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Protein utilization, hormone treatment and nutrient metabolism as they apply to culture of abalone Haliotis… Taylor, Barbara Elan 1998

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PROTEIN UTILIZATION, H O R M O N E T R E A T M E N T A N D NUTRIENT M E T A B O L I S M AS T H E Y A P P L Y TO C U L T U R E OF A B A L O N E Haliotis kamtschatkana by BARBARA ELAN TAYLOR B . S c , The University of British Columbia, 1987 M . S c , The University of British Columbia, 1990 A THESIS S U B M I T T E D IN P A R T I A L ^ F U L F I L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF D O C T O R OF  PHILOSOPHY in  T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Zoology) We accept this as conforming to the required standard  T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A December, 1997 © Barbara Elan Taylor, 1997  In  presenting  this  degree at the  thesis  in  partial fulfilment  of  University of  British Columbia,  I agree  freely available for reference copying  of  department publication  this or  and study.  of this  his  or  her  representatives.  DE-6 (2/88)  It  thesis for financial gain shall not  ^-^lAjQV  The University of British Columbia Vancouver, Canada  Da,e  4* . 2ff  that the  may be  permission.  Department of  requirements  I further agree  thesis for scholarly purposes by  the  .MX  is  that  an  advanced  Library shall make it  permission for extensive  granted by the understood  be  for  that  allowed without  head  of  my  copying  or  my written  ii  ABSTRACT Development of formulated diets for use in abalone culture demands knowledge of the animal's nutritional requirements, growth, and metabolism. This thesis addresses some important aspects of these issues with regard to the abalone Haliotis kamtschatkana. Protein requirement was investigated from the standpoint of how protein utilization is affected by dietary protein content, amino acid balance, and protein sparing, together with the possible amelioration of the last through enzymatic adaptation to diet. Focus on protein utilization stems from the fact that protein is a costly dietary ingredient and is an essential nutrient for protein growth. With respect to abalone growth, I assessed the efficacy of administering vertebrate growth hormones which enhance growth in other cultured aquatic animals. I also investigated metabolism of cells isolated from the abalone digestive gland (a primary site of metabolic conversions) from the standpoints of seasonal variation in metabolic activity, and relationships between digestive gland and gonad activity during the reproductive cycle. My data show that optimal utilization of dietary protein in Haliotis kamtschatkana occurs when diets are formulated with about 20 % dry mass of protein, and with carbohydrates, rather than fats, comprising the energy source since enzymatic adaptation to diet does not alter protein-sparing effects. With respect to growth, I found no enhancement from treatment with recombinant bovine or porcine somatotropin, or somatostatin (vertebrate growth hormones). Furthermore, I found that metabolism in these animals is seasonal and possibly inter-related with the competitive needs of reproduction and somatic growth. These findings contribute to the general understanding of abalone biology and provide information useful for culture of abalone.  iii  TABLE OF CONTENTS Abstract  ii  List of Tables List of Figures  v  :  .-  y  Acknowledgments  i  viii  General Introduction  1  Chapter 1: Effect of dietary protein content on protein utilization by abalone Haliotis kamtschatkana 1.1 Abstract.... 1.2 Introduction 1.3 Methods and Materials 1.4 Results 1.5 Discussion  8 8 9 11 16 22  Chapter 2: Effect of dietary amino acid balance on amino acid utilization by abalone Haliotis kamtschatkana 2.1 Abstract 2.2 Introduction 2.3 Methods and Materials 2.4 Results 2.5 Discussion  27 -.27 28 30 36 40  Chapter 3: Effect of protein sparing on protein utilization by abalone Haliotis kamtschatkana 3.1 Abstract 3.2 Introduction 3.3 Methods and Materials 3.4 Results 3.5 Discussion :  48 48 .....49 51 58 68  Chapter 4: Effect of vertebrate growth hormone on growth of adult abalone Haliotis kamtschatkana 4.1 Abstract 4.2 Introduction 4.3 Methods and Materials 4.4 Results  •  74 74 75 76 78  iv  4.5 Discussion Chapter 5: Seasonal changes in nutrient utilization and response to growth hormones by isolated digestive-gland cells of abalone Haliotis kamtschatkana 5.1 Abstract 5.2 Introduction 5.3 Methods and Materials 5.4 Results 5.5 Discussion  82  86 86 87 89 92 99  General Summary  103  References Cited  106  List of Tables Table 1.1 Composition of diets formulated to determine the optimal level of dietary protein for abalone Haliotis kamtschatkana 12 Table 2.1 Amino acid balances of whole body- and ovary-proteins of abalone Haliotis kamtschatkana, and of kelp Nereocystis leutkeana Table 2.2 Composition of diets formulated to test the effect of varying dietary amino acid balance on amino acid utilization by abalone Haliotis kamtschatkana  32  34  Table 2.3 Correlations of amino acid balances in diets formulated to match the amino acid compositions of abalone whole body, abalone ovary, or kelp 37 Table 3.1 Gross nutrient and energy content of four diets for abalone Haliotis kamtschatkana: kelp Nereocystis leutkeana, and three formulated diets with fat either less than, equal to, or greater than carbohydrate 53 Table 3.2 Specific nutrient composition of diets formulated for abalone Haliotis kamtschatkana, to test the effect of varying carbohydrate:fat ratio on protein utlization 54 Table 4.1 Mortality of abalone Haliotis kamtschatkana treated with weekly injections of bovine serum albumen, somatostatin, recombinant porcine growth hormone, recombinant bovine growth hormone, or left untreated 80  vi  List of Figures Figure 1.1 Food intake by abalone Haliotis kamtschatkana fed formulated diets with 0, 10, 20, and 30 % dry mass casein 17 Figure 1.2 Live mass of abalone Haliotis kamtschatkana fed formulated diets with 0,10, 20, and 30 % dry mass casein 18 Figure 1.3 Nitrogen excretion by abalone Haliotis kamtschatkana fed formulated diets with 0, 10, 20, and 30 % dry mass casein 20 Figure 1.4 Protein utilization by abalone Haliotis kamtschatkana fed formulated diets with 10, 20, and 30 % dry mass casein 21 Figure 2.1 Food intake by abalone Haliotis kamtschatkana fed formulated diets with varying amino acid balances  38  Figure 2.2 Live mass of abalone Haliotis kamtschatkana fed kelp or formulated diets with varying amino acid balances 39 Figure 2.3 Nitrogen excretion of abalone Haliotis kamtschatkana fed formulated diets with varying amino acid balances 41 Figure 2.4 Protein utilization of abalone Haliotis kamtschatkana fed formulated diets with varying amino acid balances 42 Figure 3.1 Food intake by abalone Haliotis kamtschatkana fed formulated diets with fat content less than, equal to, or greater than carbohydrate content 59 Figure 3.2 Live mass of abalone Haliotis kamtschatkana fed kelp or formulated diets with fat content less than, equal to, or greater than carbohydrate content 60 Figure 3.3 Nitrogen excretion by abalone Haliotis kamtschatkana fed kelp or formulated diets with fat content less than, equal to, or greater than carbohydrate content. 61 Figure 3.4 Protein utilization by abalone Haliotis kamtschatkana fed formulated diets with fat content less than, equal to, or greater than carbohydrate content 63 Figure 3.5 Lipase activity in the gut of abalone Haliotis kamtschatkana fed a kelp diet or formulated diets with fat content less than, equal to, or greater than carbohydrate content 64 Figure 3.6 Alginate lyase activity in the gut of abalone Haliotis kamtschatkana fed a kelp diet or formulated diets with fat content less than, equal to, or greater than carbohydrate content 66  Vll  Figure 3.7 Amylase activity in the gut of abalone Haliotis kamtschatkana fed a kelp diet or formulated diets with fat content less than, equal to, or greater than carbohydrate content 67 Figure 4.1 Live mass of abalone Haliotis kamtschatkana treated with weekly injections of bovine serum albumen, somatostatin, recombinant porcine growth hormone, recombinant bovine growth hormone, or left untreated 79 Figure 4.2 Water content of abalone Haliotis kamtschatkana treated with weekly injections of bovine serum albumen, somatostatin, recombinant porcine growth hormone, recombinant bovine growth hormone, or left untreated 81 Figure 4.3 Gonad indices of abalone Haliotis kamtschatkana treated with weekly injections of bovine serum albumen, somatostatin, recombinant porcine growth hormone, recombinant bovine growth hormone, or left untreated 83 Figure 5.1 Oxygen consumption by isolated digestive-gland cells of male and female abalone Haliotis kamtschatkana  93  Figure 5.2 Percent change in oxygen consumption by isolated digestive-gland cells of male and female abalone Haliotis kamtschatkana after administration of either glucose, amino acids, or glucose + amino acids 95 Figure 5.3 Percent decrease in oxygen consumption by isolated digestive-gland cells of male and female abalone Haliotis kamtschatkana after administration of 100 u M recombinant bovine somatotropin 96 Figure 5.4 Percent decrease in oxygen consumption by isolated digestive-gland cells of male and female abalone Haliotis kamtschatkana after administration of 100 u M somatostatin 97 Figure 5.5 Gonad and digestive-gland indices of abalone Haliotis kamtschatkana over a 15-month period 98  Acknowledgments  I gratefully acknowledge the assistance of several individuals without whom this thesis could not have been completed. I thank my supervisor Dr. Tom Carefoot, for his aid and advice through every stage of these investigations, from diving to collect research animals, to wading through the world's longest sentence, and everything between. Research animals were collected at Bamfield Marine Station, with the assistance of Dr. Andy Spencer, Director, and his staff. Growth hormones were generously provided by Drs. Ewen McLean and Ed Donaldson, who also participated in the study along with Deborah Donovan. I thank my supervisory committee: Dr. Dave Higgs, Dr. Peter Hochachka, Dr. George Iwama, and Dr. Dave Randall, for their participation. Especially noted are Drs. Higgs and Hochachka who each allowed me extended use of their laboratories. Drs. Steve Land and Jim Staples provided much advice during development of the digestive-gland cell isolation technique. Dr. Bill Milsom generously allowed me access to computer equipment, and Dr. Steve Reid gave me comprehensive critiques of the early drafts of this thesis which were invaluable. I am also grateful to my husband Mike Harris for encouraging me throughout this work, to my son Cailean Harris for , inspiring me to finish, and to my parents Louise and Bill Taylor for making it a possibility. Throughout this work I was financially supported by NSERC, and University Graduate Fellowships and Teaching Assistantships from the University of British Columbia.  1 G E N E R A L INTRODUCTION Abalone (genus Haliotis) are herbivorous marine gastropods which have been harvested for centuries and are highly prized for their meat and shells. The abalone Haliotis kamtschatkana (Jonas) once supported a small fishery in British Columbia; however, the local population has been extensively depleted and fishing (commercial, sport, and native) has been suspended since 1990. Increased efficiency and intensification of commercial harvesting has led to similar stock depletion in many areas of the world and, thus, there is wide-spread interest in abalone culture. Proper nutrition is an essential component in the culture of any animal, and wellestablished techniques of animal culture typically utilize formulated feeds. At present, about 14 formulated diets for abalone are produced in five different countries and research groups in another eight countries are actively investigating abalone nutrition (Fleming et al., 1996). Many extant abalone culture operations rely on abundant natural stands of seaweed to feed abalone. This practice imposes major constraints because of the time and money costs involved in the harvest, preparation, and storage of seaweed. Formulated diets are more consistent in nutrient composition, less environmentally destructive, and afford greater flexibility in terms of culture location than natural diets. More importantly, growth efficiency can be enhanced by using formulated diets tailored to fit the nutritional needs of the cultured animal. Before diets can be formulated to enhance growth efficiency, however, the nutritional requirements of abalone must be known. Many of these requirements are still in question. My first three studies reported in this thesis determined the nutritional requirements for optimizing protein utilization. Protein utilization, rather than growth, was selected as the measure of diet performance in these investigations because the diets were formulated with an intent to investigate metabolism of dietary protein rather than to maximize growth. The focus on protein metabolism  2 stemmed from the fact that protein is an expensive dietary component, and efficient utilization demands that it be reserved for protein synthesis. The efficient utilization of dietary protein depends upon several factors: the dietary content of protein, the digestibility of the protein, the balance of its amino acids, and the proteinsparing effects of non-protein nutrients. Ideally a formulated diet should supply enough amino acids to support ample production of tissues, organs, enzymes, mucus, and gametes, as well as sufficient energy to fuel such production. Some investigators feel that optimal dietary protein content cannot be determined without knowing the optimum ratios of dietary energy to protein for abalone growth (Britz, 1996a; Fleming et al, 1996). However, diets which are high in lipid are unpalatable to abalone (Mai et al, 1995; Chapter 3) and, thus, lipid cannot be used as a main energy source. Indeed, laboratory- and commercially-produced diets for abalone generally contain a level of lipid within the range found in natural kelp foods (1 - 5 % dry mass lipid; Jeong et al, 1994; Virtue and Nichols, 1994). Thus, varying the dietary protein:energy ratio is accomplished mainly by altering the ratio of protein to carbohydrate. Determination of the optimal amount of protein in diets for abalone was made in this manner, and the results are presented in Chapter 1. Numerous studies have investigated the optimal level of dietary protein for cultured fish (tilapia: Newman etal, 1979; goldfish: Lochman and Phillips, 1994; milkfish: Sumagaysay and Borlongan, 1995), crustaceans (shrimp: Colvin and Brand, 1977; Fenucci etal, 1980; Robertson etal, 1993; prawn: Deshimaru and Yone, 1978; Millikin etal, 1980; crayfish: Huner and Meyers, 1979; Koshio etal, 1993), and molluscs (mussels: Kreeger and Langdon, 1993; abalone: Ogino and Kato, 1964; Uki etal, 1985a and 1986a; Mai et al, 1994; Britz, 1996a). These studies generally indicate that increased dietary protein supports increased growth until a plateau is reached,  3 and that excessive levels are inefficiently utilized (i.e., catabolized for energy). My study aimed to identify the dietary protein content which would maximize protein utilization. In my first and third investigations (Chapters 1 and 3) casein was used as the protein source. Casein, as a protein source for abalone, has been reported to have nutritional quality superior to that of the protein in macroalgae {Haliotis discus hannai: Uki et al., 1985a, b, and 1986a; and H.fulgens. Viana et al., 1993). Britz (1996b) demonstrated, however, that casein is inferior to proteins from fish meal and the microalgae Spirulina spp. for H. midae. Nonetheless, since protein source has not been investigated for H. kamtschatkana, casein was selected as the protein source in my first and third investigations for its availability, because it contains all 20 of the amino acids which typically comprise tissue proteins, and because it has been used extensively as a protein source in experimental diets for abalone and other aquatic animals. Numerous studies have investigated the nutritional quality of proteins from different sources for culturedfish(Nose, 1971; Ogino and Chen, 1973, Takeda et al, 1975; Winfree and Stickney, 1981), crustaceans (prawns: Balazs and Ross, 1976; shrimp: Smith et al, 1985; Lim and Dominy, 1990; Das etal, 1995; crabs: Bordner, 1989; Sudaryono etal, 1995), and abalone (Uki etal, 1985a, b, and 1986a; Viana et al, 1993; Mai et al, 1994; Britz, 1996b; Viana et al, 1996). In such studies the different amino acid balances as well as different digestibilities of protein were compared. The second study of my thesis investigated the effect of dietary amino acid balance on protein utilization by Haliotis kamtschatkana. Digestibility was not a factor because crystalline, rather than protein-bound amino acids, were included in the diet. Dietary protein is digested into individual amino acids which are absorbed by the animal and reformulated into body proteins for purposes of either maintenance or growth. Body proteins are typically composed of 20 amino acids. About half of these are considered essential because they cannot be synthesized by most animals, while non-essential amino acids can be synthesized by transamination reactions. Allen and Kilgore (1975) established that 10 amino acids are  4 essential for abalone (the same ten that are essential for most animals: arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine). A formulated diet which supplies both essential and non-essential amino acids in an optimal balance would be used most efficiently because the use of nitrogenfromessential amino acids to synthesize non-essential amino acids would be minimized. Thus, assuming a sufficient supply of dietary energy, the relative quantities of available amino acids required for growth and maintenance defines the optimal balance of amino acids, and determines the utilization of dietary protein. The third study of my thesis compared the protein-sparing effects of carbohydrates and fats. Specifically, I determined the ability of abalone to extract energy from carbohydrates and fats, thereby sparing proteinfromcatabolism and preserving it for anabolism. Protein-sparing effects vary among fish (chinook salmon: Buhler and Halver, 1961; rainbow trout: Atherton and Aiken, 1970; De la Higuera etal, 1977; Beamish and Medland, 1986; brook trout: Ringrose, 1971; catfish: Page and Andrews, 1973;plaice: Cowayetal, 1975;turbot: Adronetal, 1976;tilapia: De Silvaefa/., 1991; lungfish: Sakthivel and Baskaran, 1995), crustaceans (shrimp: Andrews etal, 1972; Clifford and Brick, 1978; lobsters: Capuzzo and Lancaster, 1979; crayfish: Ackefors etal, 1992; prawns: Sedgwick, 1979; Shiau and Peng, 1992; crabs: Kucharski and De Silva, 1991; Linton and Greenaway, 1995), and molluscs (scallops: Whyte et al, 1990). Furthermore, protein sparing would be expected to vary with enzymatic adaptation to diet, if such adaptation enhanced the animal's ability to extract energy from fats and/or carbohydrates. Thus, the activities of the abalone digestive enzymes were monitored in the third study of my thesis to determine whether differences in protein sparing would be ameliorated by enzymatic adaptation to the diets. That the activities of digestive enzymes can be manipulated by changes in diet has been shown for rainbow trout (Kim et al, 1992), shrimp larvae (Le Moullac and Van Wormhoudt, 1994; Rodriguez et al, 1994), crab larvae (Harms et al, 1994), and abalone (Knauer et al, 1996).  The fourth study of my thesis was the only component which investigated growth in abalone, and involved a test of the effect of hormone treatment on growth of adult abalone. Growth of abalone has been well studied (Sloan and Breen, 1988), and is known to be influenced by the type and quantity of food available, and by characteristics of habitat such as water motion, substrate, temperature, and season. Breen and Adkins (1979) suggested that Macrocystis integrifolia and Nereocystis leutkeana are the optimal algal foods for Haliotis kamtschatkana, and that protected or semi-exposed habitats afford better growth than exposed sites. Smooth, regular substrates, to which abalone can adhere and locomote with least energy expenditure, should also provide better opportunities for growth. Laboratory studies (Paul and Paul, 1981) have determined that growth of H. kamtschatkana is best at 13 -14 °C. Studies of field populations ofH. kamtschatkana (Larson and Blankenbeckler, 1980; Paul and Paul, 1981) have suggested that growth is slowest in winter/spring and fastest in late summer. Rather than repeat studies on these foregoing factors which are known to influence growth; attention was given in this thesis to a new technique for growth enhancement in aquaculture. Since hormone treatment has been shown to enhance growth of teleosts (reviewed by McLean and Donaldson, 1993), my study determined the efficacy of employing recombinant bovine and porcine somatotropins, and somatostatin as anabolic agents. The first four investigations utilized whole live abalone as test subjects and, as such, encountered some problems. Specifically, these included slow growth rates and difficulty in formulating diets which varied in nutrient composition but were still palatable to abalone. For the fifth study of my thesis these problems were circumvented by using an in vitro preparation of isolated digestive-gland cells. With this method I investigated the seasonality of metabolism and nutrient utilization, and the effect of hormone treatment. In this regard, I quantified seasonal variation in digestive gland and gonad size, as well as seasonal variation in the response of  6 abalone digestive-gland cells to nutrient substrates, and to treatment with recombinant bovine somatotropin and somatostatin. The digestive gland was the obvious source of cells for an in vitro investigation due to this gland's multiple functions. One function, of course, is digestion which for abalone includes extracellular digestion, absorption, and intracellular digestion of nutrients. This gland facilitates digestion through production of enzymes (e.g., lipases, proteases, and carbohydrases). The specific complement of these enzymes appears to correspond with the dietary habits of abalone. Haliotis kamtschatkana feeding on kelp consume a diet in which dry matter is composed of 38.5 % carbohydrates, 15 % protein, 3 % lipid, and 44 % ash (Wort, 1955). Thus, the need for lipases and proteases in abalone is less than in animals which consume high lipid or high protein diets. For example, carnivorous octopus (Boucaud Camou and Roper, 1995) and lobsters (Barkai et al., 1996) exhibit higher protease than carbohydrase activity, while herbivorous scallops show greater carbohydrase activity than protease or lipase activity (Teo and Sabapathy, 1990). Abalone possess numerous polysaccharide digestive enzymes which are tailored to the types of algae consumed (Anzai et al., 1991). The abalone digestive gland also functions as a storage site for glycogen (Livingstone and de Zwaan, 1983; Carefoot et al, 1993; Takami et al, 1995), and has a protective role in filtering environmental toxins and pollutants (Hyne et al, 1992). Also, since the digestive gland of pulmonate snails is involved in the production of growth hormones (Bride and Gomot, 1989) and the oocyte protein vitellogenin (Bride etal, 1992), similar functions may exist in abalone. Formulated diets for abalone are currently being developed worldwide.  