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J HIMEIWWWlmflfilmlflflfiflil'llflfl L 93 01090 7743 (“5‘5 Lunar W State University This is to certify that the thesis entitled EFFECT OF FEED COLOR ON GROWTH AND FEED ACCEPTANCE PARAMETERS 0F WALLEYE FRY AND FINGERLINGS presented by Michael Francis Masterson has been accepted towards fulfillment of the requirements for Master Mange—degree in F1 sherigg & Wildlife Major professor D 8 Dr. Donald ate _-i-_8_4___ 0-7639 MSUi: m- arm-mm» ‘ ‘ z m" ', Institution MSU LIBRARIES W \— RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. EFFECT OF FEED COLOR ON GROWTH AND FEED ACCEPTANCE PARAMETERS OF WALLEYE FRY AND FINGERLINGS By ‘~Michael Francis Masterson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Fisheries and Wildlife 1984 3/7‘iL/(s/ ABSTRACT EFFECT OF FEED COLOR ON GROWTH AND FEED ACCEPTANCE PARAMETERS OF WALLEYE FRY AND FINGERLINGS by Michael Francis Masterson Studies were conducted with walleye fry and fingerlings and lake whitefish fry to determine whether feed color contributed to artificial diet acceptance. Specially designed upwelling rearing systems were used to prolong feed suspension time. Walleye fry were not successfully started on an artificial diet. Walleye fingerlings were successfully converted from live food to artificial feed. No significant difference between survival of fingerlings fed zooplankton and those fed yellow artificial diet were observed. Significant differences in survival (P<=0.0S) were observed between yellow, red, and green artificial diets. Average survival was 90.0% fed yellow diet, 84.5% fed zooplankton, 48.9% fed red diet, and 8.9% fed green diet. Relative growth was not signi- ficantly different between any of the colored artificial diets or zoo- plankton reference diet. First feeding lake Whitefish fry accepted diets regardless of color. Study results indicate that dietary acceptance based on feed color is species specific. ACKNOWLEDGMENTS I am deeply grateful to Dr. Donald Carling, Dr. William Taylor, and Dr. Martin Balaban, my committee members for their committment, support, and guidance through the course of this study. Sincerest l appreciation to Dr. Carling for always managing to provide financial assistance. I am much indebted to Doug Sweet, Tony Ostrowski, Shawn Abbot, Valisa Dowell, Mike Feeley, Don HOOpes, Jeff Mackenzie, Val McSweeney and Bob Rice for volunteering the services during the experiments. I especially want to thank Doug Sweet for assisting in the feeding of the fish and the cleaning (Df the rearing units and the lab. Doug's interest was as invaluable as his help. I would also like to thank Mr. Harry Westers, Michigan Department of Natural Resources, Fisheries Divsion,for his guidance and support, Mr. Jim Copeland, MDNR, Wolf Lake State Fish Hatchery, for providing walleye fry, Mr. Ken Dodge and Mr. John Trimberger, MDNR, Fisheries Division, for supplying walleye fingerlings, and Mr. Gary Lamb, commercial fisherman, for allowing us to accompany him and strip whitefish eggs and milt from a portion of his catch. A special appreciation to Susan Hazard for contributing her clerical expertise in the organization of this thesis. Finally, I would like to eXpress my sincerest love and appreciation to my parents and Gigi Valentine for their patience and support. ii TABLE OF CONTENTS Page LIST OF TABLES ................................................ 1‘7 LIST OF FIGURES ............................................... V INTRODUCTION .................................................. 1 LITERATURE REVIEW ............................................. 4 MATERIALS AND METHODS ............................ . ............ 15 I. General ..... ........................................... 15 II. Experimental Diet Preparation .......................... 17 A. Water soluble colors ................................ 17 B. Water unsoluble colors ............................. 21 III. Experimental Systems .................................... 21 A. Fry rearing trays .................................. 21 B. Fry and Fingerling Rearing Units ................... 23 C. Automatic feeders .................................. 27 D. Lighting ........................................... 31 IV. Experimental Procedure ................................. 31 A. Walleye Fry ........................................ 31 B. Walleye Fingerling ................................. 34 C. Lake Whitefish Fry ................................. 37 V. Statistical Analysis ................................... 38 RESULTS ....................................................... 39 I. General ................................................ 39 II. Walleye Fry ............................................ 39 III. Walleye Fingerling ..................................... 42 IV. Lake Whitefish ......................................... 46 DISCUSSION .................................................... 49 CONCLUSIONS ................................................... 60 REFERENCES .................................................... 61 APPENDIX A .................................................... 70 APPENDIX B .................................................... 74 APPENDIX C .................................................... 78 APPENDIX D .................................................... 79 iii LIST OF TABLES Number Page 1 Size and survival of walleye fingerlings fed different types of practical feed ................................. 13 2 Percent composition of experimental diets on a dry weight basis ............... . ............................ 18 3 Vitamin mixture for use in purified fish diets (NRC 1978) .................. . .......... . ................ l9 4 Mineral mixture for use in purified fish diets (NRC 1978) ............................................... 20 Appendix C Number and percent of walleye fingerling mortalities containing food in their stomachs during fingerling Experiment 2 ...................... . ............ . ...... 78 iv LIST OF FIGURES Number Page 1 Diagram of upwelling tray (38 x 38 x 10 cm) used in walleye fry Experiment 1 ............................ 22 2 Diagram of one stack of three upwelling trays used in walleye fry Experiment 1 ............................ 24 3 Diagram of upwelling rearing unit (4 liter) used in walleye fry Experiments 2 and 3, fingerling Experiment 2, and the lake whitefish fry experiment .... 25 4 Photograph of system used in walleye fry Experiments 2 and 3, fingerling Experiment 2, and the lake whitefish experiment ................................... 26 5 Diagram of upwelling rearing unit with funnel assembly used in walleye fingerling Experiment 1 ....... 28 6 Photograph of one automatic feeder suspended over four upwelling rearing units ........................... 29 7 Relative survival of first feeding walleye fry in Experiment 1 ........................................... 41 8 Average survival of walleye fingerlings (4.0 cm TL) fed zooplankton or a yellow, red, or green artificial diet in fingerling Experiment 1 ........................ 4S 9 Average survival of first feeding lake whitefish fry fed Artemia nauplii or a red, green, or yellow artificial diet ........................................ 48 A1 Survival of walleye fingerlings (6.3 cm TL) fed a zooplankton diet ....................................... 70 A2 Survival of walleye fingerlings (6.3 cm TL) fed a yellow colored artificial diet ....................... 71 A3 Survival of walleye fingerlings (6.3 cm TL) fed a red colored artificial diet .......................... 72 A4 Survival of walleye fingerlings (6.3 cm TL) fed a green colored artificial diet .......................... 73 Bl Survival of walleye fingerlings (4.0 cm TL) fed a zooplankton diet ..................................... 74 Number Page 82 Survival of walleye fingerlings (4.0 cm TL) fed a yellow colored artificial diet ....................... 75 B3 Survival of walleye fingerlings (4.0 cm TL) fed a red colored artificial diet ................. . .......... 76 B4 Surivival of walleye fingerlings (4.0 cm TL) fed a green colored artificial diet ........................ 77 D1 Survival of first-feeding lake whitefish fry fed Artemia nauplii ........................................ 79 D2 Survival of first-feeding lake whitefish fry fed a green colored artificial diet ...... .................. 80 D3 Survival of first-feeding lake whitefish fry fed a red colored artificial diet .......................... 81 D4 Survival of first-feeding lake whitefish fry fed a yellow colored artificial diet ....................... 82 vi INTRODUCTION The Great Lakes region supports one of the largest freshwater sport fisheries in the world. In Great Lakes waters, excluding inland lakes and streams, roughly three million anglers took 35.7 million fishing trips and expended approximately $373 million in 1980 (U.S.D.I. 1982). Walleye (Stizostedion vitreum vitreum) is one of the most prized species of sport fish in inland waters and portions of the Great Lakes. Due to pollution, destruction of habitat, past commercial fishing, and the increasing interest in sport fishing, existing natural stocks are being depleted. Because of increased sport fishing demand, existing stocks of walleye are being supplemented and new walleye fisheries are being created by planting walleye fingerlings. Artificial propagation of walleye will play an increasingly important role in coolwater fish management. Traditionally, walleye have been cultured extensively using larval rearing ponds (Webster et a1. 