2004 Annual Report. Preliminary Investigations into the Feasibility of Developing Conservation Aquaculture Techniques for Burbot (Lota lota)

Transcription

1 2004 Annual Report Preliminary Investigations into the Feasibility of Developing Conservation Aquaculture Techniques for Burbot (Lota lota) A program funded by the Kootenai Tribe of Idaho Submitted to the Kootenai Tribe of Idaho and the KVRI Burbot Committee August 2004 By Dr. Kenneth Cain and Nathan Jensen Columbia Lake burbot fry (4mm)

2 INTRODUCTION As part of the Kootenai River Burbot Conservation Strategy, the University of Idaho s Aquaculture Research Institute (ARI) and The Kootenai Tribe of Idaho (KTOI) are collaborating on a joint experimental burbot aquaculture project to investigate techniques for spawning and rearing burbot (Lota lota) in captivity. Both locally and internationally, hatchery practices involving burbot have successfully been performed (Hagen 1952, Fabricius 1954, Kouril 1985, Ghan and Sprules 1993, Pullianinen and Korhonen 1993, Adamek 2000, Fisher 2000, McPhail and Paragamian 2000, Paragamian 2000, Paragamian et al. 2000, Harzevilli et al. 2003, Harzevilli 2004) and references have been made to burbot culture in hatcheries in the Czech Republic and Hungary that may aid in our efforts (Adamek 2000, Harzevilli et al. 2003, Harzevilli 2004). Early work by the Wyoming Game and Fish Commission investigated burbot propagation in the 1930 s and 1950 s (Hagen 1952). Burbot were also pond cultured for the purpose of stocking into natural water bodies and reports indicate that cuture technologies have been improved as a result (Adamek 2000). Adamek (2000) made reference to pond farming experiments suggesting deliberate aquacultural conservation efforts. Burbot culture has been investigated for the past 5 years in the Czech Republic and Hungary (Dr. A. Harzevilli, personal communication); however, only a limited number of reports have been published on this subject. Examples of hatchery spawning techniques aimed specifically at rearing this species have not been reported to our knowledge. This study utilized existing knowledge to complete preliminary trials at the University of Idaho s Aquaculture Research Institute (ARI) with regards to burbot broodstock husbandry, spawning, egg incubation, and feeding (protolarval, larval and juvenile) techniques. The goal of this study is to develop protocols and methods for rearing and propagation of burbot in an aquaculture laboratory setting, and to ultimately evaluate the feasibility of aquaculture as a conservation tool for burbot. This report summarizes results from preliminary (first year) trials focusing on system design, broodstock holding and spawning, fertilization, embryo incubation, protolarval and larval rearing, and juvenile rearing and feeding. 2

3 METHODS System design Various methods were used for maintaining and rearing burbot during various life stages in four independent rearing systems: 1) a recirculating chilled water system for adult fish, 2) an incubation and early life stage system, 3) a juvenile grow out/feed trial system, and 4) two outdoor circulars used as pond cultures. One recirculation aquaculture system was constructed at the ARI solely for the purpose of coldwater rearing of brood fish. This system has the capability to maintain temperatures of 3-4 C. Manually opening and closing lids designed for complete darkness when closed controlled photoperiod. Day length was mimicked to their native environment by opening the lids in the morning and closing them at night. This system was also partially insulated to meet thermal demands set forth by prior researchers (Kjellman et al 2000, Paragamian et al 2000, Taylor and McPhail 2000). A second semi-recirculation system was designed for low flows necessary to incubate burbot eggs through hatching and early larval rearing. This system was designed to recirculate chilled water and provided a constant supply to a head box. Recirculation of water in the system allowed low temperatures to be maintained, an important parameter of successful hatching (Paragamian et al 2000, Taylor and McPhail 2000). Chilled recirculated water exited from the head box to the incubators and provided developing burbot embryos and larvae a flow through environment. A third flow through system was built for a feed trial and juvenile isolation. Fifteen 20- liter aquaria equipped with air stones and screens to prevent escape were assembled to complete the trial. This system held juveniles that were transitioned to artificial diets and juveniles from outdoor tanks transitioning from live to dry diets. To mimic natural conditions, two outdoor circular tanks with 1900 L capacity were also used to raise developing juveniles. These test systems provided preliminary indication of their effectiveness, were partially insulated to reflect heat, and were equipped with an air stone for circulation. When temperatures rose to levels we felt were unacceptable, based on other researchers findings (Paragamian et al 2000, Taylor and McPhail 2000), ice was added. All systems (except the broodstock system) were designed to be low budget, utilizing all available resource we had at our disposal. Future experiments will require system improvements based on findings presented in this report. Broodstock Broodstock selection was based on cooperative agreements with the British Columbia Ministry of Fisheries, whose collected and provided available wild stocks. Duncan River (British Columbia) fish served as broodstock in this study. When permitting was in place 3