My thesis offers  guidance regarding the optimal content and amino acid balance of dietary protein, and the optimal source of non-protein energy required to insure that a maximal amount of dietary protein is retained in abalone tissues. Since the advent of recombinant D N A technologies, intensive study  has been devoted to manipulating growth in aquacultured organisms with hormone treatment. Accordingly, the applicability of this technique to abalone culture is assessed here. The in vitro studies on abalone digestive-gland cells reported in this thesis provide insight into the seasonal variation in abalone metabolism and into digestive-gland function. The overall results of my studies will be helpful in the formulation of diets for abalone in that they will contribute to the understanding of nutrient metabolism in these commercially prized animals.  C H A P T E R 1: E F F E C T O F D I E T A R Y PROTEIN C O N T E N T O N P R O T E I N UTILIZATION B Y A B A L O N E Haliotis kamtschatkana 1.1 A B S T R A C T Diets formulated for abalone should meet, but not exceed, protein requirements to insure that dietary protein is used for protein synthesis rather than energy production. This criterion demands identification of an optimal content of dietary protein, a level at which protein utilization (the proportion of absorbed dietary nitrogen that is retained in body tissues) is maximized. Adult abalone Haliotis kamtschatkana were held in individual cages floating in an aquarium tank supplied with recirculating seawater of 14 ° C and 32 %o. These animals were fed diets containing 0, 10, 20, and 30 % dry mass casein (referred to as 0%C, 10%C, 20%C, and 30%C, respectively) for 16 weeks to determine optimal dietary protein content. Protein utilization was significantly different, with the data resolving into two overlapping groups: abalone fed the 30%C diet (54 % nitrogen retention) were similar to those fed the 10%C diet (68 % retention); and abalone fed the 10%C diet were also similar to those fed the 20%C diet (77 % retention). Protein utilization was significantly influenced by time, averaging 88 % retention over the first 6 weeks but decreasing significantly to 44 % at 10 weeks. Only abalone fed the 20%C and 30%C diets exhibited a positive mass gain, and the 0.42-g gain in live mass by abalone fed 30%C diet was significantly higher than that of the other diet groups. Differences in mass gain did not correlate with food intake which was significantly lower for abalone fed the 0%C casein diet (0.38 g' d") but similar for all other diet groups (0.55 g d" ). Therefore, 1  the 20%C diet was optimal for adult H. kamtschatkana.  1  9 1.2 I N T R O D U C T I O N In formulated diets protein content should be set at a level which maximizes the efficiency with which dietary proteins are transformed into body proteins. Utilization of dietary protein as an energy source rather than for tissue growth constitutes waste of this nutrient. Various studies have investigated the effects of dietary protein content on growth, survival, and feed conversion of several invertebrates: shrimp (Clifford and Brick, 1978; Fenucci etal, 1980; Robertson et al, 1993), prawns (Kitabayashi et al, 1971; Deshimaru and Yone, 1978; Millikin et al, 1980), lobsters (Gallagher et al, 1979; Capuzzo and Lancaster, 1979), crayfish (Hunter and Meyers, 1979; Koshio et al, 1993), and molluscs (mussels: Kreeger and Langdon, 1993). Abalone too have been subjects of such investigations (H. discus: Ogino and Kato, 1964; H. discus hannai: Uki et al, 1986a; H. discus hannai and H. tuberculata: Mai et al, 1994; and H. midae: Britz, 1996a). Two general trends regarding optimal dietary protein content are apparentfromthese and other studies. First, as an animal's age and size increase, protein requirement decreases relative to energy requirement (Maynard and Loosli, 1969). The requirement for amino acids shiftsfroma high-growth, low-maintenance requirement in early life stages to a low-growth, high-maintenance requirement in later stages (Pike and Brown, 1967). This phenomenon has been demonstrated in penaeid shrimp (Colvin and Brand, 1977) and freshwater prawns (Balazs and Ross, 1976). Thus, optimal dietary protein content for abalone would be expected to be highest for young, rapidly growing abalone and to decrease with age and size. In my study, the potentially confounding effect of this trend was minimized by selecting adult abalone of a narrow size range. A second trend regarding optimal dietary protein content is that regardless of whether an animal is an herbivore, omnivore, or carnivore, growth is enhanced by formulated diets that contain a protein concentration greater than that found in natural foods. Ogino and Ohta (1963) documented this for the abalone H. discus which exhibited enhanced growth when fed formulated diets containing 30 % dry mass crude protein, relative to their growth on natural algal foods containing 11  10 % dry mass crude protein. In addition, Uki et al. (1986a) reported an optimal protein content of 20 30 % in formulated diets for H. discus hannai containing casein, and Mai et al. (1994) reported an optimal protein content of 30 % in formulated diets fori/, discus hannai and H. tuberculata, diets which contained a mix of casein, gelatin, and crystalline amino acids. These are the only studies on abalone which based optimal protein content on protein utilization rather than on growth. Based on these findings, I compared protein utilization in abalone fed diets with protein contents of 0 to 30 %. Although growth generally increases with increased dietary protein concentration, the efficiency of protein utilization depends upon the extent of conversion of dietary proteins to new body tissues, and does not continue to increase with increasing levels of dietary protein. This has been demonstrated for humans (Calloway and Spector, 1955a and b), rats (Sibbald et al., 1956; Yoshida et al., 1957), chickens (Donaldson et al., 1956; Summers et al, 1964), fish (sea bass: Metailler et al, 1980; chinook salmon: McCallum and Higgs, 1989), and crustaceans (lobsters: Capuzzo and Lancaster, 1979; prawn: Hewitt, 1992). Inefficient protein utilization can result from increased catabolism of protein as an energy substrate when animals consume diets with excess protein and with inadequate energy from lipid and carbohydrate. The reduced efficiency of these diets is possibly due to the metabolic costs of deamination and subsequent production of ATP from amino acids rather than from carbohydrates and lipids. Thus, optimal growth and protein utilization cannot be achieved by simply maximizing protein consumption. Given the above concerns, formulation of diets must aim to maximize not only growth but also protein retention (measured as nitrogen retention). One method to determine nitrogen retention involves monitoring nitrogen excretion. Oxidation of protein as an energy substrate increases nitrogen excretion, and occurs when dietary protein content exceeds protein requirement or when non-protein energy sources in the diet fail to meet an animal's energy requirement. Both circumstances lead to inefficient protein utilization and, therefore, to poor rating of the diet. Thus, the focus of my study was  11 to assess protein utilization in an attempt to determine optimal levels of dietary protein. Growth was expected to increase with increasing dietary protein content. Protein utilization, however, was expected to be optimal below 30 % dry mass protein based on knowledge that this protein level maximizes growth in other abalone species, and the fact that optimum protein utilization is expected to occur at a lower protein content than maximum growth.  1.3 M E T H O D S A N D M A T E R I A L S Collection and maintenance of abalone Abalone Haliotis kamtschatkana were collected by SCUBA divers in shallow subtidal areas near the Bamfield Marine Station on the west coast of Vancouver Island, British Columbia. The abalone were transported to the University of British Columbia where the study was conducted. Sixty abalone were divided into four groups of 17 g mean individual live mass. Animals were housed in individual 250-ml plastic beakers fitted with mesh windows for water circulation, and suspended from a styrofoamframein a recirculating seawater system maintained at 14 °C (the optimal temperature for growth of this species: Paul and Paul, 1981) and 32 %o. Each group of 15 animals was fed ad libitum on one of four formulated diets that contained varying casein content. The study ran for 16 weeks from October 1992 - January 1993. Dietformulation andfeeding The test diets were formulated to contain 15 % dry matter which was approximately double the dry mass composition of kelp (Wort, 1955), a natural food for abalone. Each diet contained either 0, 10,20, or 30 % dry mass casein, (respectively referred to as 0%C, 10%C, 20%C, and 30%C). The precise compositions of these diets are given in Table 1.1. In the diets where casein was less than 30 % dry mass, a consistent dry mass composition of the diet was maintained by including an appropriate ammount of cellulose. Cellulose was not a filler since it is digestible to abalone, rather it contributed an  12 Table 1.1 Composition (g per dry kg diet) of formulated diets used to determine the optimal level of dietary protein for abalone Haliotis kamtschatkana. These diets were formulated to contain 15 % dry matter. All diets contained common components represented by 33 % dry mass Protein-Free Diet obtained from ICN Ltd., the composition of which is as defined below.  Diet Dietary Component  0%C 270 0 400 330 1000  agar casein cellulose common components total  1  1  10%C 270 100 300 330 1000  20%C 270 200 200 330 1000  30%C 270 300 100 330 1000  Common components (g dry kg" of formulated diet) contributed by the Protein-Free Diet were: 221.4 % sucrose, 49.5 corn starch, 26.1 cellulose, 16.5 corn oil, 13.2 mineral mix , and 3.3 vitamin mix . 1  2  3  2  Composition of mineral mix (g kg" Protein-Free Diet): 20.0000 calcium phosphate dibasic, 2.9600 sodium chloride, 8.8000 potassium citrate monohydrate, 2.0800 potassium sulfate, 0.9600 magnesium oxide, 0.1400 manganous carbonate, 0.2400 ferric citrate (16 - 17 % Fe), 0.0640 zinc carbonate, 0.0120 cupric carbonate, 0.0004 potassium iodate, 0.0004 sodium selenite, 0.0220 chromium potassium sulfate, 4.7200 sucrose.  3  Composition of vitamin mix (mg' kg" Protein-Free Diet): 0.006 thiamine hydrochloride, 0.006 riboflavin, 0.007 pyridoxine hydrochloride, 0.030 nicotinic acid, 0.016 D-calcium pantothenate, 0.020 folic acid, 0.002 D-biotin, 1 X 10' cyanocobalamin, 0.016 retinyl palmitate, 10.200 D L - a - tocopherol acetate, 0.025 cholecalciferol, 5 X 10" menaquinone, 9.730 sucrose.  1  1  5  5  13 amount of energy which facilitated maitenance of a similar gross energy among the diets, as well, it played a phagostimulatory role (pers. observ.). It should be noted that these test diets did not contain a source of n-3 fatty acids which are required by most marine species. Of the fatty acids found to be essential in most animals, corn oil provides only those of the n-6 (linoleic) family. The lipid requirements of abalone have not yet been completely defined; however, abalone appear to require small amounts of n-3 fatty acids (Uki et al, 1986b), and their tissues contain a high proportion of n-6 fatty acids (Dunstan et al, 1994). Thus, there may have been a deficiency of n-3 fatty acids in the diets used in this study. In subsequent studies (Chapters 2 and 3) I mixed corn oil and menhaden oil to provide both n-3 and n-6 fatty acids. Also, the diets used in this and subsequent studies (Chapters 2 and 3) contained a general mix of vitamins and minerals because the requirements for these micronutrients in abalone are not known. To prepare the diets, agar was added to boiling distilled water in a 4 % mass/volume ratio and mixed with a hand-held blender until homogenous. Before casein and the common components (Protein-Free Diet obtained from ICN Ltd.) were added, the mixture was cooled to 65 - 70 °C to prevent denaturing of casein which has been shown to be nutritionally detrimental for abalone (Uki and Watanabe, 1986). After addition of all components the mixture was blended to an homogenous consistency. The diets were left to cool and set, and stored at 4 °C until used. A known mass of fresh food was provided daily to each abalone, and uneaten food was collected and weighed the following day. The loss of dietary nutrients through leaching was assessed by comparing the percent dry mass of food particles which had been held in seawater for 24 h with that offreshlymade diet. There was less than 3 % ± 0.1 (n = 10) loss of dry matter. Digestibility The digestibility of each diet was assessed in order to calculate the amount of protein assimilated. Five abalone from each diet group were held individually in 250-ml plastic beakers, each  14 filled with seawaterfromthe recirculating system, and individually aerated. On seven consecutive days each abalone was fed a known mass of food, and 24 h later uneaten food was collected and weighed. The entire 250 ml of seawater within the beaker, together with the faeces it contained, was collected daily for assessment of faecal mass and protein content. Seawater from the beaker was homogenized for 2 min to distribute faecal material evenly throughout the volume. Ten-ml aliquots of the homogenized seawater-faeces mixture were used to determine the protein content of the faeces. Since faecal protein was dissolved in the seawater by the homogenization, a spectrophotometric technique (Bio-Rad protein assay) was used to assess the protein mass dissolved in 10-ml aliquots. These measurements were made in triplicate and were used to determine the total mass of protein in the seawater. Triplicate 10-ml aliquots of seawaterfromthe recirculating system were also assessed for protein content to correct for any dissolved protein resident in the seawater. Protein digestibility was then calculated as: protein digestibility = (protein eaten - faecal protein)(protein eaten)"  1  Nitrogen excretion Nitrogen excretion (NEX) of each abalone was measured every 2 - 4 weeks by sealing the abalone in a 250-ml plastic container (taking care to exclude air bubbles) for a 3-h period between 0800 - 1300 h. A sample of seawater was then drawn and analyzed for ammonia content using the method of Solorzano (1969). The total amount of ammonia excreted over the 3-h period was calculated from the known volume of seawater in each container. Since over 90 % of waste nitrogen from prosobranch gastropods is eliminated in this form (Cockcroft and McLachlan, 1990; Clarke et al, 1994), ammonia was the only nitrogenous waste measured. In order to extrapolate N E X to an amount of nitrogen excreted per day, a time series of ammonia assays was carried out. Excretion was measured once every 4 h for 28 h.  15 Protein utilization Protein utilization (PU) was assessed as the percentage of absorbed dietary nitrogen retained by the body tissues of the abalone. P U = (absorbed dietary nitrogen - N£x)(absorbed nitrogen)" (100 %) 1  Absorbed dietary nitrogen was calculated from the amount of casein consumed by correcting for protein digestibility (determined here to be 0.65 which is within the range of values obtained in other studies on abalone: see Fleming et al., 1996 for review) and multiplying by 0.16 which is the proportion of nitrogen in casein . This measure equates N 1  E X  with catabolism of dietary protein,  however, it should be noted that turnover of tissue protein and catabolism of any protein or amino acid acquired from non-dietary sources would also contribute to N E X , although to what extent is unknown. This method of assessing protein utilization differs from those typically applied in aquaculture nutrition (Wilson, 1989). It was, however, uniquely designed to accommodate my focus on protein retention rather than growth (as is measured by protein efficiency ratio: PER) and on changes in protein utilization over time which precluded the carcass analyses necessary for measures of net protein utilization (NPU). Statistical analyses The datafromall measures of food intake, mass change, N E X , and P U were analyzed using two-factor Repeated Measures ANOVA, coupled with Student-Newman-Keuls Multiple Comparison Tests (SNK). Values presented in the text are means + standard error. Data were not corrected to body mass because the same animals were measured repeatedly and all four diet groups had a similar mean mass.  Note: Kjeldahl analyses indicate that casein contains 96.7 % protein (D. Higgs, pers. comm.) and 15.8 % N (Merck Index) but for these calculations I considered casein to be 100 % protein and 16 % N .  1  16 1.4 R E S U L T S Figure 1.1 illustrates mean daily food intake over a period of 16 weeks for four groups of abalone fed diets with varying casein contents. Data are mean daily food intakes for the 15 abalone comprising each diet group: Two main trends were evident in these data, thefirstbeing a significant effect of diet on daily food intake (F3356 = 12.8; p < 0.001). Mean daily intake by abalone feeding on the 0%C diet (0.4 ± 0.02 g d' ) was significantly lower (p < 0.05, SNK) than that for the abalone fed 1  the other three diets which formed a statistically homogenous group (mean food intakes of 0.6 + 0.02, 0.6 ± 0 . 0 2 , and 0.5 ± 0.02, respectively, for abalone fed the 10%C, 20%C, and 30%C diets). The second trend was a significant effect of time on daily food intake (F 35 = 272.7; p < 0.001). 5J  4  Regardless of diet, feeding was highest in thefirst4 weeks of the experiment (0.8 + 0.02 g d" for all 1  diets combined), dropping to its lowest level of 0.3 + 0.0 at 10 weeks, and then increasing to 0.5 + 0.02 at 16 weeks. Food intake from 10 weeks onward was significantly lower than the intake at 4 weeks (p < 0.05, SNK). Data on mass change of abalone fed the experimental diets were variable but exhibited trends similar to those of the feeding data (Figure 1.2). Both diet (F 56 = 12.8; p < 0.001) and time 3;3  (F5^54 4.8,p < 0.001) had significant effects on mass change. The 30%C group (mean mass change =  of 0.4 ± 0.1 g) was significantly different from all other groups (p < 0.05, SNK). The mean mass change of -0.2 ± 0.1 g of abalone feeding on 0%C was not significantly different from the -0.1+0.1 mass change of abalone feeding on 10%C (p < 0.05, SNK). The 10%C group was also statistically indistinguishablefromthe 20%C group (mean mass change of 0.1 ± 0.1 g; p < 0.05, SNK). Mass change was affected by time in a general trend that held for all diets and paralleled the time effect previously noted for feeding. In thefirst6 weeks of the experiment, when feeding was highest (Figure 1.1), mass change was also highest (0.4 + 0.1 g for all diet groups combined), but then declined, reaching a low of -0.2 + 0.1 by 12 weeks. Mass loss continued for 2 weeks after feeding began to  17 Figure 1.1 Food intake by abalone Haliotis kamtschatkana fed formulated diets with 0,10, 20, and 30 % dry mass casein, over a 16-week period from October - January. Each point represents mean daily food intake (g" d") ± S.E. for N = 15 abalone each of 17 g live mass. 1  MONTHS NOV  DEC  JAN  o • T •  4  6  8  10  12  TIME (weeks)  14  16  0% casein 10% casein 20% casein 30% casein  18 Figure 1.2 Live mass of abalone Haliotis kamtschatkana fed formulated diets with 0, 10, 20, and 30 % dry mass casein, over a 16-week period from October - January. Each point represents mean live mass (g) ± S.E. for N = 15 abalone each of 17 g starting live mass.  MONTHS NOV  DEC  JAN o • • •  20 19 18  \  C/2  -4. r •  17  > 16  0 15 A 14  ~i— —i—1—i—1—i—1—i—1—i—1—r~ 1  4  6  8 10 12 TIME (weeks)  14  16  0% casein 10% casein 20% casein 30% casein  19 increase, but this trend reversed by the end of the 16-week period for all abalone fed formulated diets. Highest mass gain was noted for abalone fed the 30%C diet (0.3 ± 0.1 g;p < 0.05, SNK). The results of the time-series assays of nitrogen excretion (NEX) indicated that N E X was consistent over a 24-h period (0.1 - 0.5 mgN" 3-h" individual" ), so 3-h measures of N 1  1  E X  were  converted to a daily rate by multiplying the 3-h values by 8 (Figure 1.3). N E X was significantly affected by both diet (Fs^se = 62.6;p <0.001) and time (F3356 = 14.1 ;/> O.001). Abalone feeding on 30%C exhibited a significantly higher mean N E X (5.9 + 0.2 mg N ' d" ' individual" ) than the other 1  1  groups. There were no significant differences in mean N E X among abalone feeding on 0%C, 10%C, and 20%C when calculated over the 16-week period (1.3 ± 0.2 mg N" d' ' individual" , 1.3 ± 0 . 