An Evaluation of Several Feeding Methods for Enhancing Smoltification in Summer Steelhead (Oncorhynchus mykiss) at Dworshak National

Fish Hatchery, Idaho

Final Report Prepared by

Idaho Fishery Resource Office U. S. Fish and Wildlife Service Dworshak Fisheries Complex

Dworshak Fish Health Center

U. S. Fish and Wildlife Service Dworshak Fisheries Complex

Dworshak National Fish Hatchery

U. S. Fish and Wildlife Service Dworshak Fisheries Complex

Abernathy Salmon Culture

Technology Center U. S. Fish and Wildlife Service

Columbia River Research Laboratory U. S. Geological Survey

Biological Resources Division Western Fisheries Research Center

Marrowstone Marine Station

U. S. Geological Survey Biological Resources Division

Western Fisheries Research Center

Moore-Clark USA, Inc. 177 Telegraph Road #385

Bellingham, WA

Submitted to

Complex Manager Dworshak Fisheries Complex U.S. Fish and Wildlife Service

Ahsahka, Idaho 83520

August 2000

2

EXECUTIVE SUMMARY

Many agencies in the Columbia River basin cooperate to improve the health and condition of juvenile salmonids (Oncorhynchus spp.). Each agency possesses specialized expertise to research new rearing methods to improve hatchery stock performance. This report presents results of a pilot study conducted through a cooperative effort by the U. S. Fish and Wildlife Service (USFWS), the U. S. Geological Survey, Biological Resources Division (USGS-BRD), and a private feed company, Moore-Clark, USA. Within the USFWS, the Idaho Fishery Resource Office (IFRO), Dworshak National Fish Hatchery (NFH), the Abernathy Salmon Culture Technology Center (ASCTC), and the Idaho Fish Health Center (IFHC) provided fish, special feed formulations, personnel, health screening and materials to this production-level effort. Within the USGS-BRD, the Columbia River Research Laboratory of the Western Fisheries Research Center provided personnel, materials, and analytical capabilities, and Marrowstone Marine Station (MMS) provided personnel and seawater rearing capacity. Moore-Clark USA provided a special feed formulation and logistical support. The purpose of this study was to investigate the value of several feeding methods for producing a greater proportion of hatchery steelhead (O. mykiss) that successfully smolt and migrate. Factors affecting the propensity to emigrate are size, growth rate, and developmental stage. Our study addressed the factors of size or growth rate through two approaches: regulation of ration size and feeding schedule and use of enhanced feeds. In one experiment, we determined minimum ration and feeding schedules from previous growth performance for Dworshak NFH summer steelhead to reduce growth during winter months. The major assumption underlying that experiment was that hatchery fish may develop physiologically at sizes similar to wild fish and smaller than hatchery release goals. Two other experiments studied special feed formulations that have been found to increase growth rates and survival, or improve the transition to seawater. Results demonstrate that growth patterns may be manipulated by a reduction in feed followed by a return to full rations prior to release from the hatchery, with no effect on smolt development or seawater growth and survival. Small fish (< 200 mm) from the reduced ration, intermittent feeding schedule group were detected earlier at dams during emigration than large fish (> 200 mm) but were recovered at a slightly lower rate. Enhanced feeds did not appear to affect smolt development. Seawater growth, survival, and smolt condition were similar among all treatment groups. Further production-level investigations under standard hatchery conditions are essential to test innovative hatchery practices affecting growth, physiological development, and survival.

3

ACKNOWLEDGMENTS This project was a cooperative pilot project involving the Idaho Fishery Resource Office (USFWS), Dworshak National Fish Hatchery (USFWS), the Idaho Fish Health Center (USFWS), Abernathy Salmon Culture Technology Center (USFWS), the Columbia River Research Laboratory (USGS-BRD), the Marrowstone Marine Field Station (USGS-BRD), Moore-Clark USA, Inc., the Department of Food Science at the Washington State University, and the Department of Chemistry at Eastern Oregon State University. The project involved a number of complex activities requiring close coordination and cooperation that would not have been possible without the assistance of many people at these agencies. The production and maintenance staffs at the Dworshak National Fish Hatchery were responsible for rearing the steelhead and maintaining the facilities. Nelson’s Silver Cup was responsible for preparing the enhanced Abernathy Diet. Randy Bowen, Doug Burum, and Jill Olson at the Idaho Fishery Resource Office assisted in PIT-tagging steelhead smolts. We are very thankful to Billy Connor, also at the IFRO, who familiarized us with the PIT-tag interrogation-by-code facilities at Little Goose Dam and allowed us to collect our PIT-tagged steelhead in conjunction with his research project. Nancy Elder, Ron Spinek and student intern Jim Meek with the Marrowstone Marine Station provided facilities and assistance for the seawater rearing evaluations. Kathy Clemens at the Idaho Fish Health Center provided diagnostic support during a disease outbreak at Marrowstone Marine Station. We are especially thankful to Pamela Petrusso and Jennifer Coyle at the Columbia River Research Laboratory for their assistance in data analysis and document editing. Dr. Anna Cavinato (EOU) with student assistants Todd Rogers and Melissa Wenz conducted shortwave near infra-red readings and Dr. Barbara Rasco (WSU) and student Yiquin Huang ran total body lipid and tissue moisture analysis.