4 (November 2003), 20 adults were captured and transported to the KTOI hatchery. Fish were held at the KTOI hatchery for approximately two months prior to delivery to the ARI, which occurred during January Eggs from an unnamed tributary of Columbia Lake in BC were also collected and fertilized. These fertilized eggs were transported to the ARI for incubation on February 11 th, Adults from the Duncan River were evenly divided into five 1100L tanks that comprise the adult rearing system. The system s temperature was decreased according to KTOI recommendations and to be consistent with natural river temperatures. Temperatures were decreased incrementally until spawning commenced. Average temperature during the period of spawning was 2.5 C. When spawning was complete the adult Duncan River burbot were transitioned to warmer temperatures simulating natural summer temperature increases. Current temperatures are at or near 8 C (+/-1 C). The Duncan River adults displayed no obvious sexual dimorphisms upon arrival. On February 22, 2004 they were subject to an ultrasound examination by Dr. Ronald Sandy and Travis Saveraid of Washington State University. Of the 20 adults, thirteen fish were determined to be putative females. These females were divided into three treatment groups and males were combined with females in each tank. One group received an injectable form of salmon GnRHa with a dopamine inhibitor (Ovaprim, Syndel British Columbia), a second group received the salmon GnRHa in a controlled release implant (Ovaplant, Syndel British Columbia), and the final group saw no exogenous hormone. The seven males were not injected or implanted with any exogenous hormone. Implants and injections were given to females following anesthetization with MS-222. Hormones were carefully administered to adults and dosages were calculated based on recommendations from the supplier (1 ml/kg body weight). The dose for injections was divided in half and each half was delivered into the white muscle tissue directly posterior and slightly lateral behind the head. Recommendations for delivery of injected hormone in salmon were to administer the dosage interperitonealy (ip). We deviated from this recommendation due to our lack of knowledge about burbot internal anatomy. For the implant, the recommended placement was in front of the dorsal sinus using a 10 gauge needle and implant delivery gun designed by the supplier. We improvised and used a fabricated version of a passively integrate transponder (PIT) tag syringe modified for this application. The location recommended to receive the implant was the dorsal sinus immediately anterior to the dorsal fin. Dorsal sinus was indeterminable due to the fact that burbot have two dorsal fins. Implants were placed to one side of the medial dorsal line slightly lateral between the two dorsal fins. The area chosen to receive the treatments was carefully wiped dry with a soft towel and the external surface was covered with iodine disinfectant to prevent internal transmission of external pathogens. Finally, a small amount of superglue was applied over the wound and the adults were released back into the tanks of origin. Within two days all but one of the superglue bandages had fallen off. 4