1 and 1  1  2.2 ± 0.2 for 0%C, 10%C, and 20%C, respectively; p < 0.05, SNK). The trend for N E X over time was unlike the time-trends noted for the other measured parameters. Thus, overall N E X was lowest at 4 weeks (1.7 + 0.25 mg N  d' individual" ) when feeding (Figure 1.1) and mass gain (Figure 1.2) were 1  1  greatest. Over the next 8 weeks while both feeding and mass gain declined, N E X was consistent at a rate significantly higher than the rate at 4 weeks (2.1 - 3.0 mg N  d" individual" ; p <0.05, SNK). At 1  1  16 weeks there was a significant rise in N E X to an overall mean of 3.7 + 0.4 mg N ' d" individual" . 1  1  Data on food intake, protein digestibility, and N E X were combined in the calculation of protein utilization (PU), defined as the proportion of absorbed dietary nitrogen which was retained in body tissues rather than excreted. Casein digestibility was found to be consistent at a mean of 0.65 for the 10%C, 20%C, and 30%C diets, respectively ( F ^ = 1.2; p = 0.33). Figure 1.4 shows a significant effect of both diet (F^e? = 4.6; p = 0.01) and time (F , 64 = 21.3; p < 0 001) on PU. 5  2  ;  Abalone fed 20%C had the highest mean PU (76 ± 2 %) followed by 10%C (68 ± 4 ) and 30%C (54 ± 3). The results fell into two overlapping subsets (p < 0.05, SNK); with 10%C- and 20%C-fed abalone in one group, and 30%C- and 10%C-fed abalone in another. P U also exhibited variability with time. Highest levels (88 %) were recorded over thefirst4 weeks of the study. They then decreased  20 Figure 1.3 Nitrogen excretion (NEX) by abalone Haliotis kamtschatkana fed formulated diets with 0, 10, 20, and 30 % dry mass casein, over a 16-week period from October - January. Each point represents mean excretion (mg N ' d' ) ± S.E. for N = 15 abalone each of 17 g live mass. 1  MONTHS NOV  DEC  0% casein 10% casein 20% casein 30% casein  8  B 6 O H 05  u zw O O  4  2  4  6  8  10  12  TIME (weeks)  14  16  21 Figure 1.4 Protein utilization (PU) by abalone Haliotis kamtschatkana fed formulated diets with 10, 20, and 30 % dry mass casein, over a 16-week period from October - January. Each point represents mean protein utilization [(N absorbed - N excreted)(N absorbed)"^ 100 %)] + S.E. for N = 15 abalone each of 17 g live mass.  MONTHS NOV  *  DEC  JAN  20 H  4  6  8  10  12  TIME (weeks)  14  16  22 significantly (to 69 - 74 %) over the next 4 weeks, reaching a significant low of 42 % at 10 weeks (p < 0.05, SNK), before reversing this decline. P U was significantly higher at weeks 12 (63 ± 4 %) and 16 (59 + 5). The levels at weeks 12 and 16 were similar to those of week 8, but were significantly lower than P U over the initial 6 weeks (p < 0.05, SNK).  1.5 DISCUSSION Based on overall differences among the diets used in this investigation I concluded that 20 % casein is the level which optimizes protein utilization by Haliotis kamtschatkana. The data on food intake did not identify a single optimal casein content, since diets with 10, 20, and 30 % dry mass casein were consumed at similar daily rates. The 0%C diet was, however, consumed at a significantly lesser rate. Data on mass change eliminated the 0%C and 10%C diets as being optimal, since abalone did not grow while eating these diets. By contrast, the abalone fed the 20%C and 30%C diets exhibited a positive mass change. Nitrogen excretion and protein utilization data indicated that the 20%C diet was optimal for protein utilization because abalone fed the 20%C diet had a significantly lower nitrogen excretion as well as a significantly higher protein utilization than abalone fed the 30%C diet. All variables were significantly influenced by time. These trends, however, were not appreciably different between the different diet groups. Therefore, the time effects did not alter the conclusion that protein utilization by H. kamtschatkana is optimal when dietary protein content is 20 % dry mass. Values of protein utilization in my study were comparable to that of another study with abalone (Uki et al., 1986a) which evaluated use of dietary protein with measures of net protein utilization. Since net protein utilization is calculated as the ratio of protein gained by an animal fed the test diet plus protein lost by an animal fed a non-protein diet, to protein intake, it is a measure of the proportion of dietary protein retained in the body. Uki et al. (1986a) reported net  23 protein utilization of 93 % for Haliotis discus hannai fed a diet with 10 % dry mass casein, 68 % with 20 % dry mass casein, and 60 % with 30 % dry mass casein. The difference in protein utilization between these two species is noteworthy. H. discus hannai showed maximum protein retention at 5 -10 % casein and declining retention at 20 %, whereas H. kamtschatkana exhibited greater protein retention when the dietary protein content was 20 % compared to 10 % casein. These measures of protein retention can be compared with other values for cultured aquatic animals. Net protein utilization in plaice ranges from 40 - 54 %, but increases with increasing dietary protein content and increasing carbohydrate.lipid ratio (Cowey et al., 1975). In tilapia, net protein utilization ranges from 20 - 43 % depending upon whether the diet is higher in carbohydrate than lipid and protein or vice versa, respectively (De Silva et al., 1991). Capuzzo and Lancaster (1979) reported that lobsters retained 28 - 45 % of dietary protein depending upon whether they were fed low or high protein:starch ratios, respectively. Atherton and Aiken (1970) noted that retention of dietary protein ranged from 25 - 83 % in rainbow trout fed diets containing different sources of proteins and lipids, but retention remained between 30 - 37 % when the fish were fed different ratios of lipid and carbohydrate (De la Higuera et al., 1977). The similarity of protein utilization in abalone fed 30%C and 10%C, and the fact that both values were lower than that for abalone fed 20%C, suggests that the protein level in the 30%C diet was excessive whereas the 10%C diet contained insufficient protein for optimal protein utilization. Abalone fed 30%C catabolized twice as much of their dietary protein as abalone fed 20%C. Presumably, this higher protein catabolism was necessary to meet energy requirements because the abalone could not derive enough energy from the non-protein energy sources in the diet. Abalone fed the 20%C diet had the highest protein utilization, and therefore the highest protein retention. Accordingly, I concluded that among these three formulated diets 20%C had the optimal protein content. Although protein utilization could not be calculated for the abalone fed 0%C since they  24 had no protein intake, nitrogen excretion was measured and compared to that of the other diet groups. An interesting aspect of the nitrogen excretion data was that despite a range in daily consumption rates and broadly ranging growth rates, Haliotis kamtschatkana fed diets with 0-20 % dry mass casein excreted a similar amount of nitrogen (1.2 - 2.2 mg N ' d" ). Such nitrogen 1  excretion indicates a daily catabolism of 7 - 14 mg of protein, the source of which could have been either diet or body tissues. Daily nitrogen intake can be calculated from daily food intake. Combining this with nitrogen excretion provided an estimate of daily nitrogen balances. Daily nitrogen balances of -11, 1, and 5 mg N ' d" were obtained for abalone fed the 0%C, 10%C, and 1  20%C diets, respectively. A nitrogen balance over the entire 16-week period can be obtained by multiplying the daily nitrogen balances by the number of days in the study. Similarly, total mass change for the 16-week period can be obtained by adding the mass changes calculated over 2 - 4 week intervals. Comparing these two data sets demonstrated that the total mass gain of 5 % for abalone feeding on 20%C was largely a protein gain. Similarly the 9 % loss of body mass for abalone feeding on 0%C was less than the protein loss, indicating that some body constituent was being added while protein was being lost. The 2 % loss of body mass for abalone feeding on 10%C occurred despite a protein gain equal to 0.9 % of body mass. In these abalone some body constituent was being lost at a greater rate than protein growth was occurring. Thus, combining data on nitrogen excretion, mass change, and food intake indicated changes in body protein composition, and from this it was possible to resolve the ambiguity caused by the statistical similarity in food intake, mass change, nitrogen excretion, and protein utilization observed between the 10%C- and 20%C-fed abalone. The 20%C diet was optimal because it afforded the greatest increase in body protein through conversion of dietary protein into tissue protein. Data on nitrogen excretion have not been extensively reported for abalone. The range of nitrogen excretion in this study (1 - 6 mg N" d" ' individual" ) was considerably higher than 53 u,g 1  1  25 N ' d' ' individual" which can be estimated from Peck et al. (1987) for Haliotis tuberculata of 1  1  similar size. The data in my study, however, are consistent with Duerr's (1968) measures of ammonia excretion in four species of herbivorous marine prosobranch snails: 4 - 85 |ig N ' g' live 1  mass' d" versus 70 - 129 u,g N g" live mass d" measured here for H. kamtschatkana. The 1  1  1  higher protein content and higher digestibility of the casein diets as compared with natural algal diets may account for my higher values for H. kamtschatkana, or my values may simply reflect a high variability of nitrogen excretion which is evident from other molluscan studies (e.g., 81 ng N • g" • h" for Antarctic limpets: Clarke, 1990; 257 fig N • g' ' h' for intertidal littorines: Aldridge et 1  1  1  1  al, 1995). Comparison of mass change in this investigation to data from growth studies is somewhat inappropriate because the intent here was to formulate diets to optimize protein utilization rather than to optimize growth. Such comparison is also difficult because most studies report growth in terms of increased shell length. Furthermore, growth of abalone shows substantial variability both within species (Kojima et al, 1977; Mottet, 1978; Breen and Adkins, 1982; Sloan and Breen, 1988), and between species (Fleming et al, 1996). However, if one converts the length data obtained from field (Quayle, 1971; Breen and Adkins, 1979; Breen, 1980; Larsen and Blankenbeckler, 1980) and laboratory (Paul etal, 1976; Paul and Paul, 1981) studies using the shell length-total mass relationships obtained by Breen and Adkins (1982), it is apparent that growth of//, kamtschatkana in my study was about two orders of magnitude less than observed in other studies. What then accounts for the slow growth of abalone eating these formulated diets? The energy content of these diets likely exceeded the demands for growth since they were 85 % higher than that of natural algal diets (11.3 kJ' dry g' for kelp; calculated from chemical composition of 1  26 Nereocystis leutkeana obtained by Wort, 1955). Although the test diets resembled kelp in their relative proportion of carbohydrate and lipid, they did not match the precise nutrient composition of kelp. The formulated diets did not contain the same starch and sugar components, the same ratios of amino acids, or the same types and ratios of fatty acids, as do natural kelp diets. These differences may have contributed to slow growth. The intakes of the formulated diets in the present study (2.3 - 3.3 % body mass per day) were higher than the range of 0.7 - 2.5 % body mass per day reported by Fleming et al. (1996) for various species of abalone, of similar size, consuming various formulated diets. There was also considerable variability in daily intake over the course of the experiment. Daily food intake was initially high (0.73 - 0.90 g' d" ), perhaps due to the novelty of the diets (abalone seem drawn to 1  novel flavours and textures). A later decline followed by a subsequent increase in food intake (for all diets except 0%C) may represent a rejection of the diet followed by an acceptance motivated by starvation, or by an adaptation to diet facilitated by physiological change. The fact that daily intake of 0%C did not increase after the initial decline may be an indication of nutritional stress caused by protein deprivation. The results of this study indicate that protein utilization in adult Haliotis kamtschatkana is optimal when the diet contains about 20 % dry mass protein from a source such as casein. However, further work on optimization of the available levels and balance of dietary amino acids, and the ratios of dietary protein to non-protein energy sources will be necessary to maximize growth in this species under culture conditions. This investigation stands as a comparison of protein utilization when casein varies between 0 - 3 0 % dry mass in diets for Haliotis kamtschatkana, despite the differences in diet type, food intake, mass change, and nitrogen excretion between this and other studies on a variety of abalone species. These data indicate that a casein content of 20 % was optimal for H. kamtschatkana.  27  C H A P T E R 2: E F F E C T O F D I E T A R Y A M I N O ACID B A L A N C E O N A M I N O ACID UTILIZATION B Y A B A L O N E Haliotis kamtschatkana 2.1 A B S T R A C T The effect of dietary amino acid balance on amino acid utilization by abalone Haliotis kamtschatkana was investigated. Diets were formulated with amino acid balances mimicking those of kelp (KAA), abalone whole body (BAA), and abalone ovary (OAA), or with predominantly glycine (GAA). Seventy-five abalone were equally divided into five groups of 30 g mean individual live mass. Four groups were fed the formulated diets while one group received a natural diet of kelp. Abalone were held for 28 weeks in individual cages floating in a flow-through seawater system of ambient temperature (7-17 °C) and salinity (25 - 28 %o). Food intake, mass change, nitrogen excretion, and amino acid utilization were measured for abalone fed the formulated diets. The amount of food eaten varied significantly with season but not with amino acid balance in the diet. Despite differing amino acid balances, all diets except K A A supported a mass increase. Kelp-, GAA-, and OAA-fed abalone gained the most live mass (3.3 g, 2.4, and 2.2, respectively), and B A A - and KAA-fed abalone gained the least (0.7 and 0.0, respectively). Nitrogen excretion for BAA-, OAA-, and KAA-fed abalone declined significantly after the first 4 weeksfrom1.6 mg N d" individual' to 0.8, whereas in kelp- and 1  1  GAA-fed abalone it remained steady at 1.6 and 0.4, respectively. Amino acid utilization among BAA-, OAA-, and KAA-fed abalone increased significantly after thefirst4 weeksfrom-4 % to 74 %, whereas GAA-fed abalone had a mean of 66 - 92 % throughout the study. Thus, amino acid balance of the formulated diets did not influence amino acid utilization by Haliotis kamtschatkana.  28  2.2  INTRODUCTION Some 200 amino acids occur naturally but only 20 are commonly found in biological proteins.  Up to 10 of these must be exogenously supplied to animals because they cannot be endogenously synthesized, either at all or in sufficient supply to meet metabolic demand. These essential amino acids do not differ substantially among animal phyla. For example, turkeys (Hurwitz et al, 1983), flatfish (Cowey etal, 1970), prawns (Cowey and Forester, 1971), lobsters (Mason and Castell, 1980), and abalone (Allen and Kilgore, 1975) all require arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine from an exogenous source. Although there is some indication that abalone also require taurine, this is not a protein-bound amino acid but rather part of the free amino acid pool in abalone tissues (Wilburn and Manahan, 1995). Free amino acids in marine gastropods are usually considered to function in osmoregulation (Campbell and Bishop, 1970; Matsushima, 1988). The relative quantities of essential amino acids in a diet are as important as their presence or absence since the balance of essential amino acids strongly influences the efficiency of protein synthesis. This is because the degree to which dietary essential amino acids are utilized in protein synthesis is limited by the essential amino acid present in the smallest amount relative to demand. Excess amino acids cannot be stored and instead are catabolized. This is a waste of protein and contributes to lower amino acid utilization. The essential amino acid composition of dietary proteins determines, in part, their nutritional value. For efficient amino acid utilization, it is therefore optimal that an animal be fed an amino acid balance similar to that found in the animal's own proteins. The concept that the amino acid balance within an animal's body will serve as a guideline for diet formulation is supported by studies in fish  29  (Wilson and Poe, 1985; Gatlin, 1987), shrimp (Farmanfarmaian and Lauterio, 1979), and prawn (Deshimaru and Shigeno, 1972). Alternatively, it has been suggested that the optimal amino acid balance for an animal is to be found in its egg proteins since these support the animal's rapid early growth (Ketola, 1982). In this regard, formulated diets for prawn broodstock have been based upon the essential amino acid profile of their eggs (Das et al, 1996). Although optimization of dietary amino acid balance is important to maximize growth and amino acid utilization, diet is not the only source of amino acids. In many animals amino acids are supplied to some degree by symbiotic micro-organisms. For example, invertebrates such as isopods (Carefoot, 1984), sea hares (Carefoot, 1988), and sea urchins (Fong and Mann, 1980) are known to benefit from microbial synthesis of essential amino acids within the gut. Lawrence (1975) suggested that amino acid synthesis by gut bacteria of urchins is what allows these animals to live on a wide diversity of algal tissues and, accordingly, a wide diversity of amino acid intake. Since abalone are similar to urchins in their dietary habits, gut microflora may similarly play a role in meeting their amino acid needs. If such symbionts contribute significantly to the amino acid uptake in abalone, these animals would not be dependent solely upon their diet to meet their requirements for essential amino acids. It should be noted that the determination of essential amino acids for abalone by Allen and Kilgore (1975) was made by injecting radioisotopes into juvenile abalone and identifying amino acids which were subsequently radio-labeled. Thus, the role of gut microflora in amino acid production was not assessed. Many soft-bodied marine invertebrates also obtain amino acids through their capacity to absorb these nutrients directlyfromseawater (Jorgensen, 1976; Stewart, 1979; Stephens, 1981), and there is some suggestion that such uptake plays a significant role in the nutrition of these animals (Puce  30  and Stephens, 1987 and 1988; Wright, 1988). The potential to obtain amino acidsfromnon-dietary sources may decrease the influence of dietary amino acid balance on amino acid utilization by abalone. The above considerations were each accounted for in this investigation. Diets were formulated with amino acid balances mimicking either the whole body or ovary of adult abalone, or the natural kelp food. These balances were achieved by mixing crystalline amino acids as done by Carefoot (1984) who grew isopods 75 % faster on a diet formulated with a mix of crystalline amino acids patterned after kelp, than on a diet of kelp itself Additionally, a diet with an amino acid content of glycine was included to determine whether microbial production of amino acids or uptake from seawater play a role in the nutrition of Haliotis kamtschatkana. Survival and/or growth on this diet would indicate that essential amino acids are provided by an exogenous source other than diet. To date, the gut bacteria of abalone have only been studied with respect to their contribution to digestive enzymes (Erasmus et al, 1994), not their synthesis of amino acids, and the nutritional role of amino acid uptake from seawater has not been investigated. Therefore, the diets formulated with amino acid balances patterned after abalone whole body and ovary were expected to optimize amino acid utilization.  2.3 M E T H O D S A N D M A T E R I A L S Collection and maintenance ofabalone Abalone were collected by SCUBA diversfromshallow subtidal areas near the Bamfield Marine Station on the west coast of Vancouver Island, British Columbia. The abalone were transported to the Department of Fisheries and Oceans, West Vancouver laboratory, where the study  31  was conducted. Seventy-five abalone were divided into five groups of 30 g mean individual live mass. Each group of 15 animals was fed ad libitum on one of four formulated diets that varied in amino acid content or on kelp Nereocystis leutkeana. Animals were housed in individual Whitlock-Vibert incubation boxes (obtained from the Federation of Fly Fishers, Yellowstone, Montana) andfloatedin a flow-through seawater system. Over the 28 weeks of the study (July 1993 - January 1994) water temperature rangedfrom7 - 17 °C and salinityfrom25 - 28 %o. Dietformulation andfeeding Amino acid compositions of lyophilized samples of abalone Haliotis kamtschatkana whole body and ovary, and of kelp Nereocystis leutkeana (determined by high precision liquid chromatography at the Department of Animal Science, Nova Scotia Agricultural College) are listed in Table 2.1 and were compared using Spearman Rank Correlation Coefficient Analysis. Based on these percentage values, a mixture of crystalline amino acids was prepared for each diet. These mixtures were encapsulated in Ca-alginate microspheres to inhibit leaching. To encapsulate the amino acids, a 2 % solution of Na-alginate was prepared by dissolving Na-alginate in boiling distilled water that had been adjusted to pH 12. The amino acid stock mixture and a gelatin binder were added so as to be in a 32:1 ratio with the Na-alginate. The amino acid levels in the stock mixtures were prepared such that when combined with thosefromgelatin, the resulting amino acid balance matched the tissues being mimicked. The Na-alginate - gelatin - amino acid mixture was sprayedfroman atomizer into a bath of a 20 % CaCk solution which caused precipitation of Ca-alginate microspheres containing the amino acids and gelatin. The microspheres were then strainedfromthe bath and incorporated into the formulated diets.  