1978). This culture method generally requires extensive areas of water. Usually small harvestable ponds, sewage lagoons, and barrow pits sparsely scattered over a large region are used. These ponds are sometimes fertilized with manure, yeasts, or organic fertilizers to increase primary productivity. Fry are stocked shortly after hatching and are allowed to grow for approximately two months. The ponds may be fertilized during this time to maintain high zOOpIankton density. When the zOOplankton population crashes the walleye fingerlings must be harvested immediately and stocked in the desired areas within the state. With increasing interests in walleye as a sport fish, the need for high density intensive culture methods has become apparent. Historically, hatcheries have had limited success in intensive culture of large numbers of walleye fry and fingerlings (Beyerle 1975,1979a; Cernohous 1973a,b; Nagel 1974; White and Copper 1973). Unfortunately, there is limited information on the intensive culture of walleye (Nickum 1978) and even less about their nutritional requirements (Ketola 1978). The problem originates at the first feeding stage where high mortality occurs apparently from a lack of practical feed acceptance. The development of a practical feed acceptable to first feeding walleye would greatly increase the efficiency and production of walleye in hatcheries. Because walleye are primarily sight feeders (Mathias and Li 1982), color may have a significant effect on larval walleye feed acceptance. Little work has been done to determine whether larval fish have color vision, although a number of studies have shown that the eyes of larval fish are well deveIOped (Ali 1959; Blaxter and Jones 1967; Blaxter and Staines 1970; Guma'a 1982; Sandy and Blaxter 1980). The objective of this study was to determine whether walleye distinguish between feed colors and preferentially accept one colored feed over another. Because lake whitefish fry are available at a different time of the year, they were used in secondary experiments to determine adequacy of the system. First-feeding lake whitefish are similar in size to walleye and are also reluctant to accept artificial feeds (Raisanen and Behmer 1982). Current efforts to commercialize walleye culture are impeded by the inability to rear these fish intensively on practical diets. Results from this study will provide public and private aquaculturists with acceptable practical diets for intensive culture of walleye. LITERATURE REVIEW The first feeding stage of larval walleye is the greatest barrier to the intensive culture of this species. Fish culturists use the term "critical period" to refer to stages when mortality among captive larvae is high (May 1974). Fabre-Domergue and Bietrix (1897) first used critical period to describe the time of complete yolk absorption when they observed high larval mortality of marine fishes during laboratory rearing attempts. Not only is first feeding stage considered a critical period, but the point when fingerlings convert from zooplanktivorous to piscivorous can result in increased mortality and can also be considered a critical period. Walleye fry are 5-7 mm at hatching and grow to 7-9 mm on endogenous nutrient reserves before feeding is initiated (McElman and Balon 1979). Walleye fry will accept live food more readily than artificial feed. Mathias and Li (1982) determined first feeding walleye had a mouth gape of 1.4- 1.8 mm and suggest walleye fry could ingest prey of a size up to 80% of their mouth gape. A wide size range has been reported for food items of first feeding larvae. Beyerle (1979a) observed larval walleye feeding on 0.2- 0.4 mm Artemia. Daphnia species 0.4-1.6 mm in length have been observed in the guts of larval walleye (Beyerle 1979b; Mathias and Li 1982; Merna 1977). Bosmina 0.33-0.39 mm in length (Merna 1977), and copepods 0.4-1.1 mm in length (Beyerle 1979b), have also been observed in the guts of larval walleye. Prey size selection is more important than species selection (Mathias and Li 1982). A wide size range of artificial feed has been offered to first feeding walleye. McElman and Balon (1979) observed larval walleye feeding on 0.03 mm particles of feed, but cautioned that acceptance of the small size feed particle may be incidental due to the high concentration of feed in their system. Generally, the size of artificial diets acceptable to fry ranged from 0.2 mm to 0.5 mm diameter (Nickum 1978). The mouth, jaws, and teeth of the walleye are well developed by the time feeding is initiated (McElman and Balon 1979). First feeding walleye rely on vision to capture food and are considered "strike" feeders (Mathias and Li 1982). They make visual contact with a food particle, orient toward it to bring it into the visual field of both eyes, slowly approach to within 1.5 to 3.0 mm, then finally strike by curling into an S shape and lunging at the prey. As walleye age and the morphology of the mouth becomes more developed, prey will be captured by a combination of grasping and suction (Elshoud-Oldenhave 1979). Vision plays an important role in the feeding of larval fish but little is known how vision affects first feeding behavior. The physical properties of the water itself affect what a fish can see. In clear freshwater such as the Great Lakes, water acts as a monochromater, absorbing red wavelengths while transmitting blue and green wavelengths (Nicol 1975). In turbid coastal waters, light of yellow wavelengths is transmitted maximally (Jerlov 1968). Fishes have eyes adapted for optimum sensitivity to transmitted light within the environment they live (Nicol 1975; Wang and Nicol 1974). Many studies have been performed to determine the morphology of the eye of larval and adult fishes. The eyes of most adult fishes are comprised of two main types of visual cells. Rod vision is generally more active in the peripheral retina and is primarily involved in light intensity (Crescitelli 1972) and movement perception (Blaxter 1975). The cone cells function in color reception and determination of visual acuity (Crescitelli 1972). In. larval fishes the retina is comprised entirely of cone cells at least until metamorphosis. Ali (1959) reported that eyes of the young stages of Oncorhynchus species contained no rods in the retina. Larval Eurasian perch, Perca fluviatilis (Guma'a 1981), haddock, nile perch, and stickleback (Blaxter and Staines 1970) also had pure cone retinas with no rods present. Larval herring and plaice (Blaxter and Jones 1967; Blaxter l968a,b) and sole (Blaxter 1969; Sandy and Blaxter 1980) are also characterized by eyes with no rods, though they are present in the adults. The eyes of adult Stizostedion have both rods and cones. The rods are extremely small (Ali et al. 1977) and the cones are enormous compared with those of other teleosts (Zyznar and Ali 1975). Apparently there has been no studies on the eyes of larval Stizostedion, but the possibility of a rod-free retina is great. The pure cone retina means that larvae presumably cannot perceive the same wide range of intensity of light as the adult (Blaxter 1975), although as the sac fry develOp, so does visual acuity (Hairston et a1. 1982). It would also seem reasonable that color perception would also increase through the first feeding stage. Morphological studies of larval fish eyes have shown that they contain the necessary visual pigments for color vision. Larval carp (Barker and Bryant 1981), goldfish (Harosi and MacNichol 1974), and herring (Blaxter 1968a) have green, blue, and red receptors in the retina. Trichromatic color vision, though, is not uniform throughout the teleost. Percids such as the European pike-perch, Stizostedion lucioperca (Burkhardt and Hassin 1978), perch, Perca fluviatilis (Cameron 1982), and walleye, Stizostedion vitreum vitreum (Ali et a1. 1977; Levine and MacNichol 1980) were found to contain only red and green absorbing cones in the retina. Few behavioral experiments have been undertaken to determine whether fish distinguish one color from another. McLeary and Bernstein (1959) demonstrated that goldfish are capable of distinguishing red from green independently of stimulus brightness. Muntz and Cronly-Dillon (1966) confirmed goldfish could be trained to discriminate between blue, green, and red using behavioral feeding tests. Bluegills reacted preferentially to red when offered food in a red or green illuminated area (Hurst 1953). In feeding experiments with carp fry using red, green, and blue diets and red, green, and blue colored tanks, Barker and Bryant (1981) found that green diets consistently outperformed other colored diets irrespective of background color, whereas increased contrast between diet color and background color was associated with poorer performance. This is not surprising since carp are omnivorous and feed on vegetation and benthos which contrast little with the turbid waters in which they live. The Opposite response was found in rainbow trout. Ginetz and Larkin (1973) observed that higher contrast between food color and background color increased the preference for the food color. Rainbow trout, when offered two different colors of dyed rainbow trout eggs while varying the background color of the system, exercised consistent preferences for one colored food item in relation to another (Ginetz