4

TABLE OF CONTENTS EXECUTIVE SUMMARY .......................................................................................................... ii ACKNOWLEDGMENTS ........................................................................................................... iii TABLE OF CONTENTS ............................................................................................................ iv LIST OF TABLES ...................................................................................................................... vii LIST OF FIGURES .......................................................................................................................x LIST OF APPENDICES ........................................................................................................... xiii CHAPTER ONE - PROJECT DESCRIPTION..........................................................................1

Introduction........................................................................................................................1 Goal and Objectives...........................................................................................................3 Site Description ..................................................................................................................3 General Methods................................................................................................................4

Fish Culture..............................................................................................................4 Data Collection ........................................................................................................5 Statistical Analysis..................................................................................................6

CHAPTER TWO - AN EVALUATION OF THE EFFECTS OF A MODIFIED FEEDING

STRATEGY ON GROWTH AND SMOLTIFICATION OF SUMMER STEELHEAD (ONCORHYNCHUS MYKISS) AT DWORSHAK NATIONAL FISH HATCHERY ......................................................................................................................7

Abstract...............................................................................................................................8 Introduction........................................................................................................................9 Methods.............................................................................................................................11

Experimental Fish ..................................................................................................11 Feed Methods and Calculations.............................................................................11 Sampling Methods .................................................................................................12 Physiological Analyses ..........................................................................................17 Transport to Marrowstone Marine Field Station ...................................................17 Seawater Rearing ...................................................................................................17 In-River Migration and Survival............................................................................18 Statistical analysis..................................................................................................18

Results ...............................................................................................................................19 Growth, Condition Factor, and ATPase.................................................................19 Feed Consumption .................................................................................................28 Seawater Performance ...........................................................................................28

5

In-river Migration and Survival.............................................................................33 PIT-Tag Interrogation Rates .....................................................................33 Migration Time ..........................................................................................33

Discussion .........................................................................................................................37 Growth ...................................................................................................................37 Condition Factor ....................................................................................................38 Size Variation and Smoltification..........................................................................38 Feed Consumption .................................................................................................39 Seawater Performance ...........................................................................................39 In-river Migration and Survival.............................................................................39 Comparison of Inventory Methods ........................................................................40 Conclusions............................................................................................................41

Appendices........................................................................................................................42 CHAPTER THREE - EVALUATION OF AN EXTRUDED ABERNATHY DIET

ENHANCED WITH (SCHIZOCHYTRIUM) AS A MEANS OF PROMOTING SMOLTIFICATION IN SUMMER STEELHEAD (ONCORHYNCHUS MYKISS) AT DWORSHAK NATIONAL FISH HATCHERY, IDAHO ....................................45

Abstract.............................................................................................................................46 Introduction......................................................................................................................47 Methods.............................................................................................................................48

Experimental Design..............................................................................................48 Fish Culture............................................................................................................48 Feed Methods.........................................................................................................49 Data Collection ......................................................................................................49 Data Analysis .........................................................................................................53

Results ...............................................................................................................................54 Smoltification.........................................................................................................54

Pre-release .................................................................................................54 Downstream Migration. ...........................................................................54 Extended Seawater Rearing.......................................................................54

Downstream Migration Survival ...........................................................................54 PIT-tag Interrogation Rates.......................................................................54 Migration Time ..........................................................................................60

Extended Seawater Rearing Survival ....................................................................60 Discussion .........................................................................................................................67

CHAPTER FOUR - AN EVALUATION OF FEEDING THE MOORE-CLARK NUTRA

TRANSFER FW DIET AS A MEANS OF PROMOTING SMOLTIFICATION IN SUMMER STEELHEAD (ONCORHYNCHYS MYKISS) AT DWORSHAK NATIONAL FISH HATCHERY, IDAHO ....................................................................70

Abstract.............................................................................................................................71 Introduction......................................................................................................................72

6

Methods.............................................................................................................................72 Experimental Design..............................................................................................72 Fish Culture............................................................................................................72 Data Collection ......................................................................................................73 Data Analysis .........................................................................................................74

