficient nutrients for optimal growth and survival of cultured bivalves (Walne, 1970b, Brown, 1991, Nell and O'Connor, 1991, Coutteau and Sorgeloos, 1992, Brown et al., 1997, Lora-Vilchis et al., 2004, Martinez-Fernandez et al., 2006, Martinez-Fernandez and Southgate, 2007). More than fifty micro-algal species have been tested as live food for bivalve molluscs, but less than twenty species are being used commercially in aquaculture (Brown, 1991, Brown et al., 1997). Some micro- algal species which are regarded as being successful for bivalve cultures include Isochrysis sp. (strain T-ISO), Pavlova lutheri, Tetraselmis suecica, Chaetoceros muelleri, Chaetoceros calcitrans, and Thalassiosira pseudonana (strain 3H). These micro-algae are usually fed to cultured bivalves (larval, juvenile, and broodstock stages) in combination of two or three species. Suitability of the algae species for culturing bivalves may be characterized by its chemical composition, digestibility, size and non- toxicity (FAO, 1996). The appropriate size for most 19

32 bivalve ranges from 1 to 15 µm (Quayle and Newkirk, 1989). M. edulis has a very fine filter with a mesh of approximate µm which allow the efficiency retention of 1-2 µm particles (Mohlenberg and Riisgard, 1979). Both filtration rate and ingestion rate are affected by the cell size. The suitability range of the feed particles may differ among the species as well as the stage of the algae. Also the acceptability of micro-algae depend on the stage of the animal, larvae needs smaller size than juvenile and adult bivalves (Webb and Chu, 1983). Thickness of the cell wall is considered to be the most leading factor which affects its digestibility. Bivalves do not digest well thick cell wall such as Dunaliella sp (De Pauw et al., 1983) chlorophyta in general Artificial diets Due to the high expenses of growing micro-algae for bivalve production, extra interest has been developed by researchers for an alternative source of feeds which will substitute the use of microalgae as the source of feed for bivalves. Many varieties of feeds have been examined as the alternative feed source for bivalves including spay-dried algae (Laing et al., 1990) while (Zhou et al., 1991) dealt with preserved algae Spirulina. Other feed include micro-particulated feed and cereals flours (Nevejan et al., 2007, Nevejan et al., 2008, Mazon-Suastegui et al., 2008). Cereals such as rice, oats, wheat, and corn are less expensive, easy to prepare and to assimilate. They are energetically rich in essential nutrients which show their potential in meeting the nutritional requirements of bivalves (Laing, 1987). Despite the fact that most of these varieties of feeds give suitable results in rearing marine bivalves, no feed has been accepted to replace fully the algal diet without affecting the nutritional balance required by the animal (FAO, 1996). Artificial diets should be produced with minimal demand of time, space, labour, cost and it should preserve for long time (Laing et al., 2004). Alternative diets to micro-algae must have the correct particle size and remain in suspension so that they can be easily assimilated by the bivalves. The food must be highly digestible with the adequate amount of required nutrients (Laing et al., 1990). 20

33 Cereals Haven (1965) used cornstarch and wheat flour to examine the effect of carbohydrate supplement on shell and tissue (meat) growth of the American oyster (C. virginica). He reported there was an increase in meat content but no effect on shell size. Dunathan et al. (1969) studies the effects of artificial foods upon oyster fattening using cornmeal diet. They observed a significant increase in condition and glycogen level. Perez-Camacho et al. (1998) worked on the effect of micro-algae and inert (cornmeal and cornstarch) diets on growth performance and biochemical composition of Ruditapes decussatus seed. The results showed an improvement in growth rate with cornstarch, when 99% carbohydrate was added to these diets. The increase was 32.3% dry in weight when a 0.5% daily ration was used. Willis et al. (1976) reported on the use of artificial diet to improve the quality of the oyster (C. virginica). They used cornmeal, hominy meals and hominy meals with yeast additive. They concluded that the use of corn meal gave the best result increasing the dry weight of the animals and level of glycogen from 0.62 to 6.68 % within two weeks Spray-dried algae Boeing (1997) reported the use of a spray-dried Schizochytrium diet (Algamac 2000, Aquafauna Bio-Marine) as a partial substitute for living algae in diets for juvenile clams Tapes semidecussatus and Pacific oysters Crassostrea gigas. He found that up to 40% of a mixed algal diet Tetraselmis suecica and Chaetoceros sp. for clams could be replaced with Schizochytrium without significantly reducing growth rate. Furthermore, he reported that the growth of clams fed T. suecica alone could be significantly improved by supplements of Schizochytrium. 21