and Larkin 1973). Higher contrasting food items in the natural environment are more likely to stimulate a feeding response in rainbow trout because they are primarily sight feeders. Presently, the most successful technique for raising larval walleye to fingerlings is extensive culture utilizing larval rearing ponds. The general procedure for raising walleye fingerlings is to place sac fry in small ponds. The key to successful pond culture of walleye is to maintain a high ratio of food organisms to fish (Westers 1980). Production of zooplankton is sometimes stimulated by fertilization of the pond. Walleyes can be raised to a size of 2.0 to 3.0 inches before starvation and cannibalism become major factors of mortality. Early attempts to extensively culture walleye yielded extremely variable results because there were no standards of monitoring the physical and environmental parameters that can affect walleye production. Smith and Moyle (1943) determined that rudimentary factors, such as cannabalism and pond fertility influenced pond production of walleye. But Miller (1952) observed low survival of walleye fry reared in constructed earthen ponds in Minnesota using many of the same techniques. In 1968, survival rates from 34 ponds in Wisconsin ranged from 0 to 40.5% and averaged 7.8% (Klingbiel 1969). In 1971, Michigan Department of Natural Resources planted walleye fry in 13 ponds and small lakes. Survival ranged from 0 to 24.4% and averaged 9.8% (Laarman and Reynolds 1974). Extensive culture techniques were refined so that by 1978 walleye fry were cultured in Michigan with survival rates as high as 90% or more (Beyerle 1979b). However, in 1983, 69 Michigan ponds, small lakes, 10 and barrow pits stocked with 11.2 million walleye fry produced only 2.4 million fingerlings for a 21.4 percent survival rate. Wide variation in survival between ponds and annual fluctuations of walleye production in the same pond have led to inaccurate estimates of fingerling production. Most studies of extensively cultured walleye have been performed under production situations rather than under controlled scientific circumstances and thus remain more "art" than science. DeveIOpment of intensive culture techniques for walleyes can serve to centralize larval and fingerling culture and increase the reliability of production parameters. Intensive culture of walleye has been unsuccessful because of the inability to rear fry to the fingerling stage on artificial diet (Beyerle 1975,1979a; Nagel 1974; Nickum 1978). Inability to feed an artificial diet to fry and fingerlings may result from problems such as palatibility, form, texture, and motility which may vary with the age and size of the fish. Walleye fry and fingerling have been reared using many different systems and techniques. Troughs, rectangular, square, and circular tanks, aquaria, and jars have all been used in attempts to culture walleye. Reported fry behavior varies between rearing systems but flow through tanks are generally more successful than static tanks (Krise and Meade 1982). 11 Bulkowski and Meade (1981) determined walleye fry up to 32 mm in total length were photopositive and sought the brightest light conditions available. As the fish grow, behavior shifts to photonegative and they seek low light levels. Scherer (1976) found that walleye fingerlings concentrated at the bottom of his test units in light of 200 lux, while decreasing light intensity distributed fish more evenly throughout the units. In troughs that were equally divided into black and white sections, 95% of walleye fry chose the white halves (Nickum 1978). Woynarovich (1960) determined that light levels of 400 lux or greater can be detrimental to the survival of 4-6 mm prolarval of the closely related European pike-perch (Stizostedion luciOperca). Nickum (1978) found that walleye fry reared under normal lighting conditions in white troughs were so attracted to the sides of the trough they ignored all forms of live and artificial feed while fry in black troughs were evenly dispersed and readily accepted live food. Therefore, flow through rearing units of a dark or neutral color illuminated with low intensity overhead lights may reduce the amount of stress the fry will experience. Although fry have not been successfully started on an artificial diet, walleye fingerlings are comparitively easy to convert from a live diet to an artificial diet. Early attempts to intensively culture walleye mainly concerned weaning fingerlings from live food to artificial feed. These trials met with varied success as each investigator 12 modified feed, feeding regime, and rearing technique. In most cases, walleye fingerling have accepted all of the artificial diets tested (Cheshire and Steele 1972; Jahncke 1979; Lemm et al. 1980; Richard 1983; Zitzow 1983). Fingerlings initially fed brine shrimp and/or zooplankton then offered increasing ratios of dry feed to live food during a conversion period lasting 5 to 14 days generally have better survival rates than those fingerlings that were directly converted from live to artificial feeds (Nickum 1978). Investigators have converted walleye fingerlings of various sizes to many types of artificial diets (Table 1). Water and air upwelling currents have been utilized to keep both live and artificial feeds in suspension allowing the fry more time to capture it. Walleye fry will not pick feed off the bottom of the tank (Krise and Meade 1982). Work at the National Fisheries Research and Development Laboratory indicated that an upwelling current produced by aeration helped keep live feed suspended in the water column and resulted in increased fish survival (Krise and Meade 1982). Corazza (1980) found that walleye fingerlings had greater growth and survival when reared in cylindrical hatchery jars with an upwelling water flow. Upwelling currents also give an artificial diet an appearance of movement which may stimulate feeding behavior. Water color may also play an important part in walleye feed acceptance. Vision may be improved when greenish pigments are present in the water (Levine and MacNichol 13 Table 1. Size and survival of walleye fingerlings fed different types of practical feed. Initial size of % fingerling, Diet Survival Reference 0.2 gm W31 2.0 Orme and Schultz 1972 0.3 gm one2 4.0.7 Nagel 1976 0.4 gm W13 50.2 Reinitz and Austin 1980 0.4 gm W7 48.2 " 0.64 gm W13 45.0 Jahncke 1979 0.7 gm W16 23.6 Bean 1983 0.7 gm Abernathy 2.4 " 0.7 gm Glance Atlantic 6.9 " Salmon diet 1.0 gm Purina Trout chow 20.0 Cheshire and Steele 1972 1.2 gm W7 13.7 Colesante and Youmans 1981 1.2 gm W14 33.5 " 4.1 gm W3 Huh 1975 29 mm 0MP 8.4 Beyerle 1979a 29 mm W7 5. " 30 mm W3 3.0 Orme and Schultz 1972 30 mm 0MP 11.0 " 41.5 mm 0MP 7.5 Beyerle 1979a 41.7 mm W3 0.3 " 45.0 mm 0MP 20.0 " 46.5 mm 0MP 61.3 " *No data given 1 Diets with a W prefix are standard practical feeds prepared by the U.S. Fish and Wildlife Service at the Diet Testing Center, Spearfish, SD. 2 0MP = Oregon moist pellet. 14 1980). Studies at the National Fisheries Research and Development Laboratory showed that walleye fry which positioned themselves against the sides Of a tank containing clear water were randomly dispersed or congregated under feed dispensers when algae laden pond water was dispensed into the tank (Krise and Meade 1982). These walleye fry appeared to feed better in green water. However, this behavior could not be replicated at the Aquaculture Laboratory of Michigan State University. Temperature is one of the most important factors affecting growth of fry and fingerling walleye. Rearing temperatures between 16 and 24 C is considered the Optimum range for growth of walleye fry and fingerlings (Hokanson 1977; Krise and Meade 1982; Smith and Koenst 1975) with 22 C as Optimum temperature for maximum growth (Beyerle 1975; Huh et al. 1976; Koenst and Smith 1976). Jahncke (1979) observed good fry survival and growth in 17-18 C water, while Nickum (1978) Observed poorer fry survival at 20 C. Nickum (1978) postulated that higher temperatures resulted in higher metabolism which led to exhaustion Of nutrient reserves before they were able to adapt to dry feeds. A sharp drop or rise in temperature had no great effect on walleye fry and juvenile survival (Koenst and Smith 1976). MATERIALS AND METHODS I. General Three colored diet performance studies were conducted with walleye fry and two colored diet performance studies were conducted with fingerling walleye. The systems used in these experiments were developed and constructed at the Aquaculture Laboratory on the campus of Michigan State University. These experiments were carried out during the spring and summer of 1982 and 1983 and the spring of 1984. The rearing units were redesigned between Fry Experiment 1 and Fingerling Experiment 1 in 1982. A minor modification was made between Fingerling Experiment 1 and Fry Experiment 2. Modifications were also made before Fry Experiments 2 and 3 to accomodate the small size of the fry. Diets were modified in 1983 by changing from water soluble dyes to water insoluble dyes. In Fry Experiment 1, red, yellow, green, or brown water soluble colored semi-purified diets containing 45% crude protein were fed to triplicate groups of first feeding larval walleye. A triplicated reference group Of first feeding larval walleye was fed zooplankton. In Fingerling Experiment 1, red, yellow, or green water soluble colored semi-purified diets 15 