Results ...............................................................................................................................74 Indices of Smoltification........................................................................................74

Pre-release .................................................................................................74 Emigration .................................................................................................74 Extended Seawater Rearing.......................................................................75

Emigration..............................................................................................................75 PIT-tag Interrogation Rates.......................................................................75 Migration Time ..........................................................................................75

Extended Seawater Rearing Survival ....................................................................76 Discussion .........................................................................................................................76

CHAPTER FIVE - SKIN REFLECTANCE AS A NON-LETHAL MEASURE OF

SMOLTIFICATION FOR JUVENILE STEELHEAD AT DWORSHAK NATIONAL FISH HATCHERY, IDAHO ....................................................................98

Abstract.............................................................................................................................98 Methods...........................................................................................................................100

Fish Collection and Processing............................................................................100 Converting Photos to Quantitative Values of Skin Reflectance ..........................100 Data Analysis and Statistical Comparisons .........................................................101

Results .............................................................................................................................103 Skin Reflectance ..................................................................................................103 Gill ATPase..........................................................................................................103 Condition Factor ..................................................................................................103 Correlations Between Indices ..............................................................................103

Discussion .......................................................................................................................103 CONCLUSIONS ........................................................................................................................107 LIST OF REFERENCES ..........................................................................................................108

7

LIST OF TABLES CHAPTER ONE: Table 1.1. Mean monthly temperatures at Dworshak NFH from September 1997 to March 1998...........................................................................................................................5 CHAPTER TWO: Table 2.1. Mean specific growth rate (SGR, % × day-1) in length (mm) of summer steelhead at Dworshak National Fish Hatchery, Idaho, based on the Monthly Inventory Summaries (MIS), 1986-1996. ............................................................................................................................ ......13 Table 2.2. Mean specific growth rate (SGR, % × day-1) in weight (g) of summer steelhead at Dworshak National Fish Hatchery, Idaho, based on the Monthly Inventory Summaries (MIS), 1986-1996. ...................................................................................................................................14 Table 2.3. Consumption of grams of feed per grams of fish for Dworshak National Fish Hatchery System II steelhead from 1987 to 19978 calculated from the Monthly Inventory Summaries (MIS)...........................................................................................................................15 Table 2.4. Mean monthly temperatures at Dworshak National Fish Hatchery during 1986 - 1996. Derived from the Monthly Inventory Summaries (MIS)...............................................................16 Table 2.5. Number of small and large steelhead smolts from modified feeding treatment (pond 16) and control (CON 20) groups at Dworshak National Fish Hatchery, Idaho, that were PIT-tagged, released, and detected at lower Snake and Columbia River dams in 1998................................................................................................................................................34 Table 2.6. Summary of PIT-tag release and interrogation data for steelhead released from Dworshak National Fish Hatchery, Idaho, and interrogated at lower Snake and Columbia River dams from 1992 through 1997.......................................................................................................35 Table 2.7. Number of steelhead that were PIT-tagged and released at Dworshak National Fish Hatchery, Idaho, and interrogated at lower Snake and Columbia River dams, presented for each 10-mm length group released from MFS 16 and CON 20 in 1998...............................................36 Table 2.8. Mean migration time of small (< 200 mm) and large (> 200 mm) steelhead smolts from treatment pond 16 and control pond 20 at Dworshak National Fish Hatchery that were PIT-tagged, released, and detected at lower Snake and Columbia River dams in 1998................37 CHAPTER THREE:

