Extend the seasonal availability of walleye fry for stocking purposes by stimulating reproduction through manipulation of temperature, light and gonadotropic hormones.
Develop the behavioral and biochemical assays for the quality of juvenile walleye by measuring a) the swimming stamina, b) the lipid content and fatty acid characteristics of growing and fasting fish, and c) vulnerability to a predator.
Translate the results of the biochemical fitness measures into fish quality methods more readily used for a hatchery produced fish assessment, and carry out technology transfer to those most directly involved in stocking fingerlings.

Walleye is the object of considerable attention in both the commercial and recreational fishery of wild stocks and artificial propagation. However, dependence upon eggs collected from ovulated wild fish captured near their spawning grounds results in poor fertilization percentage due to unsynchronized ovulation. In order to increase the reliability of walleye culture operation it is essential to induce spawning in captive walleye broodstock and/or come up with effective out-of-season induction of maturation and final ovulation. Earlier spawning will allow hatchery operations to expand the growing season of walleye yearling, both for aquaculture purposes and stocking larger-healthier fish to the reservoirs.

Larval and juvenile fish feeding conditions are not equal in promoting growth, and size-specific lipid components presumably are reflected in differential fish survival. Analysis of lipid and fatty acids would allow evaluation of body energy reserves in a size-related fashion and prediction of fasting capability. Biochemical robustness and swimming performance might well be translated into some external indicators of percid nutritional status (such as condition factor or body density) and applied as a "fish quality" estimate. These indicators may be easily measured by hatchery personnel in the field, thereby allowing more precise selection of reservoirs to be stocked with which fish. The proposed research will also provide the first where juvenile fish of measured differences in lipid stores are tested directly for their ability to avoid predators. The results of this work will allow us to modify hatchery procedures to synchronize maximal fingerling quality with optimal lake conditions in terms of prey availability. We will determine the most reliable estimators that hatchery personnel and commercial producers can use to evaluate fingerling physiological quality. Although aquaculture production is viewed as a competitive production system to commercial wild harvest and the recreational fishery in the Great Lakes region, walleye aquaculture is expected to become an integral part of the fishery industry's management plan for long-term sustainable harvest.

Objective 1. To induce out-of-season spawning, walleye females will be exposed to an "accelerated" cooling and warming protocol beginning in August and ending in January. In December fish will be divided into 3 groups. Group 1 will be injected with microcapsules of mGnRH at a dose designed to release 2.5 µg/day for 30 days (Lee et al., 1993). A second group will be injected with hCG at 150 IU/kg on day 0 and 300 IU/kg on day two. Timing of the initial hCG injection will be determined by periodically aspirating a sample of occytes to determine when their size will reach 1000 um. Immediately prior to the two hCG injections and at regular intervals thereafter, blood will be collected from 5 females per group to determine plasma concentration of steroid hormones (testosterone and estradiol). Group 3 will be injected with vehicle only and will serve as controls. Changes in reproductive functions in fish from experimental groups relative to controls will be analyzed using oocyte diameter, steroid levels, as well as fertilization and hatching percentages for eggs fertilized with sperm derived from representative groups.

Objective 2. Fry for this objective will be obtained from the control broodstock in Objective 1, and maintained in a series of 3 ponds to be stocked at 40,000 and 3 ponds to be stocked at 200,000 fry per acre to obtain fish growing at different rates. Ponds will be filled with water from an adjacent reservoir just before stocking with 5-day old fry. To control for nutrient and productivity differences, we will fertilize the ponds once per week to bring reactive phosphate up to 30 µg P/L and inorganic N up to 600 µg/L each week. We will analyze phosphate, ammonia, and nitrate in the ponds each week prior to fertilization. Fish for quality assays will be collected by seining during weeks 2, 3, 4 and 5. Some will be analyzed for lipid and fatty acid compositions after being exposed to fasting periods of different duration. Others will be tested for stamina as determined by critical swimming speed. A final group to be evaluated for relative vulnerability to predation by largemouth bass will consist of various sizes of juveniles exposed to periods of fasting of different duration such that lipid content varies.

Objective 3: During our evaluations of physiological quality, we will examine the relationships between measures of physiological well-being, such as lipid levels, to more easily collected parameters such as condition indices and percent body water. We will then determine the most reliable estimators that hatchery personnel can use to evaluate fingerling physiological quality. Two behavioral characteristics will be used in combination with biochemical indicators.

Environmental and physical stresses can affect many physiological functions e.a. growth, reproduction, osmoregulation, homeostasis and mobilization of energy stores (Pickering, 1993; Wedemeyer et al., 1990). Walleye produced in intensive culture conditions and reared on formulated diets are stocked to many reservoirs and lakes in North America. Upon release YOY walleye need to adjust to altered conditions in order to survive. The quality of cultured walleye in terms of withstanding food deprivation, swimming capacity, and responses to physiological stress caused by natural predators have never been tested.

Juvenile walleye grown on two formulated diets were subjected successively to the same experimental conditions. The two diets fed to walleye prior to the experiments differed in protein source and added lipids (4 versus 10%). Groups of six fish were placed in triplicate rectangular wire cages and submerged randomly in three artificial stream compartments. Three treatments were designed: 1) water current plus predator, 2) water current and no predator, 3). no water current and no predator. Water velocity was maintained at 1.5 body lengths/s throughout the experiments. Six 1 year old muskellunge Esox masquinongy were used as potential predators. They were allowed to swim freely outside walleye cages and were fed with YOY walleye. The water temperature was maintained at 14oC. Fish were exposed to experimental condition for 6 weeks and were deprived of food to simulate poststocking period of fasting. Every two weeks, six fish per each treatment were removed, anaesthetized with MS-222 in less then one minute, blood sampled from the caudal vessel using heparinized syringe, weighed, measured, and frozen in liquid nitrogen for biochemical analysis. Blood plasma was separated by centrifugation at 1500 g for 10 min and stored at -20° C until assay. Total time to obtain all samples per treatment was less then 5 min to avoid stress associated with sampling procedure. Following sampling, all remaining fish were weighed, measured and returned to the artificial stream. Plasma glucose were measured using commercial Glucose HK kit (Sigma Chemical Comp.). Other parameters such as plasma cortisol (determined by radioimmunoassay, validated for use with walleye), fatty acids in total body lipids (determined gas chromatography), and proximal analysis (proteins, lipids, and ash) of fish (performed following usual laboratory procedures) are in progress to evaluate the impact of swimming and/or presence of the predator on stress and nutrients expenditure in juvenile walleye. Each parameter was first analyzed by one way ANOVA for changes that might have occurred during the experimental period. Then, each parameter was compared simultaneously among all treatments using two way ANOVA to test for treatment effects.

The weight of the fish grown on diet A (krill meal protein based, 10% lipids added) did not decrease significantly regardless of the treatment (Fig. 1A), whereas the weight of fish grown on diet B (fish meal based, 4% lipids added) decreased significantly (p < 0.05) in all three treatments during the experiment (Fig. 1B). In both experiments, walleye forced to swim coupled with the presence of potential predator lost more weight then those subjected only to swimming or resting non-exercised controls. However, no significant differences were found among the three treatments.

The plasma glucose levels in the fish grown on diet A decreased significantly (p < 0.05) in each treatment during experimental period (Fig. 2A). However, there were no significant differences between treatments. Alternative patterns of plasma glucose levels were observed in fish grown on diet B. After initial drop (first 2 weeks) the glucose levels were maintained at similar levels in all three treatments (Fig. 2B). Noteworthy is the fact that the initial level of plasma glucose was twice as high in fish grown on diet A comparing to fish grown on diet B.