16 were fed to triplicate groups of walleye fingerlings. A triplicated reference group was fed zooplankton. In Fry and Fingerling Experiment 2, red, yellow, or green semi-purified diet colored with a water insoluble dye were fed to triplicate groups of walleye fry and fingerling. Triplicated reference groups Of fry and fingerlings were fed Artemia nauplii and zooplankton, respectively. During Fry Experiment 3, red, yellow, or green artificial diets or a reference diet of Artemia nauplia were offered to quadruplicated groups of walleye fry. Well water at a temperature Of 11.6:0.2 C and 12.1:0.4 C was supplied to each unit in Fry Experiments 1 and 2 respectively. During Fingerling Experiments 1 and 2 the well water was heated to 20.1:0.4 C and 18.6:0.5 C respectively using two Fisher Automerse Model 199 immersion heaters. During Fry Experiment 3 the well water was heated to 18.1:0.5 C using a 75 gallon commercial gas water heater. Heated water was mixed with cool water using a Powers NO. 11 Thermal Regulator to maintain the desired temperature. The water flowed from the mixing valve through a column of activated charcoal to prevent gas supersaturation into a reservoir where it was gravity fed into the rearing units at a flow of approximately 0.25 liters per minute per unit. In the fall of 1983 a secondary experiment was conducted with larval lake whitefish to determine the adequacy of the system design. Well water at a temperature of 11.4:0.10 C was supplied to each unit. Four replicates 17 Of each treatment were Offered red, yellow, or green artificial diets colored with water insoluble dyes, or a reference diet Of Artemia nauplii. 11. Experimental Diet Preparation A. Water soluble colors Four isocaloric semi-purified test diets were prepared using red, yellow, green, and brown food coloring (Tables 2,3,4). Percent composition of dietary ingredients was based on NRC (1978) recommendation for actively growing coldwater fishes. All dry ingredients were mixed in an industrial food mixer (Univex Model M-lZB) for twenty minutes. A mixture of soybean Oil and cod liver Oil was added slowly and mixed for twenty minutes to ensure complete homogeneity. Three milliliters Of liquid food color was added to warm water (60-70 C) for each diet. This solution was slowly added with mixing until the diet clumped tO a dough-like consistency. The dough-like material was broken up by hand into small (5 mm diameter) pieces and dried in a forced air drying oven (without supplemental heating) for 12 hours. A portion of the diet was pulverized using a Wiley mill (Model 4) and passed through a 0.5 mm screen yielding 0.1-0.5 millimeter powder. The other portion was cut in a Waring blender and passed through a 1.5 mm screen yielding 1.0-1.5 18 .q oanme mom COHuHmOQEOO pomm .m manme mom cowuwmoaaoo pomH om.o oo.m oo.o~ mq.o oo.q mm.o N~.o oo.o~ om.mH mm.©m oo.m oo.o~ mq.o oo.q mm.o NN.H oo.©m om.mfi ww.©m Asa o.m v OOHOU HHO HO>HH poo HHO coon >Om mpfiuoazo ocfiaoco NxHE Hmumcwz HxHE CHEQOH> mmOHOHHOOImsaad cfinuxma :Humaoo cfimmmo HOHOO pOOw OHBmOHOmCH umumz ucww< wmwuoaoo HOHOO poo“ manmsaom umumz l Abuse Go NV ucmaemuwcH .mfimmn ucwfioz >Hp m co mumwp Hmucoefiuoaxm mo :Ofiuamoaaoo unmouom .N OHBQH 19 szcxxonum uo\p:m Aezmv OOOOHOOxoxOprfi pmumaxusmo Oufimasmfin HOCHUHEwuxaxzumEHp OcofimeOZB Emuw H ome Ou mmoasaaoomsdam Ou pmppm mmfiufiucmzc ommzam moo.o NEE chamus> m~.o OHuOHm m.o Apflom OMHOLV cfiumaom o.~ Ousmpfixowucm m.~ Ho:.:HEmH:H m.H Ow>mamonfim m.~ ocflxOpHuzm o.o mumcm5O0ucma Eafioamo o.- BM OHEmuH> o.m~ pfiom OHnuoom< m m cwemufi> o.oN HOuHmocH cow ma OHEmOH> o.oofi awomwz oom < swamuw> co.0mq Ho.oswaono w\:H swamuw> xfiswumiw\wa cflsmufi> «w .Awmmfi omzv muofip Smfiw pmamwusa CH Om: DOM OMOOxHE :HEmOw> .m oanme Table 4. Mineral mixture for use in purified fish diets (NRC 1978). 20 Mineral g/kg premix CaHP04' ZHZO CaCO3 KHZPO4 NaCl MgSO4 KCl FeSO4'7H20 MnSOA-HZO ZnCO3 CuSO4- 5H20 KIO3 NaMooa-ZHZO COCl2 Na25e03 366.046 261.714 176.834 106.100 53.050 17.683 8.842 6.189 2.653 0.531 0.177 0.147 0.030 0.004 21 millimeter pieces. The diets were then placed in separate plastic food containers and refrigerated until used. B. Water insoluble colors. Three isocaloric semi-purified test diets (Tables 2,3,4) were prepared using red, yellow, and green water insoluble food coloring (Aluminum Lake Red #3; AL Yellow #5; AL Green #1). The dry portion of the diets was mixed and the Oil was added as described above. After twenty minutes of mixing to ensure homogeneity, the powdered colors were added. The mixture was again mixed for twenty minutes to insure complete incorporation of the color into each diet. Warm water (60-70 C) was slowly added with mixing until the diet clumped to a dough-like consistency. The dough-like material was dried, cut or ground, and stored as described above. 111. Experimental Systems A. Fry Rearing Trays Fry Experiment 1 was conducted in specially constructed larval rearing trays (Figure 1) measuring 38 x 38 x 10 cm maintained at 11.6:0.2 C with an upwelling air current. These experimental units were designed to provide an upwelling current to keep feed particles suspended in the water column imparting artificial motility tO the feed and allowing the fry more time to accept and ingest the particles than if they fell directly to the bottom. The TOP VIEW WATER @ [ AIRSTONE L—r IIIIIIIIIIIIIIIIIIIIIIIIIII III I .u-IIn-I-Iu-I-I-II-l-IIIII-I—m-IIOI II-IIII-l- llllll IIIIII-.-ICIIIII'll-IIII.-l-IIIII'IIII-IIIIIIIIIIIIIIII:III’:I:'alll _-.._--.-. ---.------I--n-Cc---.--—--.--.l“l--::-Il:I-m 1.0-4- IIIII ll IIIII I Il'glu- 4's...- SIDE VIEW DRAIN Figure 1. Diagram of upwelling tray (38 x 38 x 10 cm) used in walleye fry Experiment 1. 23 upwelling current was provided by a 12 inch airstone placed along the center of the unit (Figure 1). Fifteen units were arranged in five stacks of three (Figure 2). Each unit was supplied with fresh well water at a flow of approximately 0.25 liters per minute. B. Fry and Fingerling Rearing Units Due to inadequacies of the initial design, the rearing system was redesigned. The bottoms of four liter amber colored glass jugs were cut off (Figure 3). Twelve units were arranged in the system (Figure 4). Fiberglass screen with approximately 2 millimeter mesh was glued along the circumference of the top of each inverted jug. Two holes Of the appropriate sizes were cut in rubber stoppers which were placed in the spout of the jugs. One stOpper opening was made to accomodate 3/16 inch air line tubing, the other for a 3/8 inch piece of rigid plastic drain tube. A one inch airstone was attached to the 3/16 inch hole on the internal portion Of the stopper while air line tubing was extended from the external portion of the stopper to the air supply. A short piece Of flexible Tygon tubing with a clamp was attached to the external portion of the 3/8 inch drain tube. A section of 3/16 inch rigid plastic tubing was shaped and glued to the tOp of the unit to direct incoming water downward in a counter-clockwise direction (Figure 3). Effluent water flowed through the screen at the tOp of the unit. A section Of 1/2 inch CPVC pipe was glued to a TOP VIEW W— W i f I @ .............. L ......T.R.A.Y ......... {Lt-UL El TRAY I i Ln. 1 DRAIN SIDE VIEW‘ Figure 2. Diagram of one stack of three upwelling trays used in walleye fry Experiment 1. TOP VIEW WATER k -V<‘TER I - DRAIN VALVE SCREEN xi; La: El Al SIDE VIEW Figure 3. Diagram Of upwelling rearing unit (4 liter) used in walleye fry Experiments 2 and 3, fingerling Experiment 2, and the lake whitefish fry experiment. 26 Figure 4. Photograph of system used in walleye fry Experiments 2 and 3, fingerling Experiment 2, and the lake whitefish experiment. 27 plastic funnel (Figure 5). The diameter of the Open end of the funnel was slightly smaller than the diameter of the bottle and rested on wooden pegs roughly 1/4 inch off the bottom of the unit. The height of this assembly, when placed in the rearing unit, was approximately 1/4 inch below the water surface. Feed particles that sank to the bottom were drawn to the center Of the unit by the counter-clockwise current, lifted up through the funnel assembly by the upwelling air current, and resuspended in the water column. The design of the experimental units underwent minor changes prior to Fry Experiment 2. The funnel assembly was eliminated because the counter-clockwise flow combined with the air upwelling was thought tO-be sufficient to keep feed particles suSpended in the water column. Black, 200 micron mesh screen was glued to the top of the units during Fry Experiment 2 to prevent fish loss in the effluent. This screen was removed during Fingerling Experiment 2. Prior to Fry Experiment 3, white, 530 micron mesh screening was glued to the top Of the units at the point Of overflow. C. Automatic Feeders During Fry Experiment 1, each unit was hand fed every 15 minutes. This procedure necessitated using many volunteers to help feed each replicate at different times of the day. This proved very tedious and we were compelled to amend this situation. An automatic feeder (Figure 6) was no g... .‘ w I l 'u‘ ‘. . . .. .a., '. . A I. . \ .p . ,~ I ‘,. . I ‘ . v . u i! ‘ n... I \u. . . I e . ' h- - - . o' . . ~ I ” u ‘ 0’ O ' a ‘. . c . " u o H . I . O "i _1 .. . J T '..-d “. ~- g“: I' ' -' 7 . - I . f" . Q v " v . b. . -' ' I. .-“._,-.*‘ n -. I. x \ . I \. l '- . .v w." I‘ -~l~- ‘e*‘- an” Ar--n - a ‘ . \. a. .. . l ' ‘. V‘"""" ~‘ rap ‘. «MA '. ‘ .