8

Table 3.1. Composition of the Abernathy Dry and enhanced Abernathy Dry extruded feeds used in the diet trial at Dworshak NFH, 1997-98. ...............................................................................50 Table 3.2. Proximate analysis and fatty acid profile of the Schizochytrium used in the enhanced Abernathy diet. .............................................................................................................................51 Table 3.3. Proximate analyses of the diets used in the Dworshak NFH diet trial, 1997-98.........52 Table 3.4. Mean monthly gill ATPase levels for summer steelhead in the treatment (BP28 and BP30) and control (BP24 and BP26) ponds in the Enhanced Abernathy Diet Evaluation Trial at Dworshak NFH, Idaho. ................................................................................................................55 Table 3.5. Mean monthly skin reflectance levels for summer steelhead in the treatment (BP28 and BP30) and control (BP24 and BP26) ponds in the Enhanced Abernathy Diet Evaluation Trial at Dworshak NFH, Idaho. ............................................................................................................56 Table 3.6. Mean monthly condition factor (K) for summer steelhead in the treatment (BP28 and BP30) and control (BP24 and BP26) ponds in the Enhanced Abernathy Diet Evaluation Trial at Dworshak NFH, Idaho. ................................................................................................................57 Table 3.7. Number of small and large summer steelhead smolts in the treatment pond 28 and control pond 24 that were PIT-tagged, released, and detected at down river dams as part of the Enhanced Abernathy Diet Evaluation Trial at Dworshak NFH in 1998........................................58 Table 3.8. Summary of PIT-tag release and interrogation data for steelhead released from Dworshak NFH from 1992 through 1997. ...................................................................................59 Table 3.9. Mean migration time of small (< 200 mm) and large (> 200 mm) steelhead smolts that were PIT-tagged, released, and detected at down river dams as part of the Enhanced Abernathy Diet Evaluation Project at Dworshak NFH in 1998. ..................................................65 Table 3.10. Mortalities that occurred during extended seawater rearing at Marrowstone Field Station for summer steelhead in the Enhanced Abernathy Diet Evaluation Project at Dworshak NFH in 1998. ...............................................................................................................................66 CHAPTER FOUR: Table 4.1. Mean monthly gill ATPase levels for summer steelhead in the treatment (BP32 and BP34) and control (BP24 and BP26) ponds in the Moore-Clark Nutra-Transfer Diet Evaluation Project at Dworshak NFH, Idaho..................................................................................................78 Table 4.2. Mean monthly skin reflectance levels for summer steelhead in the treatment (BP32

9

and BP34) and control (BP24 and BP26) ponds in the Moore-Clark Nutra-Transfer Diet Evaluation Project at Dworshak NFH, Idaho..............................................................................................................................................79 Table 4.3. Mean monthly condition factor (K) for summer steelhead in the treatment (BP32 and BP34) and control (BP24 and BP26) ponds in the Moore-Clark Nutra-Transfer Diet Evaluation Project at Dworshak NFH, Idaho..................................................................................................80 Table 4.4. Mean monthly gill ATPase levels for summer steelhead from treatment (BP32 and BP34) and control (BP24 and BP26) ponds in the Moore-Clark Nutra-Transfer Diet Evaluation Project during seawater rearing trials at Marrowstone Marine Station, Washington....................81 Table 4.5. Number of small and large steelhead smolts that were PIT-tagged, released, and detected at down river dams for treatment pond 32 and control pond 24 in the Moore-Clark Nutra-Transfer Diet Evaluation Project at Dworshak NFH, Idaho in 1998...................................82 Table 4.6. Summary of PIT-tag release and interrogation data for steelhead released from Dworshak NFH from 1992 through 1997......................................................................................83 Table 4.7. Mean migration time of small and large steelhead smolts that were PIT-tagged, released, and detected at down river dams for treatment pond 32 and control pond 24 in the Moore-Clark Nutra-Transfer Diet Evaluation Project at Dworshak NFH in 1998........................84 Table 4.8. Mortalities that occurred during extended seawater rearing at Marrowstone Field Station for summer steelhead in the Moore-Clark Nutra-Transfer Diet Evaluation Project at Dworshak NFH in 1998.................................................................................................................85 CHAPTER FIVE: Table 5.1. Statistical summary of skin reflectance, gill ATPase, and condition factor for each of the experimental groups sampled at Dworshak NFH, February to April 1998...........................105 Table 5.2. Pearson correlation coefficients calculated for comparisons of skin reflectance, condition factor, and gill ATPase for experimental groups sampled at Dworshak NFH, February to April 1998................................................................................................................................106

10

LIST OF FIGURES CHAPTER ONE: Figure 1.1. Map showing location of Dworshak National Fish Hatchery in the Lower Clearwater River drainage, and the location of Lower Snake River and Columbia River dams where PIT-tag interrogation and recovery facilities are located..............................................................................4 CHAPTER TWO: Figure 2.1. Mean fork length (mm) of summer steelhead in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 100-fish inventories (HFI)...................................................................20 Figure 2.2. Mean fork length (mm) of summer steelhead in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 20-fish physiology samples (PS).........................................................21 Figure 2.3. Mean fork length (mm) of summer steelhead in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 100 fish inventories (HFI) and 20-fish physiology samples (PS).......22 Figure 2.4. Mean weight (g) of summer steelhead in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 100-fish inventories (HFI)...................................................................................23 Figure 2.5. Mean weight (g) of summer steelhead in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 20-fish physiology samples (PS).........................................................................24 Figure 2.6. Mean weight (g) of summer steelhead in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 100-fish inventories (HFI) and 20-fish physiology samples (PS).......................25 Figure 2.7. Mean specific growth rate (SGR, % ⋅ day -1) in fork length (mm) of summer steelhead in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 100-fish inventories (HFI)..............................................................................................................................................26 Figure 2.8. Mean specific growth rate (SGR, % ⋅ day -1) in body weight (g) of summer steelhead in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 100- fish inventories (HFI).........27