5 Gamete collection was attempted every three to four days, from February 15 th to March 29 th. All females that had not spawned prior to the day of handling and males selected for use on a given day were examined for ripeness. The reason for the slight variation in ripeness checks was due in part to the occurrence of spawning events where we would immediately begin the gamete collection process if in-tank spawning was observed. The entire process lasted an average of 2 hours per examination. During examinations we applied a ripeness index to monitor physiological changes that would indicate readiness of the female to spawn. The index had a range from one to three. One was a female that felt firm to the touch and three was a female that was releasing gametes. On several occasions females deemed moderately ripe (two) would spawn in the tank within 36 hours. Therefore, our index was somewhat subjective and not completely accurate. Upon finding a ripe female we wiped the area around the urogenital opening free of water and mucus with soft towels to prevent contamination of gametes while stripping. Gametes were then hand stripped and the eggs were split between two different collection bags. Sub-samples of male gametes were collected and transported to a cryopreservation unit under the supervision of Dr. Joe Cloud (University of Idaho Biological Science Department) on a periodic basis. Motility of all samples determined; motility data are available from Dr. Cloud (jcloud@uidaho.edu). Fertilization procedures followed artificial spawning methods outlined for other species by Piper et al. (1982). Each female s eggs were split between two bags and then be fertilized with two separate males as standard procedure. All activation and rinsing was done with chilled system water in the bags that unfertilized eggs and sperm were combined. Fertilized eggs from individuals were then transferred to the incubation units. A cooler with ice was used to keep the gametes cool prior to transfer to the incubation units. Eggs were examined for fertilization efficiency using a dissection microscope (3x). First cell division of the germinal disc indicated success. Eggs that were unfertilized had no noticeable cell division. Fecundity was estimated for a portion of adults since external egg collection or removal of eggs from tanks rarely represented all of the eggs from a given female. Egg counts were determined by volumetric displacement method. Briefly, the number of 1mm diameter spheres that would displace 1ml of water was determined. The 1mm spheres were used since their size was similar to the eggs and this did not require additional handling of eggs. Egg Incubation Given the small size of the eggs (approximately 1mm) and the potentially high fecundity of females (McPhail and Paragamian 2000), we investigated the use of four styles of incubators: 1) conical McDonald type jars, 2) fabricated suspension baskets, 3) static refrigerated flasks, and 3) an experimental upweller. We used several approaches since the eggs are known to be demersal, semi-buoyant, and buoyant (McPhail and Paragamian 5

6 2000). Egg agitation was kept to a minimum to limit water usage and disturbance to developing embryos. Low flows ( ml/min) were maintained in all incubators that received water. Flows were determined independently for each incubator. Target flow rates were 50ml/min for every 100ml of eggs. Once eggs reached the eyed stage (indicated by a smoky grey color of the mass) flows were increased to approximately 500 ml/min. However, critical periods for burbot egg development are unknown. Flows were adjusted periodically and eyed egg masses were siphoned as dead eggs and/or fungus was observed. Disturbance of developing eggs was kept to a minimum, which may have promoted clustering and fungal growth in the conical incubator. Eggs were treated at the beginning of the incubation process to control fungus. Formalin was used and treatment concentrations ranged between 1000 and 2000 ppm on a daily basis. Formalin was injected via restricted gravitational flow (drip method). Calibration was done prior to each treatment, and target delivery rates were 50 ml/min for a period of twenty minutes. Formalin treatments were terminated when the first egg lot (from fish collected from Columbia Lake) had a prolonged hatching time (>70 days). Personnel from the KTOI hatchery sampled fertilized eggs from the two Columbia Lake lots and brought them to the ARI in February of These eggs were incubated in cylindrical McDonald type jars with a larger overall convex diameter at its base. Protolarvae and larval rearing As described earlier, two systems were assembled to rear burbot early life stages: recirculation and flow through styles. One system was set up for live feeding and the other was used for an experimental feed trail. At the onset of exogenous feeding when physiological factors such as yolk sac absorption, a functionally developed mandible, and gastrointestinal track protrusion forming the anus was evident, we began adding food items from 50 to 200 microns in size. First attempts to handle the fry also occurred at this stage of development. Initiation of the feed trial experiment began at this stage. Handling of larval fry continued through the stage when rotifers were clearly being eaten. Transferring larvae proved to be a delicate process. Temperatures were carefully manipulated to prevent thermal shock, flows were turned off prior to movement and adequate aeration was supplied to each aquarium receiving the protolarval and larval fish. To move the larvae we found that collecting them in small buckets and carefully pouring them into their designated aquaria worked best. We found the best way to move the small fish was to never allow them to leave water. The stages at which we began to move larvae were when they had begun feeding exogenously on rotifers (4-5mm). In addition, attempts were made to calculate the number of fish by pulling out a small sample of known volume and then performing actual counts on the sample. This was repeated three to five times. 6