- .~-- «II-Manor I n . ' I 9'. 'l‘ l [‘3 04 TOP VIEW DRAIN VALVE SCREEN SIDE VIEW FUNNEL Diagram of upwelling rearing unit with funnel assembly Figure 5. used in walleye fingerling Experiment 1. Figure 6. Photograph of one automatic feeder suspended over four upwelling rearing units. 30 designed and built to allow feeding of each unit every fifteen minutes during the 12 hour light period. The automatic feeders were used in Fry Experiments 2 and 3 and in the lake whitefish experiment. Each feeder was simply constructed using inexpensive automatic household light timers, a sheet of plexiglass, and wooden dowels. Eight one inch wide strips of plexiglass were cut and glued to a one inch dowel to create the sweep arms. A large enough sheet of plexiglass to cover four units was measured and cut. Each of the four units were positioned such that if the square piece Of plexiglass was bisected by four lines through the center, the feeder could be suspended over the four units so that a unit could be centered under every other section. The dowel containing the sweep arms was then glued to the center of the clock-like timer which rotated once every 24 hours. Thus, each alternate sweep arm would pass entirely over one unit in a span of three hours. Three automatic feeders were fixed above three sets of four rearing units. The area over each unit was outlined with a grease pencil. Twelve half inch holes were staggered approximately every three and a half degrees allowing each unit to receive feed at 15 minute intervals. Sheets of black plastic were clipped to the portion Of the sweep arm passing over the holes to permit an efficient sweeping action. Feed was placed at the spot in front of the hole directly perpendicular to the action of the sweep arm. Each 31 feeder required loading every three hours during the feeding portion Of the day. Another automatic feeder was constructed prior to the experiment with the larval lake whitefish and Fry Experiment 3 when four additional units were added to the system. D. Lighting In Fry and Fingerling Experiment 1 lighting was supplied by overhead coolwhite fluorescent lamps on a 12:12 light:dark photoperiod. In Fry and Fingerling Experiment 2, Fry Experiment 3, and the larval lake whitefish experiment, lighting was supplied by a GE Chroma F40-C50 multi-spectrum fluorescent grow light. A cellulose triacetate filter was used as an intensity filter and ultraviolet filter (less than 0.01% transmission at 285 nm). Light intensity Of 6.3 2 . . uE/m sec or less was maintained. IV. Experimental Procedure A. Walleye Fry Experiment 1 Two day Old larval walleye were Obtained from the Michigan Department of Natural Resources Wolf Lake State Fish Hatchery in the spring Of 1982 and transported to the Michigan State University Aquaculture Laboratory. The fry were maintained in a 20 liter holding tank for a period of one day. Approximately 3 days after hatching, fifty 32 randomly chosen fry were transferred to each experimental unit. Red, yellow, green, and brown artificial diets colored with water soluble dyes along with a reference diet of zooplankton were fed to triplicated groups of fry on the fifth day post-hatch. The tanks were cleaned every other day starting on day 8 and continuing through the duration of the 17 day experiment. Experiment 2 Larval walleye were obtained from Wolf Lake State Fish Hatchery in the spring Of 1983, transported to the MSU Aquaculture Laboratory, and maintained in a 20 liter holding tank of the same basic design as the experimental fry and fingerling rearing unit for one day. One hundred randomly selected fry were transferred to each of 12 experimental units. Upwelling in the rearing units was created using two Silent Giant air pumps. The design of the experimental unit underwent minor changes prior to Larval Experiment 2. The funnel assembly was eliminated. The automatic feeder systems were used and required loading every three hours. Water was maintained at 12.1:0.4 C and gravity fed to the rearing units. Air was supplied to the units by two Silent Giant air pumps. 33 Due to unforseen difficulties in experimental system design, initial feeding of fry was delayed until 10 days posthatch. The automatic feeder system was utilized to allow feeding at 15 minute intervals during the 12 hour light phase of the photOperiod. Red, yellow, and green artificial diets along with a reference diet Of Artemia nauplii were fed to triplicate groups of walleye fry. Mortality was examined daily. Temperature was recorded weekly. Experiment 3 Larval walleye were again obtained from Wolf Lake State Fish Hatchery and transported to the MSU Aquaculture Lab in the spring of 1984. They were maintained in a 20 liter holding tank of the same basic design as the experimental rearing units while fifty randomly selected fry were dipped from the holding tank with a small piece of screen and transferred to each of 16 experimental fry rearing units. The automatic feeder system was utilized to permit feeding at 15 minute intervals during the light phase of a 12:12 light:dark photOperiod. Four additional units were added to the larval fish rearing system, allowing four replicates of each treatment. Red, yellow, and green artificial diets along with a reference diet of Artemia nauplii were fed to four replicates Of each treatment. Mortality was recorded daily. 34 Water was gravity fed from the reservoir to the experimental units at approximately 0.25 lpm per unit. Temperature was monitored on a weekly basis. Cursory examination of the experimental units occurred on a daily basis to determine when no survival occurred. On day 3, the first trial was terminated; a second trial was initiated the following day. Forty fry were transferred to each unit using a pipette. The flow was reduced slightly to approximately 0.20 liters per minute and the aeration was reduced to minimum. The fry were provided with Artemia nauplii on day 1-3. On day 4 of the second trial, heavy mortalities were sustained in many units and the experiment was terminated. To ensure that gas supersaturation was not the cause of mortality, total gas saturation was measured using a Novatech Design Ltd. Gas Tensionometer. Oxygen and nitrogen saturation were determined from this reading. B. Walleye Fingerling Experiment 1 Walleye fingerlings (6.310.24 cm TL), were Obtained from the Michigan Department of Natural Resources, Fisheries Division extensive walleye rearing ponds July 1, 1982. These fish were transported from the Grand Rapids, Michigan area to the Aquaculture Laboratory at MSU and acclimated to 20 C aerated water in a 500 liter tank. These fish were supplied with zooplankton, mainly Daphnia spp. and Chaoborus 35 larvae. The following morning, 20 fish were randomly selected, weighed as a group, and placed in each of 12 randomly selected test units. The water in the reservoir was treated at 2 parts per million of potassium permanganate and at 2 parts per million Acriflavin as a prOphylactic treatment against disease. On day 1, triplicate groups of walleye were randomly selected in the system. One triplicate group was fed a reference diet Of zOOplankton throughout the experiment. Three triplicate groups were weaned from a live food to a red, yellow, or green artificial diet over a ten day period. These fish were fed ZOOplankton the first day, and on each consecutive day the amount of zOOplankton was decreased roughly ten percent while the amount of artificial diet was increased ten percent. Mortalities were removed and counted every other day. Each unit was fed by hand four times a day at three hour intervals during the light phase of a 14:10 light:dark photOperiod. Lighting was supplied by overhead GE Coolwhite fluorescent lights. Temperatures and flow for the entire system was measured and recorded on a weekly basis. Flow for each unit was determined by averaging the total flow. By day 14, those units fed an artificial diet necessitated cleaning. Twice weekly the funnel assembly was removed and excess feed and feces were drained from each unit. The same procedure was used with the replicates fed zooplankton although these 36 units remained much cleaner. The experiment ran 50 days including the 10 day weaning period. Experiment 2 Walleye fingerlings (4.0:0.3 cm TL) were Obtained from the Michigan Department of Natural Resources Jackson State Prison pond, Jackson, Michigan, and transported in a live box to the MSU Aquaculture Lab June 30, 1983. The fingerlings were acclimated to 15 C heated well water. The following morning, 15 randomly selected fingerlings were weighed as a group and placed in each of the 12 test units. The water in the reservoir was treated with 2 ppm potassium permanganate and 2 ppm Acriflavin to reduce stress and prevent disease. Each unit was provided with zooplankton, mainly Daphnia spp. and Chaoborus larvae, collected from ponds at the Aquaculture Lab. On day 1, triplicate groups of walleye were randomly selected. One triplicate group was fed a reference diet of zOOplankton throughout the experiment. Three triplicate groups were weaned from a live diet to a red, yellow, or green artificial diet over a ten day period. These units were fed zooplankton on day 1. On day 2 through day 10, the amount Of zooplankton was decreased and the amount Of artificial diet was correspondingly increased. After day 10, these units were fed exclusively on an artificial diet. Each