11

Figure 2.9. Mean condition factor (K) of summer steelhead in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 100-fish inventories (HFI)...................................................................29 Figure 2.10. Mean condition factor (K) of summer steelhead in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 20-fish physiology samples (PS).........................................................30 Figure 2.11. Mean condition factor (K) of summer steelhead in the modified feeding schedule treatment and control groups at Dworshak National Fish Hatchery, Idaho, based on 100-fish inventories (HFI) and 20-fish physiological samples (PS)............................................................31 Figure 2.12. Mean Na+, K+ -ATPase activity (µmol Pi ⋅ mg protein-1 ⋅ hr-1) of summer steelhead in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho......................................................................32 CHAPTER THREE: Figure 3.1. Total number of smolts marked with PIT-tags and subsequently interrogated at downriver dams for treatment pond 28 and control pond 24 in the Abernathy Diet experiment conducted at Dworshak NFH in May1998...................................................................................61 Figure 3.2 . Percent of small (<200 mm) and large (>200 mm) smolts marked with PIT-tags and subsequently interrogated at downriver dams for treatment pond 28 and control pond 24 in the Abernathy Diet experiment conducted at Dworshak NFH in May1998......................................62 Figure 3.3. Number of PIT-tagged steelhead smolts released and interrogated at downriver dams, by 10 mm length group, for the treatment pond 28 and control pond 24 in the Abernathy Diet experiment conducted at Dworshak NFH in 1998.........................................................................63 Figure 3.4. Percentage of smolts released that were interrogated at downriver dams for the treatment and control ponds, by length group, in the Abernathy Diet experiment conducted at Dworshak NFH in 1998.................................................................................................................64 CHAPTER FOUR: Figure 4.1. Total number of smolts marked with PIT-tags and subsequently interrogated at down river dams for the control and treatment groups in the Moore-Clark Nutra-Transfer Diet Evaluation Project at Dworshak NFH in May 1998......................................................................86 Figure 4.2. Percent of small (<200 mm) and large (>200 mm) steelhead smolts marked with PIT-tags and subsequently interrogated at downriver dams for the control and treatment groups in the

12

Moore-Clark Nutra-Transfer Diet Evaluation Project at Dworshak NFH in May.........................87 Figure 4.3. Number of PIT-tagged steelhead smolts released and interrogated at downriver dams, by 10 mm length group, for the treatment and control ponds in the Moore-Clark Nutra-Transfer Diet Evaluation project conducted at Dworshak NFH in 1998.....................................................88 Figure 4.4. Percentage of smolt released that were interrogated at downriver dams for the treatment and control ponds, by length group, in the Moore-Clark Nutra-Transfer Diet Evaluation project conducted at Dworshak NFH in 1998................................................................................89 CHAPTER FIVE: Figure 5.1. Photo of steelhead smolt inside a plexiglass case for measuring skin reflectance. The area used for assessing skin reflectance is outlined by the black lines. The black and gray calibration tabs are located in the upper left................................................................................102

13

LIST OF APPENDICES CHAPTER TWO: Appendix 2.1. Mean total length (mm) and weight (g) at release and adult returns for Dworshak National Fish Hatchery summer steelhead (Oncorhynchus mykiss) from coded wire tag rack returns for the Modified Feeding Schedules Project at Dworshak National Fish Hatchery. ........42 Appendix 2.2. Number of mortalities of summer steelhead in the modified feeding schedule treatment (MFS 16/Tank 7 and 18/Tank 9) and control (CON 20/Tank 3 and BP22/Tank 8) groups at Marrowstone Marine Station, Washington. ..................................................................43 Appendix 2.3. Food consumption by summer steelhead in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) ponds at Dworshak National Fish Hatchery, Idaho, based on 100-fish inventories (HFI) and Monthly Inventory Summary (MIS). .......................................................................................................................................................44 CHAPTER THREE: Appendix Table 3.1 Monthly mortality, weight, and size of steelhead in each treatment and control pond in the Moore-Clark Nutra-Transfer Diet evaluation project at Dworshak National Fish Hatchery, Idaho.....................................................................................................................68 CHAPTER FOUR: Appendix Table 4.1. Monthly mortality, weight, and size of steelhead in each treatment pond (Ponds 32 and 34) and control pond (Ponds 24 and 26) in the Moore-Clark Nutra-Transfer Diet evaluation project at Dworshak National Fish Hatchery, Idaho....................................................90 Appendix Table 4.2. Proximate analyses of the diets used in the Dworshak NFH diet trial, 1997-98....................................................................................................................................................92 Appendix Table 4.3. Daily feeding records for the treatment (BP32 and BP34) and control (BP24 and BP26) ponds for the Moore-Clark Nutra-Transfer Diet Evaluation Project for summer steelhead at Dworshak NFH, Idaho...............................................................................................93