7 Juvenile rearing; outdoor pond culture We stocked known numbers of first feeding larvae into two outdoor 1900L circular tanks inoculated with an assortment of zooplankton. On two separate dates 100 first feeding larval burbot were added to one tank (tank #1) and 50 larval burbot were added to the other (tank #2). The two most successful hatches were used (Columbia Lake female #1 and Duncan River Female #5) for this experiment. Daily observations were made on the outdoor tanks paying close attention to the temperature, behavior and live food availability. Habitat structure was added to tanks and included sticks, weeds, gravel, small logs and soil. Tanks were also partially insulated to maintain temperatures at or below recommended levels. Burbot were stocked into these tanks and allowed to remain from to (61 days) in tank #1 and to (55 days) in tank # 2. At the end of the trial, tanks were drained and remaining juvenile burbot were transferred to indoor aquaria for feed transition. Feeding of Larvae and Juveniles Feeding regimes consisting of algae, rotifer, and Artemia,which were all were cultured as first food items for developing larvae. Several feeding protocols were employed. Culture of live food followed recommendations in The Plankton Culture Manual, 5 th edition, by Hoff and Snell (2001). Enrichment reconstitutes (Rotorich, Florida Aqua Farms Inc.), Nannochloropsis sp., and Spirulina were added to live feed cultures at supplier recommended rates and timing to boost the nutritional value of food items delivered to the young burbot. Artificial dry diets selected for transition included Lansy Cold Water Marine Cod Diet from INVE, Belgium. Particulate sizes varied from 100 to 800 microns. All food items presented to larvae were introduced as early as possible based on observation of mouth development. The live-food feeding order was algae, algae and rotifers together, Artemia, killed artemia plus dry diet, and dry diet only. The amount of feed fed varied greatly over the entire feeding process and was delivered ad libitum during hours when hatchery personnel were present. Tanks with higher larval densities, based on visual observation, were fed at a higher rate. Attempts were made to start the larvae solely on artificial diets using periodic sprinkling of dry feed followed by agitation of the surface to suspend the food particles in the water column, and through the use of automatic feeders. Two attempts were made to begin a feeding trial in one of the newly completed systems. Our intentions were to stimulate early feeding and standardize feed training based on methods used in marine cod aquaculture and in reports from burbot culture experiments (Harzevilli 2003 et al, Harzevilli 2004). 7

8 Outdoor pond cultures were inoculated with a variety of plankton one week prior to the stocking of the burbot fry. Periodically dehydrated Spirulina and enrichment reconstitutes were added to meet plankton feeding demands. 8

9 Results and Discussion Broodstock All 20 adults available during 2004 were successfully spawned. Brood stock transfer and transition strategies and sex determination did not result in any observable adverse effects with regards to spawning. Results of the ultrasound procedures were 100% accurate at sexing adults. Photoperiod control may have contributed to successful spawning induction. Piper et al. (1982) suggested that photoperiod regulation should to be initiated six months prior to expected spawning date in salmonids. Fish in this study were captured from the wild and it was assumed that large alterations from natural photoperiod were not experienced prior to their arrival at ARI. Captive females began spawning on February 17, 2004 (Figure 1). On February 23, the first female spawned naturally in one of the tanks. This was one day prior to our scheduled delivery of hormone, suggesting that hormone use may not be required to induce spawning. Hormones were delivered on schedule to the remaining adults on February 24. All 13 Duncan River females were spawned by March 28. The implanted females mean day to spawn was 11 days post-treatment. This value was 16 days for the injected females and 20 days for the control females. We observed little if any signs of morphological change occurring with the adults. Noted changes were similar to those of J. Kouril et al. (1985), including differences between male and female urogenital openings and abdomens, though these predictors were not 100% reliable. 100% Treatment date Control Implant Injection Spermiation 80% Percent spawn 60% 40% 20% 0% 2/15/2004 2/20/2004 2/25/2004 3/1/2004 3/6/2004 3/11/2004 3/16/2004 3/21/2004 3/26/2004 Figure 1. Total cumulative percent spawn by treatment with spermiation of males. 9