unit was fed by hand four times at three hour intervals 37 during the light phase of a 12:12 light:dark photoperiod. Mortalities were removed, counted, and preserved every two days. The digestive tract of preserved walleye fingerlings were excised using a dissecting microscope. The digestive tract was Opened to determine whether or not it contained any food. A fish was determined to have food in its gut when a noticeable and distinguishable amount of either natural or artificial feed was found. Temperature and flow were measured and recorded on a weekly basis. Flow for each unit was determined by averaging the total flow. On day 14, a biweekly cleaning regime was initiated. Twice weekly excess feed and feces were drained from each unit. The same procedure was followed with the zooplankton replicates although these units remained much cleaner. The experiment ran 41 days including the 10 day weaning period. On day 41, the remaining fish from each unit were removed and weighed. C. Lake Whitefish Fry On October 31, 1983, Lake Huron lake whitefish eggs were stripped from gravid females, fertilized, and water hardened for 2 hours. They were then transported to the Aquaculture Lab on the campus of Michigan State University. The eggs were incubated in well water at a temperature of approximately 6 C for 68 days. At 50% hatching, the 38 temperature in the hatchery jars was slowly raised to 11.5 C. Eighty fry 11-13 mm TL were removed by pipette and placed in each of 16 walleye fry rearing units. Flow was approximately 0.25 lpm per unit. Red, yellow, or green semi-purified diet Of the same formulation as that used in Fry and Fingerling Experiment 2 were offered to four replicates of each treatment using the automatic feeder system. Four other replicates were Offered Artemia nauplii four times daily during the light phase of a 12:12 light:dark photoperiod. Mortalities were removed and counted daily during the 43 day experiment. Temperature and flow were recorded at weekly intervals. This experiment was performed to determine whether design changes made in the rearing units were adequate for use with fry of a relatively small size. V. Statistical Analysis A two way analysis Of variance was performed to test for interactions between each treatment. A Type I probability of error of 0.05 or less (P50.05) was considered statistically significant. When differences occurred, comparisons were made using Scheffe's Test (Scheffe 1959). RESULTS 1. General. Walleye fry were never successfully started on an artificial diet. Neither diet or rearing system seemed tO be the causative agent, although system design flaws in Fry Experiments 2 and 3 may have interfered with feed acceptance. Walleye fingerlings were successfully converted from live food to artificial diet in both fingerling experiments. Significant differences (P<0.005) in survival were Observed between each colored diet. The fish rearing units were cleaned at regular intervals throughout each experiment. Good environmental tank conditions were maintained in each experiment except during a power shortage which occured during Fingerling Experiment 1. II. Walleye Fry. Experiment 1 The first attempt to start first-feeding walleye fry on artificial feed was unsuccessful. The experiment lasted 17 days until there were no survival in any of the test units. 39 40 Because feed acceptance was to be based on fry survival, mortalities were not counted and only a relative indication of survivlal rate for each unit could be estimated (Figure 7). All walleyes fed zooplankton died by day 5 of the experiment. The walleyes fed green and brown diets experienced total mortality by day 10 of the experiment. One yellow diet replicate and all red diet replicates survived longer than 10 days, but the last red diet replicate experienced total mortality on day 17 of the experiment. Experiment 2 The second attempt to start first-feeding walleye fry on an artificial diet was also unsuccessful. The screen mesh size on the rearing units was too large to retain the walleye fry in the unit. Unfortunately, replacement screening of the apprOpriate size was delayed in shipping. Consequently the feeding of the walleye fry was delayed until 10 days post-hatch. All units experienced 100% mortality between age 18 and 25 days. There was no difference in the rate of mortality between any of the treatments. Even those fish fed Artemia nauplii died by age 25 days. Mortalities in each unit were found either against the effluent screen or in the drain tube at the bottom. There was no evidence the fry had accepted artificial or live feed. 41 8 8 2 I «a S a o v N a _ _ _ _ _ . _ .1 _. _ x5, ., x / I. ..v. x y / one l/a I”, t/ / as. // /H/ / ’ / I a z a /. a ,. Z ../. x. , / .. , . . .. , .. / /.. 2 / , . c s / /. A. / ’ / l/ / aa/Ia / I— / // a/ 1. n/ z/ I I // ./ ”a — y / .X // r/ Ase I. )/ lav / / ”a, It d’/ It I I Xx. .4. x: r can //v6 o/’/ ./r/ .z// E5 22:258.: 1| /... O/ ES zmmec I // a. DE 2325 It /. .2 r B3 33...”; ..... 4. 2/ 5:. RE I! x)? .» v.0 9° 'IVAIAHOS Relative survival of first feeding walleye fry in Experiment 1. Figure 7. 42 Experiment 3 The third attempt at starting first-feeding walleye fry on an artificial diet also proved unsuccessful. On day 1 Of the experiment, all units experienced high mortalities. By day 3, there was less than 20% survival in any experimental unit and the first attempt was terminated. In a second attempt, up to 75% mortality occurred in the first 24 hours after transfer to experimental units. Some units had experienced 100% mortality, and survival in other units was less than 20%. By day 4, many units had 0% survival and the experiment was terminated. Total gas saturation was determined to be 101.8% where oxygen saturation was 102.1% and nitrogen saturation was 101.8%. 111. Walleye Fingerling. Experiment 1 Walleye fingerlings (6.310.24 cm TL) were successfully weaned from live to artificial diets over a ten day period with relatively low mortality. On day 9 though, 40% of the fingerlings in one yellow replicate died due to a bacterial infection. External symptoms suggested Columnaris to be the cause. This was the only instance of mortality which could be attributed to disease in Fingerling Experiment 1. On day 19, there were mechanical difficulties with the pumps that supplied water to the system. This resulted in mortalities in all units except one. There was greater than 43 50% mortality in four different replicates. One zooplankton replicate experienced no mortality, while the other two replicates only experienced one mortality each. Higher mortality rates were Observed in units fed an artificial diet. Water quality was visibly poorer (i.e. cloudy water) in these units. On day 33 a storm caused a 16 hour power failure in the laboratory. This resulted in the elimination Of flowing water and aeration in the system. Seventy-five percent of the units experienced one or more mortalities. All remaining fish in one red replicate died due to lack of oxygen. Of the four replicates that experienced no mortalities only one had greater than two fish at the time of the power failure. Survival at 50 days ranged from 0-15% fed red diets, 0-20% fed yellow diets, 0-10% fed zooplankton, and 0% fed green diets. Total percent survival in each treatment calculated by: Survival = ( total number surviving X 100 ) total initial fingerlings was 8.3% for fingerlings fed yellow diet, 6.7% fed red diet, 5.0% fed zooplankton, and 0% fed green diets. After conversion to artificial feed, only those walleye fed red diet were observed to be actively feeding, although white feces indicated fingerlings fed a yellow diet were feeding. 44 Experiment 2 Walleye fingerlings Of a smaller size (4.0:0.3 cm TL) than those used in Fingerling Experiment 1 were successfully weaned from a live diet of ZOOplankters to an artificial diet over a ten day conversion period. On day 14-17, 60% Of the fingerlings in one yellow diet replicate died of an apparent bacterial disease. External symptoms suggested Columnaris to be the causative agent. This replicate was determined to be an outlier (Steel and Torrie 1980) and was treated as such in further statistical analyses. During the first 25 days Of the experiment 91.55% of all mortalities were emaciated. Of the 8.45% mortalities that seemed in good health, half could be directly attributable to Columnaris disease. After day 25 no mortalities were emaciated. Significant differences (P<0.005) in survival were Observed between each colored artificial diet. NO significant difference was Observed between survival of fingerlings fed zooplankton and those fed a yellow artificial diet. Survival (:SD) at 41 days averaged 0.900 (0.033) fed yellow diet, 0.845 (0.039) fed zOOplankton, 0.489 (0.139) fed red diet, and 0.089 (0.102) fed green diet (Figure 8). Upon examination of the stomach contents of moribund fish, it was determined that only 5.9% Of their stomachs contained food. When differentiated by diet, 0% of the :2. [H ov mm om m>.l-| puma mum ..... huh: zummc.lll N.o 'IVAIAHOS 'ngs (4.0 cm TL) fed ingerli f yellow, red,cn:green artificial diet in fingerling Experiment 1. J of walleye Average survival zooplankton or a Figure 8. 