32  Table 2.1 Amino acid balances of whole body- and ovary-proteins of abalone Haliotis kamtschatkana, and of kelp Nereocystis leutkeana. Values are expressed as percentages of the total dry mass of amino acids and were calculated from the mean of three replicate HPLC analyses. Amino acid alanine arginine asparagine cystine glutamate glycine histidine isoleucine leucine lysine methionine phenylalanine proline serine threonine tryptophan tyrosine valine total  Abalone whole body 6.47 10.58 11.08 1.15 13.59 9.06 0.78 4.26 4.36 7.81 1.37 3.35 7.12 5.01 4.79 3.87 2.37 2.98 100  Abalone ovary 8.03 6.44 9.19 0.49 6.58 5.50 1.50 6.50 9.19 9.98 2.16 4.45 3.34 3.69 6.54 8.37 3.47 4.58 100  Nereocystis leutkeana 8.44 7.49 10.20 0.27 3.85 5.91 1.45 6.89 8.86 7.75 2.85 4.38 1.80 3.48 5.70 11.55 2.71 6.42 100  33  The formulated diets were prepared byfirstdissolving agar (3 % mass/volume) in boiling distilled water. This binder mix was allowed to cool below 50 °C to prevent melting of the Ca-alginate microspheres, before combining it with the other dietary components in the proportions shown in Table 2.2. Algin (another binder) was added with the microspheres to help set them in the diet. Algin was added in a 3:1 ratio with the agar so that, in total, binders comprised 4 % dry matter of the diet. The diet was mixed to homogeneity, left to cool and set, and stored at 4 °C until used. Known amounts of the formulated diets were provided to the abalone every two days, and uneaten food was collected and weighed at the next feeding. The loss of dietary nutrients through leaching was assessed by comparing the percent dry mass of food particles held in seawater for 48 h with that of freshly made diet. There was less than 4 % + 0.1 (n = 10) loss of dry matter. Although loss of food particles from the cages introduced some inaccuracy in the assessment of food intake, the data nonetheless provided a comparison of relative intake of each formulated diet. Due to its structural consistency, uneaten kelp was more likely to escape the cages and so records of kelp intake were not kept. Abalone were weighed every 5-6 weeks. Nitrogen excretion Nitrogen excretion (NEX) of each abalone was measured at 5 - 6-week intervals. For this measurement individual abalone were sealed in separate 250-ml plastic containers (taking care to exclude air bubbles) for a 3-h period between 0800h - 1300h. A sample of seawater was then drawn and analyzed for ammonia content using the method of Solorzano (1969). The total amount of ammonia excreted over the 3-h period was calculatedfromthe known volume of seawater in each container. Daily N E X was estimated from these data by multiplying by 8 (see Chapter 1). Since 90 %  34  Table 2.2 Composition (g' dry kg' ) of formulated diets used to test the effect of varying dietary amino acid balance on amino acid utilization by abalone Haliotis kamtschatkana. Amino acid balances mimicked either abalone whole body (BAA), abalone ovary (OAA), or natural kelp food Nereocystis leutkeana (KAA). The amino acid content of a fourth diet was comprised mainly of glycine (GAA). Diets were formulated to contain 16 % dry matter. All diets contained common components represented by 42 % Protein-Free Diet obtained from ICN Ltd. 1  Diet BAA 30 10 250 0 190 420 30 70 1000  Dietary component calcium alginate gelatin amino acid mix glycine agar common components menhaden oil algin 1  2  total  OAA 30 10 250 0 190 420 30 70 1000  KAA 30 10 250 0 190 420 30 70 1000  GAA 30 10 0 250 190 420 30 70 1000  1  Refer to Table 2.1 for composition of amino acid mix.  2  Common components (g' dry kg" of formulated diet) contributed by the Protein-Free Diet were: 281.8 sucrose, 63.0 corn starch, 33.2 cellulose, 21.0 corn oil, 16.8 mineral mix , and 4.2 vitamin mix . 1  3  3  3  Refer to Table 1.1 for compositions of mineral and vitamin mixes.  35  of waste nitrogenfromprosobranch gastropods is eliminated as ammonia (Cockcroft and McLachlan, 1990; Clarke et al., 1994), this was the only nitrogenous waste measured. Amino acid utilization Faeces from the formulated-diet groups were examined periodically throughout the 28-week period and were never observed to contain any intact Ca-alginate microspheres. This suggested that all the encapsulated amino acids consumed were digested and absorbed by the abalone. Thus, nitrogen absorbed was considered equal to nitrogen consumed, and amino acid utilization (AAU) was calculated as the percentage of dietary nitrogen retained in the body tissues of the abalone.  1  A A U = [(food eaten % dietary amino acid' % N in amino acid) - N E X ] (food eaten % dietary amino acid • % N in amino acid)  _1  100 %  Statistical analyses Datafromall measures of food intake, mass change, N E X , and A A U were analyzed using twofactor Repeated Measures A N O V A coupled with Student-Newman-Keuls Multiple Comparison Tests (SNK). The data were not corrected to body mass because the same animals were measured repeatedly and all diet groups had a similar mean mass. The amino acid contents of abalone whole body, abalone ovary, and the kelp Nereocystis leutkeana, as well as the diets based on these amino acid balances, were compared with the Spearman Rank Correlation Coefficient Analysis (SRCCA). Values presented in the text are mean + standard error.  1  In this calculation % dietary amino acid was 25 % dry mass, or 4 % wet mass, of the diets. For ease of calculation % N in amino acid was taken as 16 % which is a standard N content in typical proteins. The amino acid balances of the formulated diets actually ranged from 14 -18 % N.  36  2.4 R E S U L T S The correlations of amino acid balances in BAA, OAA, and K A A are shown in Table 2.3. G A A was not included in these comparisons since its difference from all other balances was obvious. Regardless of whether total amino acids or essential amino acids were compared, the BAA, OAA, and K A A diets were all significantly correlated to one another. Only when the total amino acids of B A A and K A A were compared was there a significant difference in amino acid balance. Thus, it is apparent that the amino acid balances in the four test diets were not substantially different from one another. The formulated diets were not eaten at significantly different rates (F3,i96 = 1.8;/? = 0.17). Food intake was significantly different over time (F ,i95 = 455.2; p < 0.001; Figure 2.1), being greatest 4  in September (0.16 ± 0.03 g' d"), and lower in January (0.14 ± 0.05), November (0.09 ± 0.02), 1  October (0.07 ± 0.02), and August (0.07 ± 0.02), with the last three measures not being different from one another (p < 0.05, SNK). Despite similar levels of food intake by the abalone fed the four formulated diets, mass change varied significantly over the 28-week study (Figure 2.2). Significant differences occurred among the five diet groups (F^s = 4.9;p = 0.002); however, there was overlap in the data. The only consistency was that kelp-fed abalone (mean mass change of 0.65 + 0.16 g) gained significantly more mass than both BAA-fed abalone (0.14 ± 0.18) and KAA-fed abalone (-0.10 ± 0.27; p < 0.05, SNK). Similarly, significant differences occurred over time (F 4 = 3.5;/? = 0.009), but again with overlapped subsets (p 4;2  5  < 0.05, SNK). Here, mass change in January (mean for all diet groups; 1.64 + 0.08 g) was significantly greater than that in September (-0.47 ± 0.22) or in October (-0.21 ± 0.24). It is also noteworthy that abalone fed the G A A diet gained mass over the experimental period.  37  Table 2.3 Correlations of amino acid balances in diets formulated to match the amino acid compositions of abalone Haliotis kamtschatkana whole body (BAA), abalone ovary (OAA), or kelp Nereocystis leutkeana (KAA), tested with Spearman Rank Correlation Coefficient Analysis.  Correlated parameters Essential amino acids  Total amino acids  P  P  Diets compared  r  B A A and K A A  0.4  0.07  0.6  0.04  B A A and O A A  0.6  0.008  0.7  0.01  K A A and O A A  0.9  < 0.001  0.9  < 0.001  s  r  s  38  Figure 2.1 Food intake by abalone Haliotis kamtschatkana fed formulated diets with varying amino acid balances, over a 28-week period from August - January. Each point represents mean food intake (g d' ) ± S.E. for N = 15 abalone each of 30 g live mass. 1  TIME (weeks)  39  Figure 2.2 Live mass of abalone Haliotis kamtschatkana fed kelp or formulated diets with varying amino acid balances, over a 28-week period from August - January. Each point represents mean live mass (g) + S.E. for N = 15 abalone each of 30 g starting live mass.  MONTHS AUG  4  SEP  8  OCT  12  NOV  16  TIME (weeks)  DEC  20  JAN  24  o  Kelp  •  KAA  T  BAA  •  OAA  •  GAA  40  As shown in Figure 2.3, kelp-fed abalone also differedfromthe other diet groups in nitrogen excretion (NEX).  NEX  differed significantly among the 5 diet groups irrespective of time (¥4,745 = 8.7;/?  < 0.001) but again, there was overlap among homogenous subsets (p < 0.05, SNK). N E X of kelp-fed abalone was significantly higher than that for any other diet group (1.6 + 0.1 mg N ' d" ' individual" ). 1  1  The next highest N E X , for BAA-fed abalone (1.1 + 0.2 mg N d" individual" ) was significantly 1  1  higher than the lowest N E X (0.4 + 0.03) exhibited by GAA-fed abalone. A time effect was also evident (F4,245 = 3.2; p < 0.01), with August N E X (mean for all diets; 1.3 + 0.1 mg N ' d' individual" ) being 1  1  significantly greater than all other months (0.8 ± 0.2). Since all formulated diets were ingested to a similar extent (Figure 2.1), the trend for amino acid utilization (AAU) corresponded closely to that of N E X (Figure 2.4). Time significantly influenced A A U (F ,i95 = 22.3;p < 0.001). A A U in August (mean for all diets; 13.8 ± 20.2 %) was significantly 4  lower than all other months with the exception of October (60.4 ± 1 5 . 1 ) . Diet also had a significant effect on A A U (F ,  3 m  = 3.6;/? = 0.02). A A U was lowest for the BAA-fed and KAA-fed abalone  (mean of 51 %) and highest for OAA- and GAA-fed abalone (77 % ;p < 0.05, SNK).  2.5  DISCUSSION The results of this study suggested that dietary amino acid balance did not play a major  role in amino acid utilization by Haliotis kamtschatkana. Amino acid utilization had the following trend among the diet groups: G A A > O A A > K A A > B A A , but this trend was weak due to statistical overlap. However, since the amino acid balances in all the formulated diets were correlated to some degree, this overlap was to be expected. Food intake and mass change did not  41  Figure 2.3 Nitrogen excretion (N ) by abalone Haliotis kamtschatkana fed formulated diets with varying amino acid balances, over a 28-week period from August - January. Each point represents mean excretion (mg N ' d' ) ± S.E. for N = 15 abalone each of 30 g live mass. EX  1  MONTHS AUG  SEP  OCT  NOV  DEC  JAN  2.5  2.0  O a H  1.5 H  u  X  1.0  o  0.5  0.0 8  12  16  TIME (weeks)  20  24  o  Kelp  •  KAA  •  BAA  •  OAA  •  GAA  42  Figure 2.4 Amino acid utilization (AAU) by abalone Haliotis kamtschatkana fed formulated diets with varying amino acid balances, over a 28-week period from August - January. Each point represents mean A A U [(N absorbed - N excreted)(N absorbed)' (100 %)] ± S.E. for N = 15 abalone each of 30 g live mass. 1  MONTHS AUG  SEP  OCT  NOV  8  12  16  DEC  JAN  100 J 0  s  z o H  50  A  H Q 0H  o z -50 H  TIME (weeks)  20  24  43  resolve the ambiguity in identifying the optimal dietary amino acid balance. All formulated diets were consumed at equal daily rates and mass change overlapped extensively among the five diet groups. Kelp-, G A A - , and OAA-fed abalone gained most, while B A A - and KAA-fed abalone gained least. The similar mass change in G A A - , OAA-, and kelp-fed abalone suggests that dietary amino acid balance is not critical to amino acid utilization by H. kamtschatkana. This is supported by Stahl and Ahearn (1978) who reported similarfindingsregarding the specific amino acid requirements of the giant prawn Macrobrachium rosenbergii. However, such findings conflict with Farmanfarmaian and Lauterio (1979) who reported giant prawns to be dependent upon dietary amino acid balance, as well as with findings for other cultured aquatic animals (several teleosts: Ketola, 1982; and carp: Ravi and Devaraji, 1991). Since the abalone studied here did grow on the G A A diet (glycine replacing the protein source), they could not be dependent solely upon dietary amino acids to support protein growth^ and must have obtained amino acids (especially essential amino acids which cannot be synthesized by their tissues) from alternative sources such as gut bacteria and/or seawater. Thus, when formulating diets for abalone, amino acid balance may sometimes be a minor concern. However, the consistency with which these alternative sources provide amino acids must be determined before they can be relied upon to play a significant role in abalone nutrition. Overall amino acid utilization in abalone fed G A A , OAA, and K A A (60 - 80 %) were each higher than that of the B A A group (42 %), and the differences were significant for G A A - and OAA-fed abalone. These amino acid utilization values can be compared to measures of net protein utilization in another study on abalone (Uki et al, 1986a). Net protein utilization is  44  calculated as the ratio of protein gained by the test animal plus protein lost by an animal fed a non-protein diet, to protein intake; thus it is a measure of the proportion of dietary protein retained in the body. Uki et al. (1986a) reported net protein utilization of 70 % for Haliotis discus hannai fed diets with 20 % dry mass casein and net protein utilization of 37 % when fed diets with 22 % dry mass protein from fish meal (for further comparison with measures of protein retention in cultured aquatic animals see Chapter 1.). Through measures of nitrogen intake and excretion this investigation assessed the proportion of dietary amino acids retained in body tissues. Amino acids that were not incorporated into body tissues, for the most part, must have been catabolized and their amino groups excreted. Excess amino acids cannot be stored, although the level of free amino acids in abalone tissues can fluctuate to some degree (Watanabe et al., 1993; Mai et al, 1994), presumably depending upon relative levels of protein anabolism and catabolism. The results of my study indicated that 58 % of absorbed dietary nitrogen was excreted by the B A A group and 40 % by the K A A group. Nitrogen is excreted as animals rid themselves of toxic ammonia which is created when amino acids are catabolized. Amino acid catabolism occurs when the balance of dietary amino acids does not match body requirements or when the dietary energy supply is insufficient to fuel protein synthesis. The four formulated diets all contained an equivalent energy supply; therefore, the differences in amino acid utilization noted for abalone fed these diets were the result of differences in their amino acid balances. Amino acid utilization was significantly greater when glycine comprised the majority of the amino acid content, or when the dietary amino acid balance mimicked either abalone ovary or kelp, rather than abalone body. The inferior  45  utilization of amino acids of the B A A diet relative to the G A A diet may reflect extreme sensitivity to certain amino acids being supplied in surplus or deficiency; however, the amino acids which elicited this response could not be identified here. Inferior amino acid utilization in abalone fed B A A indicates the potential pitfall of using the amino acid content of an animal's whole body as an indication of amino acid requirement. As suggested by Fleming et al. (1996), a sample of the whole body provides a static ratio of amino acids which does not account for amino acids which are in high demand because the proteins which contain them have a high rate of turnover. The optimal amino acid balance is one which meets the needs of protein synthesis, which is a dynamic process not well-represented by a static measure of amino acid content. Cumulative mass change over the 28-week period for each of the formulated diet groups did not vary in the same manner as amino acid utilization. Mean mass change over the sampling intervals for GAA-, OAA-, and BAA-fed abalone averaged 0.4 g over the 28-week study. G A A and OAA-fed abalone were not significantly different than kelp-fed abalone which increased by 0.6 g, and BAA-fed abalone were not significantly different than KAA-fed abalone which lost 0.10 g over the study period. The statistical overlap in these data belies any suggestion that differences in amino acid balance had a significant impact on protein metabolism. The mass changes of the diet groups in this investigation ranged from a 2.2 % increase to a 0.3 % decrease in mean body mass. As noted in Chapter 1, such mass changes are about two orders of magnitude lower than growth rates reported for field and laboratory populations of Haliotis kamtschatkana fed natural diets. Similar growth among the kelp-, O A A - , and GAA-fed  abalone indicates that the formulated diets were not the cause of slow growth. Instead, the slow growth of abalone in this study suggests either that culture conditions were inferior or that the time-span of this study did not include a somatic growth phase. Reports on the timing of growth in abalone (Cox, 1962; Forster, 1967; Poore, 1972; Sainsbury, 1982; Keesing and Wells, 1989) make the latter unlikely. Suboptimal culture conditions are a more likely explanation for the slow growth in this investigation. In particular, the seawater salinity at the study site (25 - 28 %o) was less than that of the abalone's natural habitat (32 %o) and this may have adversely affected growth. While mass change varied among the diet groups, food intake exhibited identical fluctuations over time regardless of diet (Figure 2.1). Thus, despite differences in amino acid balance, the diets were equally palatable to abalone. Overall mean daily food intake (0.6 + 0.03 g d" ) 1  represented a consumption of approximately 2 % body mass per day which is within the range reported for intake of formulated diets by abalone of similar size but varying species (Fleming et al., 1996). Food intake fluctuated over time in a manner similar to my previous study (Chapter 1), and the similarities in fluctuation suggest a seasonal effect on food intake. Such a seasonal effect has been reported for Haliotis iris (Marsden and Williams, 1996) and may be related to the abalone's reproductive cycle and its associated metabolic fluctuations. Nitrogen excretion by Haliotis kamtschatkana did not exhibit seasonalfluctuationssimilar to those of food intake. Nitrogen excretion by abalone fed the formulated diets was greatest in August and then declined to a significantly lower level for the remainder of the 28-week period. In contrast, kelp-fed abalone exhibited highest nitrogen excretion at the final sampling in January. The total range of nitrogen excretion in this investigation (0.2 - 1.9 mg N ' d" individual' ) was 1  1  47  comparable to that of H. kamtschatkana fed a diet containing 20 % dry mass casein (0.5 - 4.1 mg N" d" ' individual" ; see Chapter 1). Overall, kelp-fed abalone had the highest rate of nitrogen 1  1  excretion and the highest mass gain whereas, among abalone feeding on the formulated diets, the G A A group had the highest mass gain coupled with the lowest nitrogen excretion. Thus, the lower amino acid content of the kelp diet as compared with the formulated diets (15 % dry mass versus 25 % dry mass), actually generated a higher protein catabolism. Despite this, the mass gain of abalone fed kelp was higher than those fed the formulated diets. This suggests that the formulated diets were nutritionally inferior to the kelp diet. Dietary amino acid balance is not the basis for this inferiority; rather, I suspect that deficiencies in essential fatty acids were most to blame, since the requirements for these nutrients are least understood for abalone, and I had no way to ensure that my diets met the requirements for H. kamtschatkana. Catabolism of amino acids by kelp-fed abalone was four times that of the formulated diet-fed groups, and anabolism may have been 1.1 - 1.3 times higher. My investigation indicated that abalone fed diets with amino acid balances that mimicked those of abalone whole body, abalone ovary, or kelp, or a diet in which glycine comprised the majority of amino acid content retained similar amounts of amino acids. Therefore, I have concluded that dietary amino acid balance was not a determining factor in amino acid utilization in this study. Abalone fed the formulated diets, however, lost differing amounts of non-protein mass. Overall, the different amino acid balances appeared to influence diet performance in the order of G A A > O A A > K A A > B A A .  48 C H A P T E R 3: E F F E C T O F P R O T E I N SPARING O N P R O T E I N UTILIZATION B Y A B A L O N E Haliotis kamtschatkana 3.1 A B S T R A C T Protein utilization is of concern in aquaculture due to this nutrient's cost and profound influence on growth. Protein sparing, the fulfilling of energy requirements by non-protein nutrients thereby sparing protein from catabolism and saving it for anabolism, was investigated using formulated diets for abalone Haliotis kamtschatkana. Of specific interest was a possible difference in the ability of abalone to utilize energy from fats versus carbohydrates, and whether this difference could be ameliorated through enzymatic adaptation to diet. Four groups of 20 abalone of 16 g mean individual live mass were individually caged in a common aquarium tank and fed ad libitum on kelp or one of three formulated diets that varied in fat carbohydrate ratio (referred to as F<CHO, F=CHO, and F>CHO, respectively). Food intake, mass, nitrogen excretion, and protein utilization were measured periodically over 44 weeks. At 0, 10, and 44 weeks, the digestive systems of five abalone from each diet group were assayed for activity of lipase, alginate lyase, and amylase. Carbohydrates had a greater protein-sparing capacity than fats because protein utilization was significantly higher in abalone fed F<CHO than in abalone fed F>CHO. F<CHO-fed abalone also had a significantly greater food intake and mass gain than F>CHO-fed abalone, but nitrogen excretion and activities of digestive enzymes were similar. Thus, carbohydrates had a greater protein-sparing effect than fats. The superior protein-sparing effect of carbohydrate was not ameliorated by enzymatic adaptation to the formulated diets, despite an increase in the activities of digestive enzymes seen in abalone fed the formulated diets.  