1

CHAPTER ONE

PROJECT DESCRIPTION

Introduction Wide length frequency distributions are a common characteristic of juvenile steelhead (Oncorhynchus mykiss) at Dworshak National Fish Hatchery (NFH). Lengths often range from 80 to 260 mm total length (TL) by the time they are released from the hatchery as smolts. Based on a hatchery pre-release inventory in 1995, almost 20% (460,000) of the smolts released that year were less than 180 mm (FL). Bigelow (1997) calculated that less than 30% of the PIT-tagged steelhead smaller than 170 mm (TL) released from Dworshak NFH in1995 were detected at a downriver dams in the Snake or Columbia rivers. These fish are assumed to be non-migrants that do not successfully complete the parr-smolt transformation process. The release of fish smaller than 180 mm and larger than 250 mm TL conflicts with the National Marine Fisheries Service (NMFS) Biological Opinion on Artificial Production in the Columbia River Basin (NMFS 1999) and The Proposed Recovery plan for Snake River Salmon (NMFS 1995). One concern is that large and small steelhead that fail to emigrate may negatively impact sensitive fish species such as the endangered Snake River fall chinook salmon, spring/summer chinook salmon, or the threatened wild Snake River steelhead, through displacement, competition for food, and/or behavioral influences (Viola and Schuck 1995). Juvenile hatchery steelhead have been collected in several tributary streams below Dworshak NFH after smolts were released in 1994, 1995, and 1996; however, the effects of residualized hatchery fish on wild steelhead in those streams has not been determined. The primary objective of the NMFS guidelines for release size of hatchery steelhead is to reduce the potential impacts of various state and federal hatchery programs on listed fish species, by attempting to minimize the number of non-migrating hatchery summer steelhead smolts released into the Snake River basin. The release of large numbers of hatchery steelhead which fail to meet NMFS size guidelines has caused state and federal hatchery managers to focus on length frequency distributions of their steelhead populations, and to look for ways to modify rearing programs in order to comply with the guidelines. The underlying assumption of this approach is that manipulation of the size distribution of a steelhead population will address the issue of insuring successful parr-smolt transformation. Evidence does suggest a critical size threshold is important in the timing of seaward migration (Folmar and Dickhoff 1980; Whitesel 1991). For steelhead, research indicates that salinity preference can be size and age dependent (Folmar and Dickhoff 1980). Conte and Wagner (1965) concluded that steelhead smaller than 120 mm had not reached the critical or optimal size for development of an effective osmoregulatory system, and suggested that the critical size for steelhead was about 140 to 150 mm. Despite focus on the role of fish size, smoltification is an extremely complex process involving a number of variables influencing a variety of physiological, behavioral, and morphological attributes (Wedemeyer et al. 1980; Folmar and Dickhoff 1980). Light and temperature both influence smoltification and the migration tendency in salmonids (Zaugg 1981; Solbakken et al.

2

1994; Muir et al. 1994), with photoperiod probably being the most important factor responsible for synchronizing seasonal changes in salmonid physiology (Zaugg et al. 1986; Jonsson 1991). In steelhead, migratory behavior; gill sodium, potassium-activated adenosine triphosphatase (Na+, K+-ATPase) activity; and condition factor were found to be influenced primarily by photoperiod (Hoar 1976). Water temperature has a significant influence on smoltification, especially development of increased gill ATPase activity (Zaugg et al. 1972; Adams et al. 1973, 1975; Zaugg and Wagner 1973; Wagner 1974a). Production strategies that meet NMFS guidelines for size at release for hatchery steelhead will not necessarily promote an increase in the proportion of actively migrating steelhead smolts. Hatchery practices can have a significant influence on the parr-smolt transformation process (Folmar and Dickhoff 1980; Shrimpton et al. 1994), and could result in fewer outmigrants if their direct influence on the physiology and behavior of steelhead prior to and during smoltification is not considered. During the parr-smolt transformation, anadromous fish are sensitive to husbandry practices including water temperature (Johnson and Saunders 1981), photoperiod (Zaugg 1981; Sigholt et al. 1998), water flow (Hosmer et al. 1979; Banks 1994) , rearing density (Banks 1992; Ewing and Ewing 1995; Ewing et al. 1998b), and release time (Bilton et al. 1982). The influence of various rearing practices within the hatchery can affect both timing of the onset of smoltification and the process of smoltification itself, and can have dramatic effects on the ultimate success of the parr-smolt transformation (Folmar and Dickhoff 1980; Hansen et al. 1989). Smoltification also involves the endocrine system and its effect on growth (Dickhoff et al. 1997) and activation of thyroid and interrenal tissues (Barron 1986; Iwata 1995). Therefore, to focus attention solely on fish size ignores the complexity of smoltification and the need to develop an approach to stimulate smoltification in hatchery steelhead of all sizes.