46 mortalities fed zooplankton had food in the stomach, 2.7% Of the mortalities fed green diet had food in the stomach, 8.7% Of the mortalities fed red diet had food in the stomach, and 11.1% Of the mortalities fed yellow diet had food in the stomach. Relative growth (%) was not significantly different (P<0.05) between fish fed any of the artificial colored diets or zooplankton reference diet. Relative growth per day (:SD) was 2.8 (0.84)% for zooplankton replicates, 3.36 (1.59)% for red, 3.33 (0.39)% for yellow, and 4.01 (0.49)% for green artificial diet replicates. There was only one mortality Observed during the experiment that was directly attributable to cannibalism. On day 15 a fingerling in one of the red replicates was Observed with the anterior portion of a cohort protruding from its mouth. The cannibal was pursuaded to egest the fish which was dead and considered a mortality. There were four other suspected cannibalistic actions. One fish in each of two green replicates and two zooplankton replicates could not be accounted for and were assumed to be cannibalized. IV. Lake Whitefish. First feeding lake whitefish fry were successfully started on an artificial diet. A separate unit containing fry that were starved experienced total mortality by day 24. On day 29 there were no significant differences in survival 47 between any of the feeding treatments. However, at day 43 Of the experiment significant differences in survival (P(0.005) were Observed between treatments. There was a significant difference in survival between fish fed yellow diet and red and green diets, and between fish fed red and green diets and Artemia nauplii (Figure 9). Survival (:SD) at 43 days averaged 0.568 (0.073) fed Artemia nauplii, 0.297 (0.022) fed green diet, 0.282 (0.057) fed red diet, and 0.182 (0.022) fed yellow diet. However, at day 29, five days after total mortality was Observed in a unit containing extra fry that were starved, no significant differences in survival existed between any Of the treatments. 48 HRTENIH NHUPLII "”"DREEN DIET "“"RED DIET YELLOW DIET Figure 9. 'IVAIAHOS 0.2 25 20 DAYS Average survival of first feeding lake whitefish fry fed Artemia nauplii or a red, green, or yellow artificial diet. DISCUSSION The inability to successfully start first feeding walleye fry on an artificial diet was not totally unexpected, yet the incapability to get even a small percentage of the fry to initially accept the diet was unanticipated. These results are consistent with past efforts to get first feeding walleye to accept an artificial diet (Beyerle 1979a; Jahncke 1979; Martin 1975; Nickum 1978; Wiggins et al. 1981). Colesante (1983), feeding fry a combination of brine shrimp, zooplankton, and artificial diet, felt 4-6% survival to 60+ days was encouraging. Attempts to start other Percid larvae, such as perch (Hale and Carlson 1972; Mansueti 1964) have also been largely unsuccessful. Nutritional quality of the diet may be one of the limiting factors to successful culture of walleye fry. Acceptability of an inert particle does not seem tO be the problem. Colesante and Youmans (1982) reported walleye accepted brine shrimp cysts. Artificial diet acceptability between 21% (Nickum 1978) and 75% (Beyerle 1979a) has been reported, however survival past 21 days was reported to be less than one percent. This phenomenon occurs in other fishes as well. Attempts to start the fry of Coregonus spp. on an artificial 49 50 diet have largely resulted in failure (Braum 1967; Gunkel 1979). Lake whitefish did not readily accept a dry diet until they averaged 40 mm TL but only achieved a survival rate of 4% (Raisanen and Behmer 1982). Lake whitefish, Coregonus clupeiformis, were used in an experiment to determine whether the design of the rearing units was adequate to rear fry of a small size. Lake whitefish were selected because the fry were available prior to the walleye fry and because of their reluctance to accept an artificial diet (Raisanen and Behmer 1982). Whitefish fry are twice as large as walleye fry at hatching and were much more visible in the system. It was surprising that of the lake whitefish fry fed three colored diets in an upwelling system, 60-70% accepted the diets irregardless Of color. When the experiment was concluded at day 43 significant differences in survival existed. However, the Observed differences in survival could be misleading. The whitefish fry fed Artemia nauplii were growing well, while all fry fed an artificial diet were emaciated and dying at a slow rate. Although the fry accepted artificial diets, as evidenced in Observations of colored feed in the gut, it seems there was either an essential compound lacking from the feed or the ingredients in the feed are too complex for the fry to digest efficiently. This clearly indicates that, in the case of the whitefish, nutritional adequacy of the diet, not feed 51 acceptance, was the limiting factor in the survival of the fry. Nickum (1978) reported no differences in acceptance Of red, brown, or yellow particles by walleye fry fed a dry diet. An effort to color the diet with an orange-red dye to improve the acceptance Of W7 feed by walleye fry also proved unsuccessful (Orme 1978). However, acceptance of these diets may have been limited due to diet texture. For most species of fish that have small fry, no suitable dry feed has been developed, even when particle size and gross composition were technically identical to live food organisms (van der Wind 1979). When formulating a feed consisting of minute particles it is extremely important that each individual particle contains all essential nutrients in the proper proportions. If each particle is not complete, the fry may not receive a nutritionally complete diet. Texture, form, and palatability all play an important role in diet acceptance. The physical size, shape, and texture Of feeds should be designed to accomodate and conform to the fishes anatomical organs for seizing, engulfing or otherwise ingesting food (Webber and Huguenin 1979). It is generally reasoned that walleye fry will accept soft textured feeds more readily than hard textured feeds (Nickum 1978). First feeding walleye fry may not have the mechanical or chemical ability to break down a hard artificial diet into digestible pieces. Recently thawed 52 Oregon Moist Pellets rolled into small "worms" spin as they sink enticing largemouth bass to sample them leading to increased acceptance Of dry feed (West and Leonard 1978). Reasons for the failure of fry to survive on an artificial diet are not very clear. It is difficult to prove the nutritional adequacy Of a feed if the feeding technique and husbandry is not Optimal. Because the fry in experiments 2 and 3 survived less than one week in the larval rearing units and no Observed evidence of active feeding occurred, it is doubtful that nutritional inadequacy of the artificial diet was the limiting factor in the mortality of the walleye fry. Even those replicates fed a live diet died at the same rate as the replicates fed an artificial diet. Although McElman and Balon (1979) first Observed food particles in the digestive tract of walleye fry at 236.2 temperature units (1 TU=l C above 0 C per day) post-hatch, they determined the mixed feeding interval (endogenous and exogenous) was between 97 TU and 132 TU. This period corresponds to between 5 and 8 days in my system. Larval rearing system design may have been the cause Of premature mortality in larval walleye. Turbulence created by minimal air upwelling and 0.25 liter per minute flow was not originally thought to be a factor contributing to the inability to start walleye fry on artificial diets. Sustained swimming ability of larval walleye (9.5 mm TL) was determined by Houde (1969) to be at a velocity less than 3 53 cm/sec. 1n the fry experiments, velocities were Observed to be less than 2 cm/sec, just enough to keep most of the artificial diet in suspension. Velocity of the feed particles was not thought to be great enough to prevent capture by the walleye fry. In each unit, a number of fry had broken the surface tension of the water at the ventral portion of the head and were Observed hanging motionless by the surface tension. When walleye hatch, they are free swimming immediately. Generally, they will swim to the surface and remain in motionless suspension just below the water surface. The fry will be in an environment of maximal oxygen concentration with a minimal expenditure of energy. This is thought to be a tranSportation mechanism, moving the fry from the spawning grounds to more productive water with the least amount of energy expended (McElman and Balon 1979). In laboratory or hatchery rearing of walleye fry, abnormal conditions may be created preventing this behavior. Due to the turbulence created in an upwelling system, more energy may be required to maintain a position in the unit than would otherwise be necessary. Assuming that for most aspects Of ontogeny there exists an optimal time and sequence for the develOpment of related features, any retardation of development caused by the expenditure of energy otherwise required for growth and development may cause asynchrony in important events of develOpment (McElman and Balon 1979). At the time the fry are ready to feed, the 54 morphology of the mouth, stomach, or digestive tract may not be develOped to the point which would allow the fry efficient handling and digestion Of an artificial or live food item. Since walleye fry begin feeding before yolk sac depletion, subsequent mortalities may not be evident until utilization of endogenous nutrient reserves has been completed. Mortalities at this time have, in the