10 Male spermiation began during the week of February 10th and February 17th (Figure 1). Every male was actively spermiating by March 2 nd. Spermiation continued through the march 28, when the last female was spawned. On March 29th, four of the seven males were still spermiating. However, since all females had spawned by this time, after which further fish handling ceased. Milt viscosity varied among males and among spawns. Motility estimates have not yet been calculated and compared. Subsamples of sperm were collected from all males and cryopreserved. Fertility rates for cryopreserved semen samples collected and frozen during 2004 will be determined in brood year Natural spawning in the tank was a common occurrence during this study. Of the 13 females in captivity, 9 (69%) spawned or at least partially spawned in the tanks prior to a scheduled handling times. When spawning was observed, behaviors were noted and were similar to observations by Fabricius (1954). Females used their tail to agitate eggs and suspend eggs and milt throughout the water column. This behavior would eventually cease shortly after lids were opened for photoperiod regulation, after which fertilized eggs settled to the bottom of the tank. On all occasions where in-tank spawning occurred, film was consistently observed on the surface of the water. This film was thought to be immobilized sperm cells and ovarian fluids, and proved to be a good indicator of a spawning event. Pheromone release within the recirculated water traveling to each tank in the system may have contributed to in-tank spawning. This combined with the fact that males and females were held together likely increased the likelihood of intank spawning. When such an event occurred, the male in the tank appeared to release all sperm. Future attempts to collect additional milt from such males were unsuccessful in all cases. When in-tank spawning events were observed, the females were removed, anesthetized, and stripped for their remaining gametes. Each time a spawning event occurred attempts were made to salvage the eggs from the bottom of the tank via siphoning. After the first uncontrolled spawning event the outflow of the tanks were modified with a top release inner standpipe to keep eggs from flowing into the sump. This modification allowed an estimated 90% recovery rate by siphoning naturally fertilized eggs, but fecal material was often present. Our calculations revealed an estimated value of approximately 1910 eggs per 1 ml of volume. It was not uncommon to strip 400,000 eggs from a single female. Egg numbers ranged anywhere from 0 to 500 ml of eggs per female, including in-tank spawned restrips. Average weight of females was approximately 2 kg. Fertilization Fertilization rates generally exceeded 90%. The artificially fertilized eggs, the field fertilized eggs from Columbia Lake, as well as eggs from in-tank spawners showed this level of fertilization. 10

11 Incubation of eggs and larval rearing Incubation flows were kept low. The purpose was to prevent eggs from overflowing or being over-addled during potential critical periods during development. It was suggested by Hagen (1952) that fungal manifestation might be controlled by agitation of the eggs during incubation in hatch trays without impacting survival. The living eggs in our incubators were surrounded by fungus. Within the fungal mass the living eggs would continue to exist. Physical appearance resembled clusters where eggs would adhere to one another with a viscous consistency. Hagen (1952) also noted similar results involving partial egg survivability to hatch even though fungus was present. We speculated that the developing eggs may be resistant to a higher degree of agitation based on flow, but this remains unknown. Agitation studies conducted by Hagen (1952) to determine impacts of transportation stress on eggs showed a decrease in fungal outbreaks and siltation as a result. No further studies on this have been reported to our knowledge. Accurately estimating egg loss and mortality was difficult. Water temperatures fluctuated from C depending on the flow of each incubator. Incubators with higher turnover rates experienced lower water temperatures to a point where it would be the same as chilled sump water. Temperatures fluctuated with the additions of new incubation units, when flows were adjusted, or as ambient lab temperatures would rise. Thermographs demonstrated these fluctuations (Figure 2). Eggs held at the KTOI hatchery also had fungal manifestations, where only small numbers of eggs survived to hatch. Fungus was also noted in previous years, for this reason we attempted chemical fungal treatments. From a hatching success and treatment standpoint, results here suggest that a conical as well as cylindrical style of McDonald jar incubators provide the greatest survival and will likely be employed in future studies. The Columbia Lake egg lot (I1) had a substantially longer hatch time (>70 days; Table 1). Interestingly, it was found that by subjecting eggs to an intentional thermal increase of 3ºC over the average water temperature for that month, hatching was enhanced and synchronized. All embryos hatched within two hours of this temperature increase. 11