Further supporting the importance of factors other than size, wild steelhead smolts outmigrating from the Snake River basin are generally smaller on the average than their hatchery counterparts (Beeman et al. 1990, 1991; Maule et al. 1994; Schrock et al. 1999). Many wild salmonid stocks exhibit osmoregulatory competence at sizes much smaller than hatchery stocks (Zaporozhec and Zaporozhec 1993; Shrimpton et al. 1994). Naturally-spawning salmonid stocks outmigrating from Columbia River tributaries exhibit a wide range of lengths and ages (Peven et al. 1994). In this study, we evaluated several methods to produce healthy, high-quality, actively migrating steelhead smolts, regardless of size. Presence of a wide range of sizes and ages at the time of smolt outmigration is a natural phenomenon in the life history of steelhead stocks in the Columbia River (Peven et al. 1994). A review of the literature indicates that high variability in size (length) within a juvenile steelhead population is, in part, a product of social interactions in which dominant individuals have greater access to food, and therefore grow significantly faster than subordinates (Abbott and Dill 1989). The same phenomenon has been observed in Atlantic salmon populations (Symons 1970; Thorpe et al. 1992). Besides experiencing slower growth rates, subordinate individuals in a population generally experience higher levels of stress than dominant individuals, which may lead to a suppression of their physiological development and a failure to complete the parr-smolt transformation. Several studies indicate that growth rate at the time of smoltification may be more critical in the parr-smolt transformation process than the

3

absolute amount of growth that occurs (Whitesel et al. 1991; Beckman et al. 1998). Therefore, we investigated two methods of promoting growth: a manipulations of growth rate with a modified feeding schedule and enhanced feeds during a period immediately preceding the release date

Goal and Objectives Three different experiments were conducted: 1) modification of feeding schedule and rations, 2) feeding with Abernathy Diet enhanced with glucans during the spring, and 3) feeding the Moore-Clark Nutra-Transfer Diet enhanced with salt, glucans, and high levels of dietary fat. The goal for all three experiments was to increase the growth rate of smaller steelhead during the spring to achieve the following objectives: 1) To increase the proportion of smaller sized (< 200 mm) juvenile steelhead that exhibit characteristics of smoltification prior to release from the hatchery, during seaward emigration, and during extended seawater rearing, 2) To increase the proportion of smaller sized (< 200 mm) steelhead smolts that successfully emigrate to down river dams on the Snake and Columbia Rivers, and 3) To increase the proportion of smaller sized (< 200 mm) steelhead smolts that survive during extended seawater rearing.

Site Description

4

Dworshak NFH is located at the confluence of the North Fork and the main stem of the Clearwater River near Ahsahka, Idaho (Figure 1.1). Construction of the hatchery was included in the authorization for Dworshak Dam and Reservoir (Public Law 87-847, enacted October 23, 1962) to mitigate for losses of anadromous steelhead caused by the dam and reservoir. The goals of the mitigation program are to return 20,000 adult steelhead to the mouth of the Clearwater River annually and to maintain the unique genetics of the stock. The hatchery was designed and constructed by the U. S. Army Corps of Engineers and has been administered and operated by the U. S. Fish and Wildlife Service (USFWS) since the first phase of construction was completed in 1969. Initial construction at Dworshak NFH included 84 Burrows ponds, 64 nursery tanks, and 9 adult holding ponds. Further construction in the early 1980's doubled the number of inside nursery rearing tanks to 128. The Burrows ponds are divided into three separate rearing systems, Systems I, II, and III. All the Burrows ponds are furnished single-pass river water from May through November when desired temperatures can be obtained through selector gates at Dworshak Dam. System I (25 ponds) continues to receive single-pass river water for the rest of the production cycle. In System II (25 ponds) and System III (34 ponds), water reuse and heating is used during the colder months of November through March enabling the hatchery to get the desired fish growth. During reuse, 10 percent new water enters the system to make up for loss. Each of the three outside ponding systems is independent of each other for temperatures when