34 Davis and Cambell (1998) as well reported the use of a Schizochytrium-based product (Docosa Gold, Sanders Brine Shrimp) and argued that mussels (Mytilus galloprovincialis) were able to grow better on suspensions of Schizochytrium and algae than on algae alone. Langdon and Onal (1999) fed juvenile mussels M. galloprovincialis by replacing living microalgae with spray-dried algae. They observed that the growth rate of mussels fed 1/4 ration of living algae plus a mixed supplement of 50:50% of Docosa Gold and spray-dried Spirulina platensis grew significantly faster than mussels fed a full live algal ration Micro-encapsulated particles Microencapsulated diets have been evaluated as a partial or complete replacement for a live algae diet for Pacific oyster (Crassostrea gigas) (Knauer and Southgate, 1997b). Laing (1987) observed that the growth rates of C.gigas and Mercenaria mercenaria spat fed microencapsulated artificial diet went up with 54% and 64% respectively compared to the rates obtained with the algal diet. The advantage of using the micro-encapsulated diets is that their biochemical composition can be accurately measured and controlled, unlike that of algal diets, which is dependent on culture age and conditions (Webb and Chu, 1982). Nevejan et al. (2007) fed mussel spat (M. galloprovincialis) with the micro-encapsulated diet MySpat (INVE Technologies, Dendermonde, Belgium), They obtained a significant increase in wet weight of mussel spat, which was almost twice as much than the mussels fed the nonsupplemented algae diet. They could replace 2/3 of the algae diet with MySpat without negatively affecting the growth of the young seed. Micro-encapsulated particles were also used to condition mussel (M. edulis) broodstock. Mussel fed a mixed micro-algae diet at cells day -1 or a 100% formulated diet MyStock (INVE Technologies, Dendermonde, Belgium) showed equally high percentages of spawning animals (80, and 85%, respectively) and large numbers of eggs (on average eggs female -1. A high 22

35 hatching rate of more than 80% was observed for the micro- encapsulated diet (Nevejan et al., 2008) Algae paste Algal pastes have been assessed as alternatives to live micro-algae (Brown et al., 1997). This product had a moderate value as a diet component for molluscs (Laing, et al., 1990). The algae paste were found to be useful as a backup diet to replace 50% of live algae in the diet of broodstock of C. virginica and M mercenaria and spat (Coutteau and Sorgeloos, 1992). The advantage of algal pastes products is that they can be stored for several months and be used as offshelf products (Brown, 2002) Single cell protein (SCP): bacteria and yeast Yeasts are unicellular eukaryotic microorganisms that are common in seawater. Yeast was considered very early on to be a potential food substitute in aquaculture due to its small size and high protein content. It has also a short generation time and can be produced more economically than photosynthetic micro-algae (Bass et al., 2007). Yeast (Candida utilis) was first used as a diet in bivalve cultures by (Epifanio, 1979). He fed juveniles of M. mercenaria, M. edulis, and C. virginica with a mixed diet of T. pseudonana and various percentages of yeast. When supplied with a 50% yeast-based diet, juvenile of M. mercenaria and M. edulis grew faster than those fed the diatom only. It was also observed that yeast diets could be supplied up to 80% as substitute to micro-algae Coutteau et al. (1994) studied the effect of algal ration and substitution of algae by manipulated yeast diets on the growth of juvenile M. mercenaria. The result shows that the substitution of 80% of the algal diet resulted in growth rates reaching 90% of those obtained for the algal-fed controls 23