12 A1-I1 A5-I /3/2004 2/13/20042/23/2004 3/4/2004 3/14/2004 3/24/2004 4/3/2004 4/13/20044/23/ /4/20 3/9/20 3/14/2 3/19/2 3/24/2 3/29/2 4/3/20 4/8/20 4/13/2 4/18/2 4/23/2 4/28/ Figure 2: Date Date Figure 2. An example of thermal graphs for the two most successful incubation vessels. A1-I1 (on left) was from the Columbia Lake Female 1 and the A5-I5 (right) graph is of the Duncan River female 5. In this study, conical McDonald jar like incubators had the best hatch rates (Table 1). The average time to first observed hatch was 135 degree days post-fertilization. The average time until estimated 90% hatch was 164 degree days post-fertilization (Figure 4). Incubator Type Identification # Success to hatch McDonald Jar A1-I1 Yes McDonald Jar A2-I2 No McDonald Jar A3-I3 Yes McDonald Jar A4-I4 Yes McDonald Jar A5-I5 Yes McDonald Jar A6-I6 Yes Hanging basket A2-I7 No Upwelling Trough 1 No Erlenmeyer flask 1 Liter beaker No Erlenmeyer flask 500 ml beaker No Table 1. Hatch success for various incubation methods. Yes indicates that a hatch was successful on at least a portion of the eggs kept in the incubator. No indicates that all eggs were lost during incubation. Of the ten incubation units employed only five resulted in protolarvae collection of any significance (Figure 3). Average hatch time for the five egg lots mentioned was 164 degree-days post fertilization. Hatch success was difficult to determine due to fungal manifestations and the lack of adequate estimations post-hatch (post-hatch abundance estimates ranged from 30,000 to 300,000). Accurately calculating the number of eggs prior to incubation by displacement is possible, but accurately estimating the number of protolarvae or larval fish immediately post-hatch proved delicate. Once first hatch was observed the incubation temperature was increased from the target range of 4 C to 9 C. This was done gradually over a period of 24 hours. Temperature increases appeared to be beneficial in decreasing the period of time required for complete 12

13 hatch to take place. This phenomenon is graphically represented by comparing Figures 4 and 5 below. Hence the thermal shocking treatment of Columbia Lake egg lot A1-I1. When comparing the degree-days to hatch for the five incubators (Figure 4) and the average incubator temperatures in those five incubators (Figure 5), the prolonged period of time for which the Columbia Lake eggs hatched can easily be seen. Also, when comparing Figures 4 and 5 the inverse relationship between average temperature (Figure 5) and length of average hatch time (Figure 1) can be seen. When egg masses reached 90% hatch, larval densities were very high in the collection aquaria. Within 15 to 30 days post-hatch, larval losses and mortality rates were substantial and agree with other reported mortality estimates (Ghan and Sprules, 1991; Paragamian, 2000). Handling stress was a factor for survival until the larvae were clearly eating enriched rotifers, visible with 3x microscopic evaluation. At this stage they began to show a slight tolerance to transfer stressors, and we stocked our outdoor pond cultures. Whenever larvae were handled with fine mesh nets it resulted in 100% mortality. Unless a size of mm can be used we would not advise using a net for transfer of larvae. It was found that larval and juvenile sizes varied widely, suggesting that grading may be important to prevent cannibalism. Handling stress and mortality associated with grading must be evaluated relative to stress and mortality associated cannibalism to maximize survival during this critical stage of burbot culture. 13

14 Degree days to hatch of burbot embryos Degree Days A1-I1 A3-I3 A4-I4 A5-I5 A6-I6 Incubator Figure 4. Degree days required for burbot embryo development from first observed hatch to estimated 90% hatch. Average incubator temperature Comparison Temperature (Celcius) Sump A1-I1 A3-I3 A4-I4 A5-I5 A6-I6 Location Figure 5. Average incubator and sump temperatures during the incubation period are represented by the bar graph. 14