49 3.2 I N T R O D U C T I O N In addition to dietary protein content and amino acid balance, protein utilization is also influenced by the efficiency with which carbohydrates and lipids fulfill the role of energy substrates. This is referred to as protein sparing, where carbon chains of carbohydrates and lipids are catabolized to produce sufficient energy to meet metabolic needs, thus sparing dietary proteinfromcatabolism so that it may be used solely for tissue maintenance and growth (Longenecker, 1968). Determination of an optimal dietary formulation with respect to protein:carbohydrate:fat ratio for maximal protein sparing would facilitate the production of diets for abalone culture which promote more efficient utilization of protein. Thus, the influence of dietary content of carbohydrate and fat on protein utilization was the focus of this part of my thesis. The effect of increasing either dietary fat or carbohydrate levels has been studied in other aquatic animals and the protein-sparing effects appear to vary with species. Most studies have focused on growth rather than protein utilization and these two processes may be inversely related (Capuzzo and Lancaster, 1979; Metailler etal., 1980; Hewitt, 1992; Chapter 1). Addition of fat to isonitrogenous (equal protein) diets was shown to promote growth in rainbow trout (Atherton and Aitken, 1970) and brown trout (Phillips et al, 1964). This growth was measured as mass gain and was not the result of increased fat storage. The superior protein-sparing effect of fat in rainbow trout is also known from studies of Lee and Putnam (1973), Steffans and Albrecht (1973), and Medland and Beamish (1985), while Adron et al. (1976) reported that in turbot the protein-sparing effect of fat was superior to that of carbohydrate. Similarly, prawns (Shudo et al, 1971; Kanazawa et al., 1979a and b) and juvenile lobsters (D'Ambro et al, 1980) exhibit improved growth when fed isonitrogenous diets with higher fat content. In contrast, Andrews et al. (1972) observed that addition of carbohydrate afforded more growth in penaeid shrimp than addition of fat and, furthermore, that starch enhanced growth more than did glucose. Andrews (1971) had previously postulated that starch was superior to  50 glucose in its protein-sparing effect in shrimps because glucose would be rapidly absorbed and catabolized and, therefore, inefficiently utilized. Starch mustfirstbe digested into its glucose constituents which may then be absorbed. Due to the time required for digestion, glucosefromstarch is taken up slowly and so it is more efficiently utilized as an energy source. The influence of carbohydrate source on protein sparing has also been demonstrated in prawns (Gomez Diaz and Nakagawa, 1990). In contrast to these studies, other investigators have observed that carbohydrates and fats do not differ in their protein-sparing effects. For example, Page and Andrews (1973) showed that fat and corn starch were equally effective as energy sources for channel catfish, and similar effects were shown for carp (Ogino etal, 1976) and yellowtail (Takeda etal, 1975). Unfortunately, most studies on protein-sparing effects have not employed isocaloric diets, or have not measured nitrogen retention so it is often impossible to determine whether the reported protein-sparing effects were unique to the inclusion of fats or if addition of carbohydrates could have achieved the same result. Protein sparing is thus an ambiguous issue. Both the existence and nature of protein-sparing effects appear to be subject to considerable interspecific variation. Differential protein sparing by fats and carbohydrates may result from differential influences of these dietary nutrients on enzyme activity. Plasticity in the production of digestive enzymes may permit enzymatic adaptation to diet (i.e., increased activity of specific digestive enzymes in response to changes in diet composition). Such adaptation may enhance the protein-sparing effects of nutrients which are provided in abundance. Enzymatic adaptation has been recorded for a variety of aquatic animals, includingfish,crustaceans, and abalone. These may be natural changes that are related to an animal's age (e.g., rainbow trout: Beamish and Thomas, 1984; lobsters: Biesot and Capuzzo, 1990; shrimp: Lovett and Felder, 1990a and b; Fang and Lee, 1992; prawn: Kamarudin etal, 1994; abalone Haliotis rufescens: Spaulding and Morse, 1991) or inducible responses due to dietary manipulations. With respect to the latter, De la Higuera et al. (1977) reported that increased lipase activity  51 improved protein utilization by rainbow trout fed on high-fat diets. Fair et al. (1980) also reported increased cellulase activity in prawns fed highfibrediets, and Van Wormhoudt et al. (1980) showed that protease activity peaked in prawns fed a medium content of dietary protein. In shrimp (Maugle et al, 1982; Lee et al, 1984) and the African giant snail (Van Weel, 1959) protease and amylase activity were observed to change with source rather than concentration of dietary protein or starch, respectively. Abalone have shown increased protease activity in response to high dietary protein content (Knauer et al, 1996). If abalone can enzymatically adapt to diet, then variation in proteinsparing effects among fats and carbohydrates may be adjustable over time and, therefore, may not be of critical concern to nutritionists when formulating diets. This investigation assessed the existence and longevity of protein-sparing effects as well as the potential for enzymatic adaptation in response to dietary change in Haliotis kamtschatkana. Formulated diets with similar energy and protein contents but varying in their carbohydrate:fat ratio, and a natural diet of kelp, were fed to abalone. Food intake, mass change, and protein utilization were assessed in each animal. The activities of alginate lyase, amylase, and lipase in abalone fed the four diets were also measured over the experimental period. My expectation was that mass gain and protein utilization would be greater in abalone fed more carbohydrate than in those fed more fat, since the nutrition of abalone is carbohydrate-based (Webber, 1970; Hayashi, 1983) and lipase activity appears to be low (Knauer et al, 1996). Also, relative to initial enzyme activities, abalone fed more fat were expected to exhibit an increased activity of lipase over time, and those fed more carbohydrate were expected to show an increased activity of amylase.  3.3 M E T H O D S A N D M A T E R I A L S Collection and maintenance of abalone Abalone were collected by SCUBA divers from shallow subtidal areas near the Bamfield  52 Marine Station on the west coast of Vancouver Island, British Columbia. One hundred abalone were divided into four groups of 16 g mean individual live mass. Twenty abalone (fivefromeach group) were immediately removed for the initial enzyme assays. The remaining 80 animals were housed in individual Whitlock-Vibert incubation boxes (obtained from the Federation of Fly Fishers, Yellowstone, Montana) which were floated in a flow-through seawater system at the Department of Fisheries and Oceans, West Vancouver laboratory. Over the 44-week duration of the study, water temperature rangedfrom7 -17 °C and salinityfrom25 - 28 %>. The seawater tank was enclosed in black plastic to prevent algal growth and to enhance food intake since Haliotis kamtschatkana are nocturnal feeders (pers. observ.). Each group of 20 animals was fed ad libitum on one of three formulated diets which varied in carbohydrate:fat ratio, or on kelp Nereocystis leutkeana. Of these 20 abalone, 10 were kept throughout the 44-week study for measurements of food intake, mass change, nitrogen excretion, and protein utilization. The remaining 10 in each group were designated for enzyme assays (see later). The study ranfromNovember 1994 - October 1995. Dietformulation andfeeding Diets were formulated to vary in carbohydrate:fat ratio, but to be similar in terms of protein and energy content. Table 3.1 provides the gross nutrient composition and energy content of kelp and each formulated diet: fat < carbohydrate (F<CHO), fat = carbohydrate (F=CHO), fat > carbohydrate (F>CHO). The formulated diets contained 15 % dry matter which was approximately double the dry mass content of kelp. The specific nutrient composition of these diets is shown in Table 3.2. To prepare the diets, agar was added to boiling distilled water in a 4 % mass/volume ratio and mixed with a hand-held blender until homogenous. Before the addition of casein, common components (Menhaden Diet obtainedfromICN Ltd.), and corn and menhaden oil, the mixture was cooled to below 70 °C to prevent denaturing of casein which would have been nutritionally detrimental (Uki and  53 Table 3.1 Gross composition and energy content of four diets for abalone Haliotis kamtschatkana: kelp Nereocystis leutkeana, and three formulated diets with fat either less than (F<CHO), equal to (F=CHO), or greater than carbohydrate (F>CHO). Values presented are contents per dry kg of diet. Nutrient levels in the formulated diets were calculated from the nutrient levels of the components used in formulation, and for kelp from the chemical composition estimatedfromWort (1955). Energy levels were estimated based on standard energy equivalents: 0.0396 MJ kg" for lipid, 0.0172 M J kg" for carbohydrate, and 0.0237 M J kg" for protein. 1  1  1  Kelp  F<CHO  Component  gkg"  MJ kg"  lipid  30  1.19  carbohydrate  350  protein  F>CHO  F=CHO MJ kg"  g kg"  MJ kg"  110  4.35  180  7.12  6.01  260  4.46  180  3.09  220  5.20  210  5.00  220  5.20  330  5.66  390  6.69  390  6.69  MJ kg"  g kg  70  2.77  6.01  350  150  3.55  fibre  30  0.51  ash  430  15  15  15  vitamin  10  10  10  10  5  5  5  1  1  antioxidant total  1000  11.26  g  V  1000  1  19.64  1000  1  1  20.50  1  1000  1  22.10  54 Table 3.2 Specific composition (g' dry kg' ) of diets formulated for abalone Haliotis kamtschatkana, to test the effect of varying carbohydrate:fat ratio on protein utilization. The diets were formulated to contain 15 % dry matter. All formulations included common components represented by either 36 % or 53 % Menhaden Diet obtainedfromICN Inc., the composition of which is as defined below. 1  Dietary component agar cellulose casein corn starch menhaden oil corn oil common components total  1  1  Fat < CHO 270 50 150 170 0 0 360 1000  3  Fat > CHO 270 110 150 0 90 20 360 1000  Common components comprised Menhaden Diet made up of (g ' kg' ): 345.4 corn starch, 200.0 casein, 170.0 menhaden oil, 150.0 sucrose, 50.0 cellulose, 40.0 mineral mix , 30.0 corn oil, 10.0 vitamin mix , 3.0 D L methionine, 1.2 D L - a tocopherol, 0.2 BHT(butylated hydroxytoluene), and 0.2 B Q T (butylated hydroxyquinone). 1  2  2  Fat = CHO 270 100 100 0 0 0 530 1000  2  Composition of mineral mix (g kg" Menhaden Diet): 20.0000 calcium phosphate dibasic, 2.9600 sodium chloride, 8.8000 potassium citrate monohydrate, 2.0800 potassium sulfate, 0.9600 magnesium oxide, 0.1400 manganous carbonate, 0.2400 ferric citrate (16 -17 % Fe), 0.0640 zinc carbonate, 0.0120 cupric carbonate, 0.0004 potassium iodate, 0.0004 sodium selenite, 0.0220 chromium potassium sulfate, 4.7200 sucrose. 1  Composition of vitamin mix (mg ' kg" Menhaden Diet): 0.006 thiamine hydrochloride, 0.006 riboflavin, 0.007 pyridoxine hydrochloride, 0.030 nicotinic acid, 0.016 D calcium pantothenate, 0.020 folic acid, 0.002 D-biotin, 1 X 10' cyanocobalamin, 0.016 retinyl palmitate, 10.200 D L - a - tocopherol acetate, 0.025 cholecalciferol, 5 X 10' menaquinone, 9.730 sucrose. 1  5  5  55 Watanabe, 1986). After addition of all nutrients the mixture was blended to an homogenous consistency. The diets were left to cool and set, and stored at 4 °C until used. Kelp and known masses of formulated diet were provided every 2 d and were always provided the day prior to measuring nitrogen excretion. Uneaten food was collected and weighed at the next feeding. The loss of dietary nutrients through leaching was assessed by comparing the percent dry mass of food particles held in seawater for 48 h with that of freshly made diet. There was less than 5 % + 0.1 (n = 10) loss of dry matter over this time. Although loss of food particles from the cages introduced some inaccuracy in the assessment of food intake, the data nonetheless provided a comparison of relative intake of each formulated diet. Due to its structural consistency, uneaten kelp was more likely to escape the cages and so records of its intake were not kept. In November, when the kelp began its seasonal die-off, a large collection was made and dried as a winter supply for the kelp-fed animals. Digestibility The digestibility of each diet was assessed in order to determine the amount of protein absorbed (see Chapter 1 for method). Protein absorption was calculated by combining the value for protein digestibility (determined for these diets to be 0.61 which is comparable to previously obtained values for this and other abalone species, see Chapter 1) with values for food intake. Mass change Abalone were weighed every 4 - 5 weeks to determine mass change throughout the 44-week period. The study period included the spawning time for these abalone; thus, to minimize the variability in mass change caused by randomly occurring spawning events, I synchronized spawning among the experimental groups. In mid-July, as soon as some animals began to spawn on their own, I induced spawning among all experimental groups using the hydrogen peroxide technique of Morse et al. (1977).  56 Nitrogen excretion Nitrogen excretion (NEX) of each abalone was measured every 4 - 5 weeks by sealing the abalone in a 250-ml plastic container (taking care to exclude air bubbles) for a 3-h period between 0800 - 1300 h. A sample of seawater was then drawn and analyzed for ammonia content using the method of Solorzano (1969). The total amount of ammonia excreted over the 3-h period was calculatedfromthe known volume of seawater in the container. Since 90 % of waste nitrogen from prosobranch gastropods is eliminated in this form (Cockcroft and McLachlan, 1990; Clark et al, 1994), ammonia was the only nitrogenous waste measured. Protein utilization Protein utilization (PU) was assessed as the percentage of absorbed dietary nitrogen retained by the body tissues of the abalone. P U = (absorbed dietary nitrogen - NEx)(absorbed nitrogen)' (100 %) 1  Absorbed dietary nitrogen was calculated from the amount of casein consumed by correcting for protein digestibility (determined here to be 0.65) and multiplying by 0.16 which is the proportion of nitrogen in casein . 1  Enzyme assays Activities of the digestive enzymes amylase, alginate lyase, and lipase were assayed at weeks 0, 10, and 44. At each of the three assay times, the entire digestive tract was dissected out of five abalone from each group and immediatelyfrozenin liquid nitrogen. Each digestive tract was weighed, placed in a flat-bottomed glass tube, and covered with nine volumes of homogenization buffer.  1  Note: Kjeldahl analyses indicate that casein contains 96.7 % protein (D. Higgs,pers. comm.) and 15.8 % N (Merck Index) but for these calculations I considered casein to be 100 % protein and 16 % N .  57 The homogenization buffer (pH 7.5) contained 20 mMNa JV-2-hydroxyethylpiperazine-A~'-2ethanesulfonic acid (HEPES), 2 mM ethylenediaminetetraacetic (EDTA), 10 mM dithiothreitol, 200 |J.M phenylmethyl-sulfonyl fluoride (PMSF) in an ethanol carrier, 200 u,M aprotinin, and 0.1% v/v Triton X-100. The immersed tissue was first minced with fine scissors, then homogenized with a polytron tissue grinder at high speed 3 times for 10 sec with 10 sec rests between grindings, andfinallysonicated 3 times for 10 sec with 10 sec rests between sonications. The homogenate was transferred to a centrifuge tube and spun at 10,000 g for 3 min. The supernatant contained the enzyme extract used for the assays. Lipase and amylase activities were assayed using diagnostic kits from Sigma Co. Alginate lyase activity was measured by monitoring the increase in absorbance at 235 nm and 15 °C of a 2-ml reaction mixture containing 0.25 % m/v sodium alginate derived from Macrocystispyrifera (Sigma Co.), 10 mM Tris-MES buffer (pH 7.5), 100 mM NaCl, 20 mM M g CI2, and 10 ill enzyme homogenate (method of Nakada and Sweeney, 1967). Enzyme activities were defined as activity units per ml of enzyme extract per min (units' ml* ' min ) where activity units referred to the breakdown of substrate moieties (4,61  -1  ethylidene (G7)-p-nitrophenyl (Gi)-a,D-maltoheptaside, 1,2-diglyceride, or alginate, for each of amylase, lipase and alginate lyase, respectively). Statistical analyses The data for all measures of food intake, mass change, N E X , PU, and enzyme activity were analyzed using two-factor Repeated Measures A N O V A or A N O V A where appropriate, coupled with Student-Newman-Keuls Multiple Comparison Tests (SNK). The food intake, mass change, N E X , and P U data were not corrected to body mass because the same animals were measured repeatedly and all diet groups had a similar mean mass. Values presented in the text are means ± standard error.  58 3.4 R E S U L T S Figure 3.1 illustrates food intake of abalone fed the formulated diets (F<CHO, F=CHO, and F>CHO). Regardless of time, the mean daily food intake of the F>CHO-fed abalone (0.13 ± 0.01 g) was significantly less than that of the other two diet groups which themselves were equivalent (0.17 + 0.01;  F2327  = 6.3; p = 0.009). There was also a significant effect of time on food intake (F10319 = 31.0;  p < 0.001), with the lowest intake occurring at the beginning of the experiment in November and December (0.07 ± 0.01 g and 0.09 + 0.01, respectively), increasing steadily to a peak intake of 0.21 + 0.02 by week 18 in March, andfinallydecreasing slightly, but not significantly, over the remaining time (p>0.05, SNK). Mass changes for abalone fed the formulated diets and the kelp diet are shown in Figure 3.2. There was a significant difference in mass change exhibited by the formulated-diet groups (¥2^27 = 6.2; p = 0.009), with the F<CHO diet producing a consistently greater mass gain than the F=CHO diet and the F>CHO diet, (p < 0.05, SNK). The overall mean mass gain was 5.8 ± 1.1 g for kelp-fed abalone, 0.2 ± 0.1 for F<CHO-fed abalone, and 0.01 + 0.1 for F=CHO-fed and F>CHO-fed abalone. Time, when considered alone, also caused a significant mass change (F ,3i9 = 6.8; p < 0.001). Abalone fed 10  formulated diets exhibited significant mass loss (-1.6 + 0.2 g) at week 39 after being spawned in July; however, kelp-fed abalone did not. This mass change for abalone fed formulated diets was significantly different from that at all other time periods (p < 0.05, SNK). The greatest mass gain among formulated diet-fed abalone was observed at week 44 (0.7 ± 0.3 g), but this was mainly due to growth in the F<CHO group. In contrast, kelp-fed abalone showed a significant decrease in mass at this time (p < 0.05, SNK). Nitrogen excretion (NEX) among groups fed the formulated diets was not influenced by diet (F2327  = 0.2;p = 0.8). As indicated in Figure 3.3,  NEX  for all diet groups was in the range 0.8 - 2.9 mg  N ' d" • individual" . Thus, even though the F>CHO diet was consumed in lesser amounts and 1  1  59 Figure 3.1 Food intake by abalone Haliotis kamtschatkana fed formulated diets with fat content less than (F<CHO), equal to (F=CHO), or greater than carbohydrate content (F>CHO), over a 44-week period from November to October. Each point represents mean intake (g' d*) ± S.E. for N = 10 abalone each of 16 g live mass. 1  MONTHS N  D  J  F  M  A  M  J  J  A  S  O  N F<CHO F=CHO F>CHO  0  10  20  30  TIME (weeks)  40  60 Figure 3.2 Live mass of abalone Haliotis kamtschatkana fed kelp or formulated diets with fat content less than (F<CHO), equal to (F=CHO), or greater than carbohydrate content (F>CHO), over a 44week period form November to October. Each point represents mean live mass (g) + S.E. for N = 10 abalone each of 16 g starting live mass.  MONTHS N  D  J  F  i—i—r  M  A  M  I  J  i  J  i  i  A  S  O  N  i r  i  22 A  /  \  1  20 C/5  >  it  18  \  I  / 16  14  _  0  i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—  10  20 30 TIME (weeks)  40  o •  Kelp F<CHO  • *  F=CHO F>CHO  61 Figure 3.3 Nitrogen excretion (NEX) by abalone Haliotis kamtschatkana fed kelp or formulated diets with fat content less than (F<CHO), equal to (F=CHO), or greater than carbohydrate content (F>CHO), over a 44-week periodfromNovember to October. Each point represents mean excretion (mg N ' d' ) ± S.E. for N = 10 abalone each of 16 g live mass. 1  MONTHS N  D  J  F  M  A  M  J  J  A  S  O  N  1  o  Kelp F<CHO F=CHO F>CHO  10  20  30  TIME (weeks)  40  62 supported less mass gain, N E X by abalone fed this diet was similar to levels observed in the other diet groups. N E X among kelp-fed abalone was also found to be similar to the other treatment groups (Fu,m = 2.0; p = 0.1). Although intake of the kelp diet was not monitored, it did yield the greatest mass gain among the diet groups. A significant influence of time on N E X was observed for all abalone regardless of diet (F i56 = 13.3;/? < 0.001), with a steady and significant increase from 1.0 + 0.1 mg N ' d'  1  3j  individual' in November to 1.8 ± 0.2 the following October (p < 0.05, SNK). The initial phase of this 1  increase (weeks 2-18) paralleled the increased food intake over the same period in abalone fed the formulated diets. Protein utilization (PU) was measured as the percentage of absorbed dietary nitrogen retained in body tissues. It was calculated for the times at which N E X was measured, but only for the three groups fed formulated diets (Figure 3.4). These calculations incorporated the results of digestibility trials which placed casein digestibility at 0.65 ± 0.08 for all formulated diets (F^n = 1-22;p = 0.33); therefore, protein absorption was 65 % of protein intake. PU in abalone fed formulated diets differed significantly (F zi = 4.7;p= 0.02), with the overall mean PU of 62 ± 2 % for F<CHO-fed abalone and V  56 + 5 for F=CHO-fed abalone (not significantly different) being significantly greater than the PU of 39 ± 5 for F>CHO-fed abalone (p < 0.05, SNK). Likewise, the effect of time was significant (F10319  =  5.8p < 0.001), with the overall mean PU of 19 ± 12 % at week 2 being significantly lower than at all other times (p < 0.05, SNK). The other 10 sampling times fell into 3 large overlapping subsets. P U was low initially (19 ± 12 % at week 2) and during the latter part of the study (40 ± 7 and 44 + 7 at weeks 39 and 44, respectively), andfluctuatedbetween 72 + 3 and 46 + 9 (mean of 59 %) for the intermediate times. Figure 3.5 illustrates that lipase activity increased in abalone conditioned to diets with a higher fat content than found in their natural diet of kelp, and this increase was significant (Fx56 = 3.6;/? = 0.02). Note that F<CHO had a 2-fold, F=CHO a 4-fold, and F>CHO a 6-fold greater lipid content  63 Figure 3.4 Protein utilization (PU) by abalone Haliotis kamtschatkana fed formulated diets with fat content less than (F<CF£0), equal to (F=CHO), or greater than carbohydrate content (F>CHO), over a 44-week periodfromNovember - October. Each point represents mean P U [(N absorbed - N excreted)(N absorbed)"^ 100 %)] + S.E. for N = 10 abalone each of 16 g live mass.  