36 CHAPTER THREE 3.0 MATERIAL AND METHODS The experiments were conducted in Yerseke (The Netherlands) at the company Le petit pecheur. The company owns a turbot grow-out facility provided with a recirculation system which has been adapted for holding shellfish till marketing. The animals are packed daily in an efficient and modern packaging and labeling line and are sold fresh in different European countries. The company was interested in this study because once they stock the shellfish in the tanks; the animals lose weight and hence market value. They were looking for means to maintain or even better increase the weight of mussels in particular, since their market value is determined by the cooked meat percentage at the mussel auction. 3.1 Experimental setup The experiments were carried out according to the recirculation principle (RAS). Each tank of 40l volume was fitted with its own bio-filter. The size of the bio-filters tanks and rearing tanks was the same (Fig.6). A biological filter is important because it maintains the water quality in the recirculation system by removing excess ammonia. Water from the big RAS system was UVtreated before it was pumped to the reserve tank, from where it was put in the experimental tanks manually. In the experimental set-up, water from the rearing tanks was pumped to the bio-filters and water from the bio-filters flowed back to the rearing tank by gravity. Water temperature was maintained between 15 and 19 0 C. A blower was installed to provide strong aeration in the biofilters and rearing tanks, and the oxygen level varied between 7 and 10mg.l -1 l during the feeding trials. The experiments counted 5 treatments and were carried out in triplicate (15 tanks in total). The water in the experimental tanks was changed at a ratio of about 30% on a daily basis and the tanks were completely emptied and cleaned on the day of sampling. 24

37 The bio-filter consisted of bio-balls which were washed with detergent (soap) and put into a disinfectant (< 5% sodium hypochlorite, Unilever Netherland). The bio-balls were rinsed three times with sea water to remove the residues of disinfectant. They were inoculated with bacteria from the big bio-filters system used by the company. The bacteria were fed with the artificial feed in order to provide them with a protein source and to allow them to adapt to the feed that will be used in the trials. The bacteria were grown for two weeks before the start of the experiments. The efficiency of the bio-filters was monitored daily by measuring nitrite and ammonia. The kit used was JBL test kit (GmbH and Co.KG, Germany). Feeding pot Biofilter Reserve tank Rearing tank Fig. 6: Experimental set-up Adult mussels were bought by the company from Scotland and kept in a big holding tank for three weeks before the start of the experiment 1. Mussels were washed by hand to remove dirt and barnacles. After washing, 1.5kg live weight of mussels, counting approximately

38 individuals, were stocked in each experimental tank. Two weeks before the start of the experiment 2, the mussels were washed and kept outside in a big tank so as to induce spawning by elevated temperatures (in the sun 22 C). A total of about 2kg of live weight (approximately individuals) was stocked in each experimental tank. A dripping system was used to deliver the food to the mussels, whereby buckets of about 8 liters were drilled at the bottom and fitted with a small silicon tube which led the feed suspension to the rearing tanks. The silicon tubes were fixed with small tape to adjust the flow of feed. Before feeding, the formulated diet, a very fine powder, was mixed with a hand mixer to homogenize the suspension. The daily amount of algae and artificial diet were put together and diluted with seawater to make up a volume of 8 liters. The feeding buckets were aerated to minimize sedimentation. Two separate feeding experiments were carried out. For each feeding experiment two diets at different feeding rates were compared, with three replicates per treatment. In experiment 1 which lasted for three weeks, the adult mussels were fed with the experimental formulated diet 101Mer (INVE Technologies, Dendermonde, Belgium) and with micro-algae produced in the hatchery of Roem van Yerseke (Yerseke, The Netherlands). A mixture of 2 algae species was used: Tetraselmis suecica which constituted 30% of the ration and Isochrysis galbana, Tahiti (T-Iso) which represented 70% of the algae diet (based on dry weight: 20pg/cell T-Iso, 275 pg/cell T.suecica). A 100% diet meant that the mussels were fed at 0.05% of their live weight being equivalent to 0.5g dry weight of food per kg of live weight of animal. Starved mussels (control) served as negative control animals. 26

39 Table 3: Treatments of experiment 1(% based on dry weight) Treatment Artificial diet (%) Micro-algae (%) Control % algae % alg:50%art % artificial % artificial In experiment 2 which lasted for two weeks, the amount of feed was increased to 0.2% which was equivalent to 2g dry weight of feed per kg of live weight of mussels, except for treatment 25% algae where the algae were fed at a level of 0.05%. In this experiment only one alga species was used, delivered by Algaelink Company (Yerseke, The Netherlands). Although claimed to be Tetraselmis suecica, shape, size, mobility and fatty acid profile did not coincide. After consultation, it is believed that the alga was indeed Nannochloris atomus. A dry weight of 200 pg/alga was assumed. Starved mussels (control) served as negative control animals. Table 3 summarizes the treatments of Experiment 2. Table 4: Treatments of experiment 2 (% based on dry weight) Treatment Artificial diet (%) Micro-algae (%) Control % algae % algae % alg:75%art % artificial