15 Juveniles Water temperature in the juvenile rearing systems remained relatively constant (14 C, +/- 2 C) because they are flow through systems. Densities were kept low due to cannibalistic tendencies and space availability. Aquaria plumbed into these systems varied from 10 to 40 Liters in capacity. As few as one burbot occupy any one of these tanks and up to four per the given volumes. Juveniles approximately 20 mm in length had a notable change in behavior: they attempted to hide when given any available structure in the aquarium: air stones, corners, screening and tank wall junctions. Gahn and Sprules (1993) suggested this behavioral change occured at lengths greater than 15.0 mm, which agreed with our findings. Due to the low fish numbers, artificial habitat structures have been added to the majority of the aquaria. No one standard structure was used for this purpose. It is obvious that the juvenile fish used the structures and show a photonegative response within the transparent aquaria. In the future, the juvenile rearing room will be covered to restrict the amount of overhead lighting and all tanks will be painted or covered to reduce stress. Primary feeding of burbot was found to be a delicate process that included small food items such as algal cells (Harzevilli 2004) and rotifers (Ghan and Sprules 1993; Harzevilli 2003). Culture of live food is time consuming and labor intensive. We experienced problems in estimating time needed to ramp-up live cultures to desirable levels. Compensation for this lag period is just another part of the feeding process that will require additional attention. Adequate time must be given to ensure cultures are consistently at levels slightly above the larval demand. In addition, more than one culture station was set up in two different laboratory facilities. On several occasions one would fail and the other was used as a backup. We investigated live feed suppliers to meet feed demands of the progeny. Costs were examined during this process and outsourcing of live feed appears to be cost-effective if space and time limitations exist. Mouth gape was described as a limiting factor in the ability for developing burbot to begin exogenous feeding (Ghan and Sprules 1993, Harzevilli 2003). We began introducing algae before complete mouth development was discernable. Close examination of mortality samples and periodic photography of live specimens helped determine appropriate timing of culture food provision. As the larvae developed they were transparent and ingested food items were observed under a dissection microscope. Feeding larvae exclusively artificial feed resulted in 100% mortality. Both attempts to begin the feed trial experiment failed due to handling mortalities due to transport and stockings into experimental tanks. Initial stocking resulted in nearly 100% mortality. Transition of pond cultured juveniles from live diet to an artificially prepared diet has thus far had limited success. The number of fish obtained from outdoor tanks has been low and about one out of twelve has been witnessed eating the dry diet. 15

16 Outdoor pond stocking success was comparable to work reported by others. Wolnicki (2001) reported 6-8 % survivability. In our tanks we retrieved 6 individuals from each tank resulting in 6% and 12% survival for an average of 9%. One thing to note about these outdoor stocked individuals is that they were on average 5 to 20 mm longer and had a noticeably better condition factor based on observation post-harvest. Pond stocking and larval grow out to the juvenile stage lasted an average of 58 days between the two tanks. There were no signs of food item depletion at the point of harvest and juveniles that were removed did not appear to have outgrown their food items. Therefore, it is feasible to conclude that fish could remain under these conditions for longer periods. If that was the case and stocking densities could be increased, then fish of a size that could be tagged for release to the wild may be obtained. One Juvenile harvested from the outdoor pond cultures reached a size of 60mm. This is the longest individual we have at this point and we speculate that a polymer or small tagging device may be possible to use on fish this size. This juvenile rearing method will require further testing but appears promising. Since the last update (presented to the KTOI and the Burbot Recovery Committee in March of 2004) we have been working to transition the Duncan River and Columbia Lake burbot progeny that remain through the juvenile stages and onto an artificial diet. Results of our attempts reveal that both strains of burbot consisting of three separate lots of eggs are currently represented. Overall survivability of laboratory-reared progeny to artificial diet was very low (estimated at >.001 %). As of August 10, 2004, 39 individuals remained in the systems designed for early life rearing. Of the 39 that remain, 28 juveniles have been witnessed ingesting artificial diet, depositing fecal matter (similar in color to the diet) and appear to be growing. At this point we have ceased all live diet culture and feeding. Live diet production ended on July 25, SUMMARY AND CONCLUSIONS Control of environmental parameters, handling procedures, sex determination and basic husbandry were essential components for initial captive broodstock management. Incubation of the eggs, hatching of larvae and rearing to the juvenile stages are components we have worked with to date. Additional juvenile culture management strategies are currently being formulated. Monitoring male and female broodstock for morphological changes, behavior and time of spawning (given three treatments) was completed during this first year of evaluation, and juvenile burbot are currently feeding on an artificial diet. All in all, results from these preliminary trials have been successful. They have provided testable criteria to build upon in relation to broodstock holding/spawning as well as larval and juvenile rearing. Through further testing of methods that showed promise during year 1, it is highly likely that development of burbot culture techniques for conservation purposes will be successful. 16