MONTHS N  2  100  T3  75  Z  o  z  J  F  M  A  M  J  J  A  S  50 * -a 5 2  25  H  O  O  N F<CHO F=CHO F>CHO  o  H  3  D  i  -25 ©  -50 0  10  20  30  TIME (weeks)  40  64 Figure 3.5 Lipase activity in the gut of abalone Haliotis kamtschatkana fed a kelp diet and formulated diets with fat content less than (F<CHO), equal to (F=CHO), or greater than carbohydrate content (F>CHO), over a 44-week periodfromNovember - October. Each bar represents mean enzyme activity (units ml" ' min' ) ± S.E. for N = 5 abalone each of 16 g live mass. 1  1  FZZ] Kelp XZZA F < C H O F=CHO F>CHO  0  10 T I M E (weeks)  44  65 than kelp. Kelp-fed abalone had a significantly lower lipase activity over the 44-week period (0.8 + 0.1 units' ml" ' min' ) than abalone fed the formulated diets which all exhibited similar overall lipase 1  1  activities (1.5 ± 0.3, 1.6 ± 0.3, and 1.5 ± 0.3 for F<CHO-, F=CHO-, and F>CHO-fed groups, respectively; p < 0.05, SNK). Time also had a significant effect on lipase activity ( F 7 = 10.6;p < V  0.001), with the lowest lipase activity (0.8 ± 0.04 units ml' min' ) being exhibited at week 0, 0, and 1  1  with a significant increase at each subsequent sampling (1.3 + 0.2 at week 10 and 1.9 + 0.3 at week 44; p < 0.05, SNK). Figure 3.6 shows the effect of diet on alginate lyase activity and, as with lipase, this effect was significant (F 6 = 3.9; p = 0.01). Despite the absence of alginate from the formulated diets, abalone 3)5  conditioned to these diets exhibited a higher alginate lyase activity than did the kelp-fed group (6.5 + 0.7 units' ml'  1  min" ) and was highest in the F=CHO-fed abalone (12.3 ± 2.2; p < 0.05, SNK). The 1  effect of time on alginate lyase activity was also significant ( F ^ = 4.4; p = 0.02), with activity being lowest (6.4 ± 0.6 units" ml" " min") at week 0, and significantly higher at weeks 10 and 44 (10.5 ± 1.5 1  1  and 10.0 ± 1.6, respectively;p < 0.05, SNK). Figure 3.7 shows the amylase activity of abalone fed the four diets. The activity of this enzyme was also significantly influenced by diet (F  3;56  = 3.7;p = 0.02). Kelp-fed abalone had a significantly  lower amylase activity (5.0 +1.6 units' ml' min") than formulated diet-fed abalone which all 1  1  exhibited similar amylase activities (7.1 ± 1.6, 9.8 + 2.1 and 9.3 + 1.8 for F<CHO-, F=CHO-, and F>CHO-fed groups, respectively; p < 0.05, SNK). Time also had a significant effect on amylase activity (F y = 4.\;p< 0.02), with the lowest amylase activity (5.5 + 1.8 units' ml' ' min" ) being 1  1  V  exhibited at week 0, and with a significant increase in enzyme activity (p < 0.05, SNK) at week 10 (10.2 + 1.9), which was not significantly different from week 44.  66 Figure 3.6 Alginate lyase activity in the gut of abalone Haliotis kamtschatkana fed a kelp diet or formulated diets with fat content less than (F<CHO), equal to (F=CHO), or greater than carbohydrate content (F>CHO), over a 44-week period from November - October. Each bar represents mean enzyme activity (units' ml" ' min ) + S.E. for N = 5 abalone each of 16 g live mass. 1  1  LZZ] Kelp V77A F < C H O ^ F=CHO &m F>CHO  0  10 T I M E (weeks)  44  67 Figure 3.7 Amylase activity in the gut of abalone Haliotis kamtschatkana fed a kelp diet or formulated diets with fat content less than (F<CHO), equal to (F=CHO), or greater than carbohydrate content (F>CHO), over a 44-week periodfromNovember - October. Each bar represents mean enzyme activity (units' ml ' min' ) + S.E. for N = 5 abalone each of 16 g live mass. -1  1  LZZ] Kelp  YZZA ^  f5553  0  10 T I M E (weeks)  44  F<CHO  F=CHO F>CHO  68 3.5 DISCUSSION The formulated diets differed in their protein-sparing effects. The F>CHO diet was not optimal for Haliotis kamtschatkana, as food intake and mass change were significantly lower on this diet than on those with higher carbohydrate:fat ratios. This was expected in that energy metabolism in abalone is dependent on carbohydrate. Abalone store energy as glycogen (Livingstone and de Zwaan, 1983), their natural dietary energy source is predominantly carbohydrate (Wort, 1955), and they exhibit low lipase activity (Knauer et al, 1996). Protein utilization of F>CHO-fed abalone (39 %) was also significantly less than F=CHO- and F<CHOfed groups. However, nitrogen excretion was similar in all groups even though food intake and mass gain were less for abalone fed F>CHO. This indicates an increased protein catabolism and poorer protein utilization by the F>CHO-fed abalone. Since protein utilization was lower when fats (corn oil and menhaden oil) were provided in greater quantity than carbohydrates (sucrose, corn starch, and cellulose), I concluded that carbohydrates were more effective than fats in sparing protein for anabolism in Haliotis kamtschatkana. All the parameters that were selected to measure diet performance varied over time. These changes all occurred in association with the induction of spawning at week 38, with the exception of food intake which generally increased over the beginning of the study period (weeks 6 and 10). In general, mass was lost as gametes were shed and there was a subsequent gain in mass during the month after spawning. Nitrogen excretion of all diet groups also increased when spawning occurred, suggesting an increased protein catabolism perhaps resulting from the decreased food intake at spawning time (although this decreased intake was not statistically significant). With decreased food intake and an allocation of energy resources to garnetogenesis, there was likely an increased emphasis placed upon protein as an energy source. This would lead to the lower protein utilization observed. Protein utilization in abalone fed the formulated diets  69 was equally low over the first 8 weeks of the study and tended to be high during the middle period. The rise in protein utilization after the first 8 weeks was synchronous with a significant increase in food intake. In previous investigations on abalone fed formulated diets composed of similar nutrients (Chapters 1 and 2), protein utilization was also found to vary over time, which provided earlier evidence that abalone might adapt to alterations in diet. The possibility that abalone can adapt to dietary changes was investigated in some detail in this part of my thesis. Specifically, I examined whether changes in diet composition would alter the activity of digestive enzymes and thereby cause a change in protein-sparing effects and protein utilization. Changes in digestive enzyme activities are known to occur when juvenile abalone begin to feed on kelp (Spaulding and Morse, 1991). However, it was unknown whether abalone possess a later plasticity in digestive capabilities, although this has been observed in other invertebrates such as the African giant snail (Van Weel, 1959), mysids (Kerambrun and Guerin, 1993), and shrimp (Rodriguez et al, 1994). It is ambiguous whether the enzymatic adaptations seen in this study represented similar adaptations to diet as those reported in these other species. Lipase activity increased significantly at weeks 10 and 44 in abalone fed formulated diets, but did not change in abalone fed kelp. Overall, lipase activity in abalone fed formulated diets was higher than in kelp-fed abalone, and this corresponded to a proportionally greater intake of dietary lipid (the lipid contents of the formulated diets were 2- to 6-fold greater than that of kelp: Jeong et al, 1994; Virtue and Nichols, 1994). This indicated that abalone exhibited enzymatic adaptation to changes in diet composition. Changes in the activities of amylase and alginate lyase, however, suggested that the adaptation was not in response to the levels of specific dietary nutrients. Alginate lyase activities in the F>CHO-, F<CHO-, and kelp-fed abalone were similar despite the fact that the formulated diets contained no alginate, while kelp is known to contain about 26 % dry mass alginate (Wort, 1955; Usov and Klochkova, 1994; Nishide et al., 1996). Amylase  70 activities were also similar among these abalone; however, the starch substrate for amylase was only present in the formulated diets, not in kelp. Furthermore, amylase activity in the F=CHO-fed abalone was significantly greater than in F<CHO-fed abalone, even though the latter group was fed proportionally more starch. Thus, the observed differences in amylase and alginate lyase activities after different lengths of time feeding on the diets suggested that enzymatic adaptation was not based upon dietary nutrient composition, but rather on some alternative driving force. The overall activities of each of the digestive enzymes increased over the 44-week period, owing mainly to increased enzyme activities in abalone fed the formulated diets. Roche Mayzaud et al. (1991) similarly observed that copepods exhibited an increased activity of digestive enzymes when food was limited. Thus, the increased activities I observed may have resulted from nutritional stress, since abalone fed formulated diets had a lower mass gain than those fed kelp. The abalone fed formulated diets may have increased their production of all digestive enzymes in an attempt to maximize the overall extraction of dietary nutrients. Since all abalone fed formulated diets exhibited similar enzymatic adaptations, these adaptations cannot explain the differences in protein utilization among the animals. The overall values of protein utilization for F<CHO- and F=CHO-fed abalone (62 % and 56 %) were significantly higher than that for F>CHO-fed abalone (39 %). This is in accordance with Mai et al. (1995) who observed that protein gain in Haliotis discus hannai and H. tuberculata was better at lower dietary lipid levels. Furthermore, all three estimates of protein utilization are consistent with those obtained in my earlier studies with H. kamtschatkana fed formulated diets (Chapters 1 and 2) and they are comparable to measures of net protein utilization obtained in other studies on abalone. For example, Uki et al. (1986a) reported net protein utilization of 68 % for Haliotis discus hannai fed a diet with 20 % dry mass casein, and 31 % when fed a diet with 22 % dry mass protein from fish meal (for further comparison with measures of protein retention in cultured  71 aquatic animals see Chapter 1). Protein utilization in abalone fed the formulated diets indicates that 60 % of the absorbed dietary nitrogen was excreted by the F>CHO-fed abalone and 40 % by abalone fed the other two formulated diets. Thus, abalone fed the F>CHO diet, as compared with the F=CHO and F<CHO diets, were forced to catabolize an additional 20 % of their dietary protein. Presumably this protein catabolism was necessary to meet energy requirements because the abalone could not access enough energy from corn oil and menhaden oil. All formulated diets supported less mass gain than kelp (mean of 1 % versus 36 %), which indicates an inferior nutritional quality of these diets relative to kelp. Since abalone possess the enzymes necessary to digest such carbohydrates as corn starch, sucrose, and cellulose (Anzai et al, 1991), it is unlikely that these carbohydrates failed to provide energy to the abalone. Possibly the lower growth noted for abalone fed the formulated diets stemmed from a deficiency in lipid metabolism. Although marine animals typically contain low levels of n-6 polyunsaturated fatty acids (PUFA), macroalgal feeders such as abalone seem to be an exception (see Fleming et al, 1996 for review). Indeed, some species of abalone are known to contain high levels of arachidonic (20:4n-6) acid (Dunstan et al., 1996) and may require this fatty acid for good growth (Floreto et al, 1996), as well as possibly requiring some n-3 fatty acids (Uki et al, 1986b; Floreto et al, 1996). Arachidonic acid is not present in either corn oil or menhaden oil, but whether abalone can synthesize this fatty acid is not known. In any case, my formulated diets contained a mixture of menhaden oil to supply n-3 P U F A and corn oil to supply n-6 PUFA. Such mixing of fish oil and vegetable oil is common in commercial and research diets for abalone (see Fleming et al, 1996 for review). Furthermore, Mai et al (1995) used a mix of corn oil and menhaden oil in formulated diets for Haliotis tuberculata and H. discus hannai, and found that these diets supported growth and protein gain. For those abalone species investigated (which does not include//, kamtschatkana) PUFA of both n-3 and n-6 families seem to be essential; however, the  72 specific PTJFA requirements are not generally known. The requirements seem to vary with species just as fatty acid composition of the body varies with species (Dunstan et al, 1996; Floreto et al, 1996; Mai et al, 1995). Since it is possible that fatty acids essential to H. kamtschatkana were lacking in the diets used here, an investigation of essential fatty acids for this species is imperative for future diet formulations. The food intakes of abalone fed the formulated diets paralleled their mass changes. F>CHO-fed abalone gained less mass and consumed less food than F=CHO- and F<CHO-fed abalone. The lesser intake of the high-fat diet suggested an unpalatability or intolerance of this diet. Mean daily intake of the F=CHO and F<CHO diets was 0.2 g d" and that of the F>CHO 1  diet was 0.1 g" d" . This represented daily food intake of approximately 1 % body mass which is 1  within the range reported for other abalone species of similar size (see Fleming et al, 1996 for review). Within this range, food intake fluctuated over the 44-week period in a manner similar to what I observed in previous studies (Chapters 1 and 2). Intake was low in winter (November February) and high in spring/summer (April - August), which may relate to the reproductive cycle of these abalone. Reproductive growth occurs in the spring/summer season and is followed by a period of somatic growth, while winter is a quiescent season (Sloan and Breen, 1988). Food intake, then, could have reflected both the energy demands of seasonal fluctuations in growth and an aversion to the F>CHO diet. Unlike the trends noted for mass change, food intake, and protein utilization, the trends found for nitrogen excretion were similar among abalone fed all diets and were consistent with my previous investigations on Haliotis kamtschatkana fed diets of similar composition (Chapters 1 and 2). The similarity of nitrogen excretion regardless of diet did not, however, negate the conclusion that fats were less effective than carbohydrates in their protein-sparing effects. Indeed, because the intake of F>CHO was less than that of the other formulated diets, the similar  73 nitrogen excretion suggested poorer utilization of the protein component (casein) of this diet. This in turn suggested that the abalone were forced to catabolize dietary protein for energy, presumably because energy from dietary fats was insufficiently accessible. This inaccessibility may stem from a deficiency in absorption since a low absorption rate of the lipid from kelp (Macrocystis spp.) has been reported for Haliotis rufescens (Leighton, 1968), although Wee et al. (1992) reported effective digestion of lipids in H. rubra and H. laevigata. Thus, despite equal nitrogen excretion, protein utilization was less for F>CHO-fed abalone than those fed the other formulated diets and, concurrently, protein sparing by corn oil and menhaden oil was less than that of corn starch, sucrose, and cellulose. In conclusion, the results of my study suggested that protein utilization in Haliotis kamtschatkana is most efficient when they are fed formulated diets which rely on carbohydrate as the source of energy. Carbohydrates had a greater protein-sparing effect than fats. Furthermore, superior protein sparing by carbohydrate was not ameliorated by enzymatic adaptation to the diets, despite the increased activity of some digestive enzymes seen in abalone fed these diets.  74  C H A P T E R 4: EFFECT OF VERTEBRATE GROWTH HORMONE ON GROWTH O F A D U L T A B A L O N E Haliotis kamtschatkana  4.1 A B S T R A C T Enhancement of growth in cultivated animals through hormone treatment is of interest in aquaculture due to this technique's potential for increasing production. In this study, injection of vertebrate growth hormones was investigated as a means of enhancing growth in adult abalone Haliotis kamtschatkana. Fifty abalone were held in individual cages within a common aquarium tank that was supplied with a constant flow offreshseawater. The abalone were fed ad libitum on kelp Nereocystis leutkeana. The abalone were divided into five groups of ten animals of similar mean individual live mass (78 g) and length (7 cm). Four groups received weekly intramuscular injections (5 ug' g body mass' ) of either: 1) recombinant bovine growth hormone (rbGH), 2) recombinant porcine 1  growth hormone (rpGH), 3) somatostatin (SST), or 4) a sham injection of bovine serum albumin (BSA). Thefifthgroup served as an untreated control. The abalone were weighed biweekly throughout the 10-week study. Water content and gonad index were assessed for each group at the end of the study period. There were no significant differences in mass gain, water content, or gonad index among the five groups. The results suggested that injection of vertebrate growth hormones does not enhance growth in adult abalone.  75  4.2 I N T R O D U C T I O N Previous parts of my thesis (Chapters 1, 2, and 3) focused on protein utilization by abalone fed formulated diets, since protein is a costly dietary nutrient and the only one which can facilitate protein growth. In commercial culture of abalone, muscle growth is preferable to non-muscle growth (i.e., gonad growth or shell growth) since it results in a more commercially valuable product. Means of enhancing growth, be they dietary or otherwise, are of interest. Use of recombinant bovine and porcine growth hormone has been shown to enhance growth of cultured salmonids (Gill et al, 1985; Agellon etal, 1988; Down etal, 1988; McLean etal, 1990,1991, and 1992; Devlin etal, 1994), and here the efficacy of this technique for the abalone Haliotis kamtschatkana was assessed. Techniques for manipulating growth in aquacultured organisms have received intensive study over the last decade. In part, this has occurred due to the advent of recombinant D N A technologies. In these technologies exogenous genes are incorporated into bacteria as a means for producing large quantities of biologically active proteins such as growth hormones. Growth hormones produced in this manner are chemically and functionally similar to those naturally produced in vertebrates (Gill et al, 1985). In teleosts, specifically, numerous studies have demonstrated the growth-accelerating effects of recombinant growth hormones (see McLean and Donaldson, 1993 for review). In contrast, comparatively few studies have examined the endocrine basis for enhancing growth in molluscs. That molluscs possess growth hormone has been known since Lubet's (1971) work with the gastropod Crepidulafornicata. Later, Geraerts (1976) characterized a growth hormone produced by neurosecretory cells in the cerebral ganglia of another gastropod Lymnaea stagnalis. This hormone was subsequently shown to stimulate shell growth (Dogterom etal, 1979; Dogterom and Jentjens, 1980) and to influence metabolic pathways (Dogterom, 1980; Dogterom and Robles, 1980). In molluscs the main growth hormone is an insulin-like peptide often referred to as molluscan insulin-like  76  peptide (MTP). MTP has been isolated and sequenced (Smit et al, 1988), and the D N A sequence coding for MTP in Lymnaea stagnalis has been identified (Smit et al, 1992). Two growth hormones have been purified from Haliotis discus hannai (Moriyama et al, 1989). Additionally, gastropod molluscs appear to possess a somatostatin-like molecule which promotes growth (Grimm-torgensen, 1983a and b; Marchand et al, 1989). This contrasts with vertebrates in which somatostatin is a pancreatic hormone which inhibits the release of growth hormone. In any case, the role of hormones in growth regulation in molluscs is an area of active research. The influence of exogenous hormones on molluscan growth was demonstrated by Morse (1981) who observed enhanced growth of post-larval Haliotis rufescens following combined treatment with mammalian insulin and growth hormone. More recently, Kawauchi and Moriyama (1991) reported that recombinant salmon growth hormone stimulated growth in H. discus hannai, while Paynter and Chen (1991) demonstrated that a recombinant rainbow trout growth hormone stimulated growth of larval oysters Crassostrea virginica. To date, studies on growth manipulation of molluscs have focused upon juvenile stages. My study examined the effects of recombinant growth hormone treatment using adult abalone, employing both recombinant bovine and porcine growth hormones. Additionally, since a molluscan growth hormone similar to somatostatin has been reported, response to vertebrate somatostatin was also investigated.  