past, been attributed to failure to make the transition to active feeding. Oxygen supply in the larval rearing system was assumed to be adequate. Large walleye do not show signs of distress until the dissolved oxygen level is only 2 mg/liter (Hoff and Chittenden 1969). With constant aeration, oxygen levels near saturation were always maintained and would not have contributed to walleye fry mortality. Walleye fry are one of the most difficult creatures to work with. Although the culture system may have influenced the survival of the walleye fry, there is great potential in using a system of this type. It was difficult for the fry to find refuge within the smaller jars. In systems utilizing larger jars, such as our 20 liter holding tanks, the upwelling air can be increased while the flow near the jar walls will be slow enough to allow refuge from the greater flow in the center. Once a system is designed to keep feed in suspension without influencing survival, nutritional requirements of walleye fry can be properly 55 examined. Until then, extensive culture of walleye fry seems the only viable method of culture. Variations in color and texture have been used to increase the acceptance Of dry feeds by walleye fingerlings, however, most such tests have lacked adequate replication and controls (Nickum 1978). The preference Of a yellow diet in Fingerling Experiment 2 was surprising. In Fingerling Experiment 1 an apparent preference for red diet was noted, but due to a power failure could not be verified. The change from water soluble feed coloring to water insoluble dyes affected the color of the diets and how they were perceived by the fingerling. Water soluble dyes leached quickly and paled within ten minutes of contact with the water. The only other things changed between Fingerling Experiment 1 and 2 was the removal Of the funnel assembly and a change from fluorescent illumination to a multi-spectrum fluorescent grow light. The diets colored with water insoluble dyes retained their color and seemed more intense or brilliant under the grow light. Thus, color cued responses in Fingerling Experiment 2 were more congruous. Cladocerans are one of the main natural food items Of walleye fry and fingerlings up to about 80 mm (Li and Ayles 1981; Mathias and Li 1982). Fingerling walleyes may even select for certain daphnids (Beyerle 1979b). Cladocerans can range in color from gold to reddish-brown, gray, or 56 almost black (Pennak 1953). During the latter half of the period natural food was collected for the walleye fingerling replicates, the predominant color of the daphnia was dark red. ZOOplankton blood plasma can be yellowish but during periods of low oxygen is reddish in color due to the formation Of erythrocruorin (Pennak 1953). Color receptors are present and well develOped in retinas Of fingerling walleye. The vast majority of pike-perch photopigment sensitivity recordings were Obtained from orange sensitive cones (Burkhardt and Hassin 1978). A red colored diet seemed likely to be preferred over other colored diets when these factors combined with the tentative results of Fingerling Experiment 1 were considered. Red may have been favored in Fingerling Experiment 1, not due to color cued acceptance but due to contrast cues. In the first fingerling experiment an Opaque white funnel assembly was used in the upwelling system. With direct overhead lighting and a predominantly white rearing unit interior, red diet may have contrasted more with the environment than did the yellow diet. When this assembly was eliminated, the interior of the unit was darkened except for the overhead lighting. In this environment the yellow diet would have greater contrast than the red or green diets. In the natural environment, underwater light scatters severely reducing contrast and promoting development of dominant red-sensitivity and opponent color processes in 57 aquatic animals (Easter 1975). Natural diet contrast has been shown to be important in the feeding of many carnivorous fishes. Pumpkinseed sunfish and yellow perch preyed selectively on ephippeal daphnia presumably due to greater visibility of daphnia with ephippia (Mellors 1975). Zaret (1972) showed that the amount Of pigmentation in the black compound eyes of relatively transparent Cladocerans can affect the rate of predation by planktivorous fish. Higher contrast between food color and background color increased the preference for the food color when different colored trout eggs were Offered to rainbow trout (Ginetz and Larkin 1973). If a neutral or dark colored rearing unit is desired (Nickum 1978), then feeding an artificial diet that greatly contrasts with the surrounding environment should increase acceptance Of that diet by fingerling walleye. An interesting factor in this experiment is the low incidence of cannibalism except in replicates of zooplankton and green artificial diet. Cannibalism is considered an inherent characteristic of walleye (Eschmeyer 1950). Cuff (1977,1980), working with larval walleye, found that starving walleye were cannibalized much more often because feeding walleye were able to escape cannibalistic attacks. The fingerlings in the green artificial diet replicates that did not make the transition from feeding on live food to an artificial diet were starving, and would therefore make easy prey for healthier, more aggressive fingerlings. The unaccounted mortalities in ZOOplankton replicates may have 58 been the result Of increased competition between the larger individuals and the smaller individuals. Smaller individuals more readily fall prey to the cannibalistic actions of larger individuals. The fact that only about 6% of the digestive tracts examined contained feed was not surprising. Most of the mortalities examined were emaciated indicating the fingerlings died Of starvation. The fingerlings that contained food in their stomachs may have died from stress possibly caused during cleaning or from acute symptoms of disease. Growth was not significantly different between any Of the treatments. Relative growth rate per day averaged 2.80% for those fed ZOOplankton, 3.33% fed yellow diet, 3.36% fed red diet, and 4.01% fed green diet. Although not significantly different, relative growth rates per day increased with decreasing diet acceptance. This trend could have been caused by density dependant factors. Surviving fish in units that experienced high mortality had more feed available and less competition for that feed, enabling the fish to grow slightly better in an uncrowded environment. But these values are still somewhat less than the relative growth rate of 5.4% per day determined for larger walleye fingerlings (Huh 1975). The results of the experiments with walleye fingerlings suggests that small fingerlings (4 cm TL) can be converted from live food to a semi-purified artificial diet with a 59 high survival rate in an upwelling system. More work is needed to confirm whether these results can be repeated consistently. If so, then standardized techniques can be employed to determine specific nutritional requirements of walleye fingerlings. Walleye could be raised intensively by combining culture techniques. Fry could be raised with high survival in rearing ponds to a length Of approximately 4 centimeters. They can then be harvested and concentrated in a hatchery utilizing an upwelling culture system and converted to a practical diet. This technique would provide fisheries management personnel with larger fish to stock and could possibly, through refinements in techniques, provide private aquacultural entrepreneurs a viable means of culturing coolwater fishes such as walleye and perch. CONCLUSIONS Until a system is designed to keep feed in suspension without influencing survival, nutritional requirements Of walleye fry cannot be examined. The upwelling system designed in this experiment is suitable for nutritional experiments with fish larger than one centimeter. 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Valley City National Fishery Complex, Valley City, ND 58072. Mimeograph report, 11 pp. Zyznar, E.S., and M.A. Ali. 1975. An interpretive study of the organization of the visual cells and tapetum lucidum Of Stizostedion. Can. J. 2001. 53(2): 180-196. 69 Appendix A. Walleye Fingerling Experiment 1. m>' I t’ N < / r a / ,’ ’ I a/ .' I/ x’ — D ,/ ’I N //. ....... I"..- I/ _ ID I / F‘ II ’ I ,/ .' / I, / — o /' "‘ I a II I/ I; ’/ /' .- ,’ I If) I, I/ I. ’/ I ’1 l T I T D —- a: (o «r cu CD 0 D D O "IVAIAHnS Survival of walleye fingerlings (6.3 cm TL) fed a red colored A3. artificial diet. 73 CD ‘ ID I / ’ I I/ I'_ U) / I V I I / I I " v~cuoo z’ ‘ ’/’ I, I-- 0 BEE / I’ " 888 / x I “Ht—O ’/ ’l _I._J..I / ,x _ In ill .'o‘.‘ m LIJUJUJ I'¢‘- xxx x“ r I I I ' 1 . ‘ ' I l l I. F— o I i’ g ('0 , .' / I' 0) I I, .‘ _ In >' I : N < l : 0 / .’ ,I' ' /’/’ I: _ 8 o I I/ II” / ,’ ' ’, ... u: / g I x’ / ,' I I / 1’ — o /’ " I ’I’ I/ ’,” ’/ x” __ m I/ ,” ’/:.' 4'" a I I I I O .— O (O ‘1' N C CD CD CD C3 A4. Survival of walleye artificial diet. 'IVMAHflS fingerlings (6.3 cm TL) fed a green colored Appendix B. Walleye Fingerling Experiment 2. 74 m>- N < '6‘va / D ,/ uJLouuJ /' P—F—F—F— ,/’ macro: 0000 ..—L0 b—dt—IO—‘b—d F. _J._J_I_J xxxx LIJLLJUJLLI 0:0:azc: i | I __ CD ' ' I v". i I I '_LD I I I I O v-l co (0 ‘3' N O D O O 0 DZ Survival of first-feeding lake whitefish fry fed a green colored artificial diet. 81 r l l l J ,7 // z’f' —40003