17 References Adamek, Z The Applicability of Feed Mixtures in Burbot, Lota lota, Farming. Krmiva 42, 2: Breeser, S. W. & F. D. Stearns Observations of the Movements and Habitat Preferences of Burbot in an Alaskan Glacial River System. Transactions of the American Fisheries Society 117: Fabricius, E Aquarium observations on the spawning behavior of burbot, Lota vulgaris L. Report of the Institute of Fresh water Research, Drottningholm 35: Fisher, S. J Early life history observations of Burbot utilizing two Missouri River backwaters. American Fisheries Society: Burbot biology, ecology, and management. 1: Ghan, D., & W. G. Sprules Distribution and abundance of larval and juvenile burbot (Lota lota) in Oneida Lake, New York. Verhandlungen der Internationalen Vereinigung der Limnologie 24: Ghan, D., & W. G. Sprules Diet, prey selection, and growth of larval and juvenile burbot (Lota lota). Journal of Fish Biology. 42: Hagen, G. O The Experimental Hatching of Burbot (Lota lota M.) in Wyoming. Wyoming Fish and Game Commission, Wyoming. Harzevilli, A.S., et al First feeding of burbot, Lota lota larvae under different temperatures and light conditions. Aquaculture Research 35: Harzevilli, A. S., et al Larvae rearing of burbot (Lota lota) using Brachionous calyciflorus rotifer as starter food. Journal of Applied Ichthyology. 19(2): Hoff and Snell Plankton Culture Manual. Fifth edition. Florida Aqua Farms Inc. ISBN: Kjellman, J., et al Occurance of Burbot Larvae in a Eutrophic Lake. American Fisheries Society: Burbot Biology, Ecology and Management 1: Kouril, J., et al The Fertility of Female and Male Burbot (Lota lota) Reproduced by stripping. Prace VURH Vodnany 14: McPhail, J.D A review of burbot Lota lota life-history and habitat use in relation to compensation and improvement opportunities. Report prepared for the Habitat Management Division, Department of Fisheries and Oceans, Vancouver, British Columbia. p

18 McPhail, J. D. & V. L. Paragamian Burbot Biology and Life History. American Fisheries Society: Burbot Biology, Ecology and Management 1: 11-2 Paragamian, V. L The Effects of Variable Flows on Burbot Spawning Migrations in the Kootnay Lake, British Columbia, Canada, After Construction of the Libby Dam. American Fisheries Society: Burbot Biology, Ecology and Management 1: Paragamian, V. L., et al Collapse of Burbot Fisheries in the Kootenai River, Idaho, USA, and Kootenay Lake, British Columbia, Canada. American Fisheries Society: Burbot Biology, Ecology and Management 1: Piper, R.G., I.B. McElwain, L.E. Orme, J.P. McCaren, L.G. Fowler and J.R. Leonard Fish Hatchery Management. United States Department of the Interior, Fish and Wildlife Services. Washington D.C. Powell, et al Induced and Synchronized Spawning of Captive Broodstock Using Ovaplant and Ovaprim. Proc. Aquaculture Assoc. of Canada, St. John s Newfoundland. Canada. Pulliainen, E. and K. Korhonen Does the burbot, Lota lota, have rest years between normal spawning seasons? Journal of Fish Biology 43: Sorokin, V.N The Spawning and Spawning Grounds of the Burbot (Lota lota). Journal of Ichthyology. 11(6): Taylor, J. L., & J. D. McPhail Temperature, Development, and Behavior in the early life history of Burbot from Columbia Lake, British Columbia. American Fisheries Society: Burbot biology, ecology and management 1: Vatcha, R The food spectrum and growth of burbot (Lota lota L.) fry in experimental condition. Bulletin of the Vodnany Research Institute of Fishery and Hydrobiology 26: (in Czech with English summary). 18