4.3 M E T H O D S A N D M A T E R I A L S Abalone Haliotis kamtschatkana were collected by SCUBA divers from shallow subtidal waters near Bamfield Marine Station, Vancouver Island and transported to the Department of Fisheries and Oceans, West Vancouver laboratory. Fifty of these abalone were individually caged in WhitlockVibert incubation boxes (obtained from the Federation of Fly Fishers, Yellowstone, Montana) and held  77  in a common aquarium tank with a constant flow offreshambient seawater at 12 °C and 27 %o. Animals were fed ad libitum on the locally available kelp Nereocystis leutkeana. The abalone were divided into five groups of ten animals (five male and five female). Each group had a similar mean individual live mass (78 g) and length (7 cm). Four groups received weekly intramuscular injections (5 ug' g body mass") of either: 1) recombinant bovine growth hormone (rbGH, obtained from AMGen 1  Inc.), 2) recombinant porcine growth hormone (rpGH, obtained from AMGen Inc.), 3) somatostatin (SST, obtained from Sigma Co.), or 4) sham injection with bovine serum albumin (BSA), each in saline solution. Bovine serum albumin was selected as the sham injection material to provide an equivalent dose of protein, but one not potentially active in stimulating growth. Thefifthgroup of abalone received no injection and thus served as an untreated control group that was handled in the same manner as the other groups. Since previous studies on hormone therapy in abalone employed immersion rather than injection (Morse, 1981; Kawauchi and Moriyama, 1991), no guidelines for injection dosage were available. Accordingly, the 5 p.g' g body mass" dosage used here was based on 1  previous work on salmon (McLean and Donaldson, 1993). Injections were made intramuscularly at 1 cm depth in the center of the foot sole and consisted of 100 u,l total volume of a saline solution containing hormone or BSA. Live mass of each abalone was measured biweekly throughout the 10-week study (April July). Water content and gonad index were assessed for five animals from each treatment group at the end of the experimental period. Water content was determined by the difference in soft tissue mass of abalone before and after being dried to constant mass at 60 °C. Prior to drying the abalone the gonad was separated from the rest of the soft tissues so gonad index could be measured as the proportion of dry grams of gonad per dry grams of soft tissue.  78  Statistical analyses Data were analyzed using two-factor A N O V A or Repeated Measures ANOVA, coupled with Student-Newman-Keuls Multiple Comparison Tests (SNK). Values presented in the text are means ± standard error.  4.4 R E S U L T S Figure 4.1 illustrates mean live mass of abalone undergoing the various hormone treatments. Only the data for abalone which survived the 10-week duration of the study were included in the graph and the statistical analyses. Mortalities among the treatment groups are reported in Table 4.1. Mortalities were higher than expected, with four and five animals dying in the rbGH and rpGH treatments, respectively, and two in each of the other treatment groups. The data suggested that stress from handling and needle-injection may have caused some of these mortalities, perhaps exacerbated by effects of the rGH's themselves. While there was a trend for differences in final mass among the various treatment groups, these were not significant (F ,i2o = 0.2;p = 0.93). There was, however, a significant time effect 4  (F ,i2o 4  - 5.9;  p < 0.001), with animals gaining mass over thefirst5 - 7 weeks of the study (overall mean mass increased from 77.8 ± 3.1 g to 82.3 ± 3.1). Over the remainder of the study, however, the abalone lost this mass, and by 10 weeks they were not significantly different from their initial mass (p > 0.05; SNK). Water content offiveabalone from each treatment group at the end of the study is shown in Figure 4.2. Results of A N O V A with mass factored out indicated that no treatment had a significant influence on water content of the abalone (F  4>2  o  = 3.7;/? = 0.06; overall water content was 79.2 ± 0.9  % live soft tissue mass). Thus, differences in water content could not have hidden any differences in live mass Since adult abalone can exhibit erowth as a eain in either somatic tissue or renroductive  79  Figure 4.1 Live mass of abalone Haliotis kamtschatkana treated with weekly injections of bovine serum albumen (BSA), somatostatin (SST), recombinant porcine growth hormone (rpGH), recombinant bovine growth hormone (rbGH), or left untreated (control), for a 10-week period from April - July. Each point represents mean live mass (g) ± S.E. for N = 5 - 8 abalone.  100  >  A  80  60 2  3  4  5  6  TIME (weeks)  •  BSA  T  SST  A  rpGH  •  rbGH  O  control  80  Table 4.1 Mortality of abalone Haliotis kamtschatkana treated with weekly injections of bovine serum albumen (BSA), somatostatin (SST), recombinant porcine growth hormone (rpGFi), recombinant bovine growth hormone (rbGH), or left untreated (control), for a 10-week period from April - July. Each group initially consisted of 10 abalone.  Treatment bovine serum albumin (BSA) somatostatin (SST) recombinant porcine growth hormone (rpGH) recombinant bovine growth hormone (rbGH) control  % mortality 20 20 50 40 20  81  Figure 4.2 Water content of abalone Haliotis kamtschatkana treated with weekly injections of bovine serum albumen (BSA), somatostatin (SST), recombinant porcine growth hormone (rpGH), recombinant bovine growth hormone (rbGH), or left untreated (control), for a 10-week period from April - July. Each bar represents mean body water content (as % total soft tissue mass) ± S.E. for N = 5-8 abalone.  82  tissue, gonad indices of abalone which had undergone the different treatments were also measured. Figure 4.3 shows gonad indices measured at the end of the 10-week period. Results of A N O V A with mass factored out indicated that no treatment significantly influenced gonad index (F 2o = 0.7;/? = 4>  0. 60). There was, however, a significant difference in the gonad indices of male versus female abalone (F13  =  8.2;/? = 0.02), with females having a higher mean gonad index than males (24 ± 1 % and 20 ±  1, respectively). Live mass and water content were not affected by sex of the abalone.  4.5 DISCUSSION Administration of growth hormone for the purpose of growth enhancement has been investigated in teleosts (McLean and Donaldson, 1993), lobsters (Charmantier etal, 1989), shrimp (Toullec etal, 1991), and abalone (Morse, 1981; Kawauchi and Moriyama ,1991). Morse (1981) demonstrated enhanced growth in post-larval Haliotis rufescens immersed in mammalian insulin and growth hormone, and Kawauchi and Moriyama (1991) reported enhanced growth of small (1 g) juvenile H. discus hannai when immersed in recombinant salmon growth hormone. Results of my investigation, however, indicated that growth of adult abalone did not benefit from injection (at a dose of 5 ug' g body mass* ) of either recombinant porcine growth hormone (rpGH) or recombinant bovine 1  growth hormone (rbGH), nor were adult abalone responsive to vertebrate somatostatin (SST). The study took place during spring and early summer which corresponds to the time of gametogenesis and negligible somatic growth in Haliotis kamtschatkana (Paul et al, 1976). This time was chosen intentionally to test the effectiveness of hormone therapy when the animals were least predisposed to somatic growth. Thus, the protocol was subjected to greatest challenge. Commitment to reproductive growth over the time-course of the experiment may have been irreversible, thus explaining the lack of somatic growth in response to the hormone treatments. Such a competitive  83  Figure 4.3 Gonad indices of abalone Haliotis kamtschatkana treated with weekly injections of bovine serum albumen (BSA), somatostatin (SST), recombinant porcine growth hormone (rpGH), recombinant bovine growth hormone (rbGH), or left untreated (control), for a 10-week period from April - July. Each bar represents mean gonad index (as % total soft tissue mass) + S.E. for N = 5 - 8 abalone.  BSA  SST  rpGH  rbGH  TREATMENT  control  84  inhibition between reproductive and somatic growth in abalone is suggested by growth rates which decrease exponentially with age (Leighton and Boolootian, 1963; Quayle, 1971; and Paul etal, 1976). Decreased growth appears to be due not to general decline in metabolism among older animals but, rather, to reproductive growth's competitive demand for energy. In support of this, Shepherd and Heam (1983) observed slower growth rates for H. laevigataand H. ruber specifically during periods of gonad production, with the decrease being especially evident in larger/older animals. Furthermore, Keesing and Wells (1989) reported a slowing of growth following sexual maturity in H. roei. The latter investigators also reported that growth is negligible during most of the year and only occurs after spawning. A post-spawning somatic growth spurt occurs in several abalone species including H. iris and//, rufescens (Cox, 1962;Poore, 1972; Sainsbury, 1982). Likewise,//, tuberculata is known to attain 70 % of its yearly growth increment in a single month after spawning (Forster, 1967). In light of this, further studies on the response of abalone to exogenous hormones should be done in the postspawning period or perhaps over several seasons, in order to determine the effect of reproductive status on response to hormone treatment. Another possible explanation for the observed lack of somatic growth in response to hormone treatment is suggested by Toullec et al. (1988) who noted that the open and lacunar nature of molluscan circulatory systems may be unfavorable for in vivo injection of hormonal substances due to sluggish and inefficient movement of hemolymph. Consequently, this led me to conduct an investigation of molluscan growth control by treating in vitro dissociated-cell preparations with growth hormone and somatostatin (see Chapter 5). Protocols which employ dissociated-cell cultures may be appropriate for continued investigation of growth-controlling substances in abalone. If growthcontrolling molecules can be identified, it is evident that they will have to be administered by means other than injection. Even though the distribution of mortalities among the treatment groups indicated  85  that needle puncture was not a chief contributing factor to mortality, it would be necessary for culture purposes that an alternative, less time-consuming method of hormone delivery be adopted. Immersion of adult abalone is not feasible for mass culture but incorporation of suitable growth-promoting substances into the animal's diet may have potential. In this preliminary investigation hormones were administered by injection since this was the only method which insured that a known quantity of hormone entered the abalone's tissues. If, indeed, adult abalone are irreversibly committed to reproductive growth at the expense of somatic growth during their reproductive period, it was also evident from this study that reproductive growth was not enhanced by exogenous vertebrate growth hormones. Regardless of treatment, females had an average gonad index of 24 % and males 20 % (these values are comparable to another abalone species on the Pacific coast of North America: H. cracherodii, whose gonad at maturity constitutes 20 % of its soft body mass; Webber, 1970). Reproductive growth of abalone may be controlled by a different physiological mechanism than somatic growth, one not influenced by exogenous vertebrate growth hormone. In this case gonad indices would not be expected to have been influenced by the administration of hormones. Investigation of hormonal control of reproductive growth could, however, lead to useful improvements during the broodstock-conditioning phases of abalone culture and, thus, would be a productive line of research to follow.  86 C H A P T E R 5: S E A S O N A L C H A N G E S IN N U T R I E N T U T I L I Z A T I O N AND RESPONSE T O G R O W T H H O R M O N E S B Y I S O L A T E D D I G E S T I V E - G L A N D C E L L S O F A B A L O N E Haliotis kamtschatkana 5.1 A B S T R A C T Metabolic activity, measured as oxygen consumption, of digestive-gland cells of Haliotis kamtschatkana was studied in relation to seasonal changes in nutrient utilization and response to growth hormone, as well as in relation to relative sizes of digestive gland and gonad. At 3 - 4month intervals between May 1995 - July 1996, data were collected on soft tissue mass, digestive gland and gonad indices, and oxygen consumption of isolated digestive-gland cells alone (representing a baseline), after addition of a nutrient substrate, and after subsequent addition of hormones. Oxygen consumption of isolated cells increased by 75 % after addition of glucose and glucose + amino acids, but did not change with addition of amino acids, which was reflective of the carbohydrate-based metabolism of abalone. Lack of response to amino acids was consistent in all seasons, but responses to glucose and the mixture of glucose + amino acids substrates fluctuated with season, being higher in spring/summer. Somatotropin and somatostatin did not increase the metabolic activity of the cells. There was no sex effect on digestive gland index, but seasonal differences in this index (9 -12 %) correlated with metabolic activity of digestive-gland cells in response to glucose and glucose + amino acids. Thus, when digestive glands were largest relative to body size the metabolic activity of their cells was greatest. Gonad index was significantly higher in males than in females (10 % and 7 %, respectively). Seasonal changes in gonad index were also observed, with values being generally high in spring prior to spawning and low in winter, but with overlap between these and the summer values.  87 5.2  INTRODUCTION Questions which arose from earlier chapters of my thesis, and which may relate to function  of the digestive gland in abalone, were addressed in this study. Specifically investigated were seasonal variations in the effects of nutrient substrate and hormone treatment on energy metabolism by the digestive gland, and the function of this gland in relation to gametogenesis in Haliotis kamtschatkana. The abalone digestive gland performs multiple metabolic functions, such as being involved in energy storage, growth, and the manufacture of enzymes. As the digestive gland is also known to produce oocyte proteins (Flari and Charrier, 1992) and a somatostatin-like molecule in pulmonate snails (Bride and Gomot, 1989), the range of function of this gland is broad. It was therefore relevant to investigate the role of the digestive gland in gametogenesis since reproductive growth in abalone appears to be mutually exclusive of somatic growth (Cox, 1962; Poore, 1972; Sainsbury, 1982; Shepherd and Hearn, 1983). However, since study of the abalone digestive gland is hampered by difficulty in gaining access to the gland due to its intimate juxtaposition with the gonad, a technique involving in vitro preparations of isolated digestivegland cells was employed. A similar in vitro technique has been used to study function of the hepatopancreas in prawns (Toullec et al, 1992) and crabs (Lallier and Walsh, 1992). The reproductive cycle in abalone of temperate regions generally follows the pattern: gametogenesis during winter and early spring, maximally ripe gonads in late spring and early summer, and spawning during summer and early autumn (Boolootian et al, 1962; Webber and Giese, 1969; Poore, 1973; Shepherd and Laws, 1974; Hayashi, 1980; Shepherd and Hearn, 1983). However, considerable variation in this cycle may exist within and between species, as well as geographically and temporally (Boolootian etal, 1962; Poore, 1973; Shepherd and Laws, 1974; Giorgi and DeMartini, 1977; Sloan and Breen, 1988). Haliotis kamtschatkana typically has highest gonad index in late spring and early summer, with spawning in mid- to late summer (Breen  88 and Adkins, 1980). However, there is evidence among some populations of "dribble" spawning throughout the year (Sloan and Breen, 1988) as is seen with H. roei (Wells and Keesing, 1989), and H. iris and H. australis (Wilson and Schiel, 1995). Energy resources which fuel gametogenesis are mostly stored in the foot muscles as glycogen (Webber and Giese, 1969; Webber, 1970). Hayashi (1983) showed in Haliotis tuberculata that the glycogen content of the foot tissue decreases from 40 % to zero as the gonad matures and, inH cracherodii, Webber and Giese (1969) and Webber (1970) showed a drop in the total dry mass polysaccharide (glycogen) in the foot from 23 % to 6 %, the lowest value coinciding with late-summer spawning. In H. cracherodii, as polysaccharides are drawn from foot stores there is an attendant increase in their concentration in the gonad, and this is somewhat more prominent in males than females (males increase 7 % dry mass and females 6 %; Webber 1970). The role of the digestive gland in gametogenesis has not been investigated in abalone. However, the digestive gland of the pulmonate snail Helix aspersa was shown by Bride and Gomot (1989) to play a role in oogenesis, specifically as the site for synthesis of vitellogenin (oocyte protein). A seasonal division between somatic and reproductive growth in Haliotis kamtschatkana was interpreted by Paul et al. (1976) from their observation of decreased growth rates during periods of gonad production. A seasonal difference in nutrient metabolism would be expected from such a seasonal bias toward either somatic or reproductive growth, since the high fat stores of eggs, and the specific proteins of both eggs and sperm, are not found in non-reproductive tissues of adults. Accordingly, one phase of this study investigated possible seasonal variation in nutrient utilization. A previous investigation of fat utilization by H. kamtschatkana during a presumed time of gametogenesis (Chapter 3) indicated that fat is not efficiently utilized as an energy source by H kamtschatkana. Thus, only glucose and amino acids were used as nutrient  89 substrates in this study. Previously I demonstrated (Chapter 4) that injection of vertebrate hormone did not enhance either somatic or reproductive growth in adult Haliotis kamtschatkana. I suggested that either the invasive stress of weekly handling and hypodermic injections negated enhancement of growth or that adult abalone were not responsive to vertebrate growth hormone. Application of these hormones to an in vitro preparation of cells was a possible means to resolve this issue. Repetitive sampling over different seasons was used to determine whether abalone have a seasonal bias against somatic growth during periods of reproductive growth. In summary, this study examined the seasonal effects of nutrients (glucose and amino acids) and hormones (somatotropin and somatostatin) on metabolism of digestive-gland cells, and correlated this metabolic activity with relative size of gonad and digestive gland. I expected: 1) that glucose substrate would be metabolized more readily than amino acid substrate as suggested by the carbohydrate-based nutrition of abalone (Webber, 1970; Hayashi, 1983), 2) that the metabolic activity of isolated cells would correlate seasonally with the reproductive cycle, and 3) that hormones would increase the metabolic activity of isolated cells.  5.3 M E T H O D S A N D M A T E R I A L S Collection and maintenance of abalone On five occasions between May 1995 - July 1996, adult abalone were collected by S C U B A divers from shallow subtidal waters near Bamfield Marine Station, Vancouver Island. Each time, ten abalone (five males and five females) ranging in size from 10 -15 cm in shell length, were collected and transported to the University of British Columbia. Abalone were temporarily housed in a recirculating seawater system at 5 - 12 °C and 32 %o until their digestivegland cells were isolated. They were fed ad libitum on fresh kelp Nereocystis leutkeana except in  90 December when this seasonal alga was not available. Thus, the laboratory feeding regime paralleled food availability in the field. Cell isolation and determination of digestive gland index Abalone were chilled in an ice slurry prior to being removed from their shells. Soft tissue masses and shell lengths were recorded. The membrane encasing the gonad was slit with fine scissors to expose the gonadal tissue, which was then removed by aspiration into 100 ml of seawater within a pre-weighed flask. Once the gonad was removed the digestive gland was cut from the animal and weighed. Digestive-gland index was calculated as the percentage of soft tissue mass represented by the mass of this gland. The digestive gland was then immersed in icecold buffer composed of (in mM) 469.5 NaCl, 10 Na A -2-hydroxyethylpiperazine-A"'-2r  ethanesulfonic acid (HEPES), 7.1 glucose, 6.9 KC1, 5.3 NaHC0 ,0.6 K H P 0 , and 0.4 N a H P 0 . 3  2  4  2  4  The buffer had been gassed to saturation with a 95%0 :5%C0 mix, adjusted to pH 7.2 (pH of 2  2  abalone hemolymph), and had 0.5 % bovine serum albumin (BSA) added. The digestive gland was minced with razor blades to release individual cells and the resulting slurry of cells and buffer was filtered through coarse (253 |0.m), then fine (73 |j.m) nylon-mesh screens. Thefilteredcell suspension was transferred to a 50 ml centrifuge tube and spun at 98 g for 7 min, at 3 °C. Three spins were performed, with the buffer and suspended debris being decanted and the cells resuspended in fresh buffer after each spin. After the final spin the cells were resuspended, transferred to a 250 ml flask, and rested overnight at 3 °C. After 14 h the cells were again centrifuged (98 g, 7 min, 3 °C) and resuspended in buffer similar to that described above but without glucose. The resulting cell suspension was used for respirometry. Cells were isolated from each of ten abalone (five of each sex) at each of five experimental times. Determination of gonad index Following aspiration of the gonad into the pre-weighed flask containing 100 ml of  91 seawater, the wet mass of gonad was determined by weighing the flask and its contents, and subtracting the initial mass of the flask and seawater. The gonad index was calculated as the percentage of total soft tissue mass represented by this gland. Cell viability The viability of the isolated digestive-gland cells was tested by the trypan dye-exclusion method (Harris and Cornell, 1983) immediately after isolation and again after resting overnight. To assess trypan blue exclusion, 2 vol of cells were incubated with 1 vol of 0.6 % trypan blue and then an aliquot was counted for cell-staining using a compound microscope. In this test the dye is excluded from intact, viable cells and only stains the nuclei of ruptured cells. Cell suspensions invariably had less than 5 % staining. Respirometry A 500 p.1 aliquot of cell suspension (10 - 30 mg cells' ml" ) was diluted two-fold with 1  glucose-free buffer in a 2-ml Gilson oxygen cell maintained at 15 °C, and equipped with a Clarktype O 2 electrode linked to a Sable Systems Data Acquisition program. Wet mass of the cells was determined by centrifuging a known volume of cell suspension in a pre-weighed Eppendorf tube at 10,000 g for 5 min, decanting the supernatant and removing excess liquid with a Kimwipe tissue, andfinallyre-weighing the tube and cells to determine wet cell mass by difference. Oxygen consumption ( V O 2 ) was expressed as u,g 0 ' min' ' g cells' . V 0 of the cell suspension was 1  2  1  2  determined over an initial 5-min period prior to the addition of a nutrient substrate, either 100 mM glucose (GLU), 50 |il of a saturated solution of a crystalline mix of casein amino acids (AA) (obtained from ICN Ltd.), or 50 (ii of a 50/50 v/v mix of the stock glucose and amino acid solutions (GLUAA). After substrate addition, oxygen consumption was monitored for a further 5 min. In a subsequent set of trials using the G L U A A substrate, following the V O 2 measurement of  92 cells plus substrate a hormone was added (100 u M of either recombinant bovine somatotropin obtained from AMGen Inc., or somatostatin obtained from Sigma Co.) and V O 2 was monitored for an additional 5 min. All nutrients and hormones were delivered in 50 u,l aliquots of a stock solution via a syringe inserted through a plug in the top of the oxygen cell. The concentrations of the stock solutions were such that the end concentrations cited above were produced by addition of 50 u,l to the 2-ml oxygen cell. The dosages were based upon a dosage-series run prior to the study (results not presented), where nutrients and hormones were administered in concentrations of 1, 10, 100, and 1000 mM and 1, 10, 100, and 1000 p,M, respectively.  The 100 mM nutrient  dose achieved the best response. Owing to a lack of response to each of the hormones at all dosages when the dosage trial was conducted, a middle dose (100 pM) was chosen. Each combination of cells-substrate or cells-substrate-hormone was run in triplicate. Statistical analyses Data from all measures of cellular oxygen consumption, digestive-gland index and gonad index were analyzed using two- and three-factor A N O V A , coupled with Student-Newman-Keuls Multiple Comparison Test (SNK). Digestive-gland index and gonad index were also compared using Spearman Rank Correlation Coefficient Analysis (SRCCA). Values presented in the text are means ± standard error.  5.4 R E S U L T S Baseline V O 2 (prior to addition of any substrate or hormone) in isolated digestive glandcells from male and female Haliotis kamtschatkana did not differ (Fi was influenced by season (F  ;4g  = 0.002; p = 0.97), but  = 29.2; p < 0.001). As illustrated in Figure 5.1, baseline V 0 was  4>48  2  highest in May (8.5 ± 0.4 pig 0 ' min" " g cells" ) and July (8.1 ± 0.4), lowest in December (3.1 + 1  2  1  93 Figure 5.1 Oxygen consumption ( V O 2 ) by isolated digestive-gland cells of male and female abalone Haliotis kamtschatkana, over a 15-month period. Each point represents mean V O 2 (pig 0 • mg cells" • h") ± S.E. for N = 5 abalone. 1  1  2  • •  10  WD  S  6A  •  O  wo =t  <s O  >  0  1 MAY  1  1 — r AUG  MONTHS  male female  94 0.4), and intermediate in August (6.3 + 0.4) and April (6.1 + 0.4), and these differences were significant (p < 0.05, SNK). Figure 5.2 illustrates the effects of the three nutrient substrates, which were significantly different (F  2>47  = 48.2; p <0.001). G L U and G L U A A exerted similar effects on V 0  2  (increases of 75 + 3 % and 74 + 7, respectively) which were significantly higher than A A (increase of 4 ± 7;/? < 0.05, SNK).  V 0 after addition of G L U or G L U A A was significantly increased 2  from the pre-substrate level (Fi g = 375.6;p < 0.001). There was a significant seasonal difference )4  in the metabolic responsiveness of digestive-gland cells (F s = 87.1;/? <0.001), with cells being 4j4  most responsive in spring/summer and least responsive in winter (p < 0.05, SNK).  There was also  a significant sex effect on the responsiveness of the cells to nutrients which did not depend upon the specific nutrient added (Fi, g = 23.9;p <0.001). With % change in V 0 for all nutrients and 4  2  seasons combined, cells from female abalone were significantly more responsive to addition of nutrients (78 ± 3 %) than were cells from males (55 ±3;p<  0.05, SNK).  As shown in Figures 5.3 and 5.4, when somatotropin and somatostatin were added to cells which had already been provided with G L U A A substrate there was a significant decline in V 0 (-39 %; Fi g = 57.6; p < 0.001), and the effect did not differ between the two hormones (Fi )4  ;48  2  =  0.4; p = 0.88). The inhibitory effect of hormone addition, however, was greater in females than in males (Fi, = 4.9;p = 0.03) and was significantly influenced by season ( F 4g  4j45  = 133.6;p < 0.001).  Abalone digestive-gland cells were least negatively affected by hormones in December, affected to a significantly greater degree in May and August, and were most significantly affected in April (p < 0.05, SNK). Figure 5.5 illustrates seasonal changes in gonad and digestive-gland indices. Gonad index showed a strong seasonal effect (F  4>45  = 4.4; p = 0.005), with lowest values occurring post-  spawning in late summer through winter (July, August, December: 6 - 9 %) and highest values in  95 Figure 5.2 Percent change in oxygen consumption (VO2) by isolated digestive-gland cells of male and female abalone Haliotis kamtschatkana after administration of either glucose (GLU), amino acids (AA), or glucose + amino acids (GLUAA). Trials were conducted five times over a 15month period. Each point represents mean % change in VO2 ± S.E. for N = 5 abalone.  male - GLUAA male - GLU male - AA female - GLUAA female - GLU female - AA  200  150  100  50  0  A "i MAY  1  1—r  1—1—1—1—1—1—r  AUG  DEC  MONTHS  APR  JUL  96 Figure 5.3 Percent decrease in oxygen consumption ( V O 2 ) by isolated digestive-gland cells of male and female abalone Haliotis kamtschatkana after administration of 100 u M recombinant bovine somatotropin (cells were provided with 50 mM glucose and 50 \il of saturated amino acid solution prior to administration of the hormone). Trials were conducted five times over a 15month period. Each point represents mean % change in V 0 2 ± S.E. for N = 5 abalone.  • •  MAY  1  1 1 1 1 AUG  r  DEC  MONTHS  male female  97 Figure 5.4 Percent decrease in oxygen consumption ( V O 2 ) by isolated digestive-gland cells of male and female abalone Haliotis kamtschatkana after administration of 100 p\M somatostatin (cells were provided with 50 mM glucose and 50 p.1 of saturated amino acid solution prior to administration of the hormone). Trials were conducted collected five times over a 15-month period. Each point represents mean % change in VC>2± S.E. for N = 5 abalone.  70 A 60 50 A  n  40 30 20 10 0 MAY  1  r  AUG  1  1  1  r  DEC  MONTHS  1  1 APR  1  r JUL  •  male  •  female  98 Figure 5.5 Gonad and digestive-gland indices of abalone Haliotis kamtschatkana collected five times over a 15-month period. Each point represents mean indices (% total soft tissue mass) ± S.E. for N = 5 abalone in the case of gonad index and N = 10 abalone (5 male and 5 female) in the case of digestive-gland index.  GONAD INDEX 10 H  \  ^  5H  X  /  / /  / \  \  0  DIGESTIVE-GLAND INDEX 10 H  i MAY  i  i i AUG  i r  i DEC  MONTHS  i APR  i  i  i JUL  •  male  •  female  99 spring prior to spawning (April, May: 10 - 11 %;p < 0.05, SNK). There was also a strong sex effect on gonad index ( F  i;4g  = 9.7; p < 0.004), with the mean index in males (10 %) being  significantly higher than in females (7 %; p < 0.05, SNK). In contrast, there was no sex effect on digestive-gland index (Fi, = 0.5; p = 0.47; the figure represents values for males and females 48  combined). A strong seasonal effect on digestive-gland index (F  4>45  = 9.6; p < 0.001) resolved  into a generally low value in winter (December 1995: 7 %) and higher values in summer (August 1995 and July 1996: 9 and 12 %, respectively), with some statistical overlap between these and the spring values (p < 0.05, SNK). There was no significant correlation, either positive or negative, of the seasonal variation in gonad and digestive-gland indices (r„ = 0.3, p > 0.20, SRCCA). There was, however, a perfect correlation between digestive-gland index and metabolic response of the cells to G L U and to G L U A A (r = 1.0;/? < 0.05, SRCCA). Thus, when digestive s  glands were smallest in winter 1995, metabolic response was least and, conversely, when digestive glands were largest in summer 1996, metabolic response was greatest.  5.5 DISCUSSION Metabolic activity of abalone digestive-gland cells, as measured by oxygen consumption, followed a seasonal pattern. Oxygen consumption of isolated digestive-gland cells of Haliotis kamtschatkana (both male and female) was highest in spring/summer and lowest in winter. This pattern paralleled seasonal changes in water temperature, food availability, and gametogenesis, all of which would be expected to affect metabolism. Seasonal changes in metabolic activity of the digestive gland were complemented by the changes in metabolic activity which occurred in response to addition of nutrient substrates in vitro (Figures 5.1, 5.2, and 5.3). Glucose, as well as a mixture of glucose and amino acids, elicited the same increase in cellular oxygen consumption (70 % over baseline). This response was most  100 pronounced in spring and summer when metabolic activity was greatest. In contrast, amino acid substrate alone did not elicit an increase in cellular oxygen consumption during any season. Abalone store energy as glycogen rather than fat or protein (Livingstone and de Zwaan, 1983; Carefoot et al, 1993; Takami et al, 1995). The use of stored glycogen to fuel maintenance, as well as reproductive and somatic growth, indicates a carbohydrate-based metabolism (Webber, 1970; Hayashi, 1983; see also Chapter 3), and explains the greater responsiveness of the cells to carbohydrate substrates. The response to glucose substrates was also greater in female abalone than in males. This greater metabolic responsiveness cannot be explained as greater gametogenic growth in females, since gonads in both sexes increased by the same mass during spring 1996. Webber (1970) has shown, however, that immediately prior to spawning the female gonad in Haliotis cracherodii consists of about 35 % dry mass lipids, as compared with only about 10 % in male gonads. Most of this lipid is allocated to the large, lecithotrophic eggs which female abalone produce by the millions (Poore, 1973; Giorgi and DeMartini, 1977; Mottet, 1978; Hayashi, 1980), and which contain sufficient energy to sustain the larvae for two weeks without feeding (Mottet, 1978; Morse, 1984; Morse and Morse, 1984). A similar increase in lipid content of the digestive gland which is transferred to the ovary during vitellogenesis has been reported for brachyuran crabs (Mourente etal, 1994), shrimp (Spaargen and Haefner, 1994), and prawns (Joshi and Diwan, 1996). Since biosynthesis of lipids is more energetically costly than biosynthesis of either protein or carbohydrate, this may have accounted for the difference measured in metabolic activity of the digestive glands of the two sexes. The metabolic activity of isolated digestive-gland cells from adult abalone was not enhanced by addition of growth hormones. These results agree with those from an in vivo trial of hormone treatments, where neither somatic nor reproductive growth in Haliotis kamtschatkana  101 were enhanced by injection of either somatotropin or somatostatin (see Chapter 4). My in vitro trial circumvented the tissue damage from repeated hypodermic injections which I cited in Chapter 4 as a possible confounding factor in the experimental protocol. Instead, digestive-gland cells were directly exposed to somatotropin and somatostatin. The abalone digestive gland performs such growth functions as storage and metabolism of glycogen, and manufacture of proteins. Furthermore, metabolism of the digestive gland of other gastropods has been shown to be under neuroendocrine control (Reddy et al, 1989) and its tissues contain growth hormones (Bride and Gomot, 1989). Nonetheless, metabolic activity of isolated digestive-gland cells was not enhanced by the hormone treatments. There were, in fact, significant inhibitory effects which corresponded to periods of high gametogenic activity. Therefore, unlike growth in salmonids (Devlin etal, 1994) and juvenile abalone (Morse, 1981; Kawauchi and Moriyama, 1991), growth in adult abalone does not appear to be enhanced by exogenous somatotropin and somatostatin. Gonad indices in reproductively mature abalone range generally from 18 - 20 % of the soft tissue mass (Webber and Giese, 1969; Webber, 1970). Although some species spawn more or less completely, leading to gonad indices of essentially zero (e.g., Haliotis cyclobates and H. laevigata: Shepherd and Laws, 1974), others show less dramatic or non-significant seasonal changes (e.g., H. rufescens: Young and DeMartini, 1970; and H. ruber: Shepherd and Laws, 1974), and gonad indices for these species may not be indicative of either gametogenic or spawning activity. In my study on H. kamtschatkana, gonad indices for both males and females showed summer lows corresponding with spawning, but values remained above 5 %. Since seasonal highs were only 7 and 10 % for females and males, respectively, spawning was neither complete nor of high magnitude during 1995-96 for this population of H. kamtschatkana. The results of my study did not show a clear relationship between the sizes of gonad and digestive gland in Haliotis kamtschatkana. Indeed, the few studies in which the two glands have  102 been measured simultaneously offer varying views of the interrelationship of these indices. For example, Boolootian et al. (1962) found a strong inverse correlation of the sizes of gonad and digestive gland in H. cracherodii, and a less evident but nonetheless reciprocal relationship in H. rufescens. Webber (1970) also showed gonad and digestive-gland indices to be reciprocally out of phase in H. cracherodii, but to a lesser extent than observed in the former investigation, and my investigation showed an out-of-phase relationship in H. kamtschatkana. Boolootian et al. (1962) interpreted their data to mean that the digestive gland of//, cracherodii stores nutrients for gametogenesis, but this was later shown by Webber (1970) not to be the case. My data suggest an alternative explanation, namely, that the digestive gland of H. kamtschatkana and perhaps other species of abalone is not a repository for nutrients destined for reproductive growth, but rather for those destined for somatic growth. Somatic growth in many temperate abalone species occurs as a brief post-spawning growth episode (Cox, 1962; Foster, 1967; Poore, 1972; Sainsbury, 1982; Keesing and Wells, 1989). Thus, the peaks of gonad index and digestivegland index are out of phase with one another because reproductive growth precedes somatic growth. Thus, garnering the nutrients to sustain gametogenesis may actually be competitive with somatic growth in abalone.  103 GENERAL SUMMARY Development of formulated diets for abalone demands knowledge of their nutritional requirements, growth, and metabolism. This thesis has addressed several aspects of these issues. Protein requirement was investigated from the standpoint of how protein utilization was affected by dietary protein content, amino acid balance, and protein-sparing effects together with the possible amelioration of the last through enzymatic adaptation to diet. My focus on protein utilization stemmed from the fact that protein is a costly dietary ingredient and the only one which can supply the materials needed for protein growth. With respect to growth, I assessed the ability of vertebrate hormones to enhance growth in adult abalone. In consideration of metabolism, I investigated the abalone digestive gland (a primary site of metabolic conversions) from the standpoints of seasonal variation in metabolic activity and the relationships between size and metabolic activity of the digestive gland and size of the gonad over the reproductive cycle. The dietary protein content required for optimum protein utilization (maximal retention of dietary protein in body tissues) was determined to be approximately 20 % dry mass (Chapter 1). This was similar to previously reported findings for abalone. Higher dietary protein content is currently employed in four of the 12 existing commercial diets for abalone (see Fleming et al, 1996 for review); however, formulation of these diets is based upon optimizing total growth rather than protein utilization. Whereas growth in abalone is positively related to increasing amounts of dietary protein, protein retention is negatively related (Uki et al, 1986a). Results of my investigation indicated that when dietary protein is 30 % dry mass, utilization is inefficient because protein is catabolized to produce energy. The effect of amino acid balance on protein utilization was determined in my second investigation (Chapter 2). Similar amounts of dietary amino acid were retained by abalone fed diets with amino acid balances mimicking either abalone whole body, abalone ovary, or kelp, or a  104 diet with a balance of mainly glycine. The fact that the mainly glycine diet could sustain abalone suggested that these animals obtained amino acids from exogenous sources such as symbiotic gut bacteria or seawater. I therefore concluded that amino acid balance may not be a major concern when formulating diets for Haliotis kamtschatkana. The greater protein-sparing effect of carbohydrate relative to fat for abalone was demonstrated in my third study (Chapter 3). I also noted that the superior protein-sparing effect of carbohydrate was not ameliorated by enzymatic adaptation to the diets, despite the increase in activities of digestive enzymes observed in abalone fed the formulated diets. From myfirstthree investigations I concluded that to insure efficient use of dietary proteins by Haliotis kamtschatkana a formulated diet should contain 20 % dry mass protein and should contain sufficient carbohydrates to meet the animal's energy needs. Mass gain by abalone in my investigations was low by aquaculture standards. However, a comparison of the mass changes observed in Haliotis kamtschatkana to a culture situation is inappropriate because here the diets were formulated to optimize protein utilization rather than to optimize growth. The slow growth of abalone in my studies may have resulted from deficiencies in lipid metabolism (see Chapter 3) since previous studies indicate that the carbohydrate and protein sources were appropriate (see Chapter 1), or from difficulties in husbanding this species. The slow growth observed in abalone fed my formulated diets, however, does not mean that protein utilization was not taking place since any growth (even tissue maintenance) requires the retention of dietary protein. Growth is of vital concern in animal culture so, accordingly, my fourth investigation (Chapter 4) evaluated the efficacy of hormone treatment for enhancing growth in cultured abalone. While such hormone treatment is known to enhance growth in cultured salmonids, (see McLean and Donaldson, 1993 for review) injection of recombinant vertebrate growth hormones did  105 not enhance growth of adult abalone. Thus, I concluded that injecting adult abalone with vertebrate growth hormones is inadvisable. Seasonal study of the abalone digestive gland (Chapter 5) provided insight into seasonal variation in metabolism. Metabolic activity of abalone digestive-gland cells followed a seasonal pattern which paralleled seasonal changes in water temperature, food availability, and gametogenesis, all of which would be expected to influence metabolism. These seasonal changes were complemented by the in vitro cellular response of the digestive gland to nutrient substrates, and these responses were reflective of the carbohydrate basis of abalone metabolism (see Chapter 3). When vertebrate growth hormones were administered to digestive-gland cells isolated from adult abalone they caused a decrease in metabolic activity. Abalone gonad and digestive-gland indices were not related to one another nor to sex, and I concluded that garnering nutrients to sustain gametogenesis is likely competitive with somatic growth in abalone. This finding is relevant to abalone culture. In general, my investigations have contributed to the understanding of abalone biology and, in particular, they have provided information pertinent to abalone culture. With respect to formulation of diets for Haliotis kamtschatkana, I have determined that to optimize protein utilization diets should contain about 20 % dry mass protein and have carbohydrates rather than fats as their energy source. I also determined that treatment with vertebrate growth hormones is not a means to enhance abalone growth. Finally, since nutrient metabolism in abalone is seasonal and likely coupled with competition between reproductive and somatic growth, abalone culture may benefit from investigation of techniques to curtail reproductive growth.  106 REFERENCES Ackefors, H., J.D. Castell, L.D. Boston, P. Raty, and M . Svensson. 1992. Standard experimental diets for crustacean nutrition research JJ. 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