5

reuse and heated water are available. General Methods Fish Culture The study used Dworshak NFH B-run summer steelhead progeny from spawning takes 11 and 12, spawned in April, 1997. Fish were reared under routine hatchery conditions. The eggs were incubated in Heath trays until transfer from the incubation room to nursery tanks in May 1997. The fish were stocked at densities of about 26,500 to 27,000 fish per tank. Nursery rearing temperatures varied, but 12 oC was the targeted average. Fish were fed BioMoist Starter during nursery rearing. Tanks were cleaned once or twice weekly as necessary and mortalities were collected and counted daily. The fish were transferred to outside Burrows ponds in System II during August 1997 at an average size of 95 to 100 fish per pound. About 28,500 fish were stocked into each Burrows pond. All fish were hand-fed BioDiet Grower (2.5-mm feed) from the time of ponding until November 11, 1997. At that time, the fish were converted from hand feeding to Babington demand feeders, and were converted from BioDiet Grower to Nelson’s Silver Cup steelhead diet (heat-extruded dry feed). Demand feeders were located at both ends of each pond, and were kept constantly full to provide the maximum rate of growth possible. Fish size at conversion to demand feeders was about 25 fish per pound. All ponds were supplied with ambient river water on a single-pass system until November 19, 1997 when the reuse system was started. Average temperatures varied between 47.5 oF (8.61 oC) for October 1997 to 54.1 oF (12.28 oC) for September 1997 (Table 1.1). The fish were released into the Clearwater River on April 29, 1998. On April 24, 1998 about 75 fish from each of the treatment and control ponds were transferred from Dworshak NFH to the seawater rearing facilities at the Marrowstone Marine Field Station on Puget Sound, Washington, for extended seawater rearing trials. The fish were kept in 100-gallon totes supplied with oxygen during transfer. Temperatures in the totes were maintained near 13 oC with ice. At Marrowstone, the fish were measured for fork length, weighed, and transferred to freshwater circular rearing tanks. Fish were allowed to acclimate in freshwater for three days and on May 3, 1998 conversion to seawater was initiated. Fish were held at 1/3 seawater for two days, 2/3 seawater for two days, and were converted to full strength seawater on May 7, 1998. The fish were fed the same diet used at Dworshak NFH. Table 1.1. Mean monthly temperatures at Dworshak NFH from September 1997 to March 1998.

Temp. Units

Sept.

Oct.

Nov.

Dec.

Jan.

Feb.

Mar.

Celsius 12.28

8.61

10.44

11.15

12.22

12.22

11.04

Fahrenheit

54.1

47.5

50.8

52.1

54.0

54.0

57.9

6

Data Collection The hatchery production staff conducted monthly sample counts on representative ponds in System II and used that data to project growth for all ponds in the system. Sample counting consisted of dipnetting a random sample of fish from the pond, taking the total weight of the fish in the net, and counting the number of fish to estimate average weight. Mortalities collected from each pond were recorded daily. Starting in September, staff from the Idaho Fishery Resource Office (IFRO) collected random samples of about 100 fish from each pond to measure mean fish length and weight. Each fish was measured for total length (nearest mm) and weight (nearest 0.1 g). In February, March, and April 1998, staff from the Columbia River Research Laboratory (CRRL) collected random samples of 20 fish from each pond to measure gill ATPase activity, condition factor, skin reflectance, body fat, and lysozyme. Fish were non-lethally anesthetized using tricaine methanesulfonate (MS-222) and were measured for fork length and weight. Gill ATPase activity was measured using the method described by Schrock et al. (1994). Skin reflectance was measured using a modification of the technique described by Haner et al. (1995). Condition factor (K) was calculated as (105 ⋅ [weight ⋅ length-3]). To measure migration rate, estimate survival during migration, and assess smolt development after release, 400 fish from one replicate treatment pond in each experiment (total of 3 ponds) and from 2 replicate control ponds were marked with passive integrated transponder tags (PIT-tags, 2000 total). To assess differences in migration rate, survival, and smolt development between smaller and larger fish, the PIT-tags were placed into 200 fish less than 200 mm (TL) and 200 fish greater than 201 mm (TL) in each pond. Tagging was conducted on April 22, 1998. Migration time was measured to Lower Granite, Little Goose, Lower Monumental, and McNary dams on the lower Snake and Columbia rivers (Figure 1.1). Survival to Lower Granite Dam during migration was estimated using the cumulative number of unique interrogations at each of the dams listed above. To compare pre- and post-release smolt development, we recaptured our PIT-tagged fish using the NMFS interrogation-by-code facilities at Little Goose Dam, and measured gill ATPase activity for comparison with activity levels prior to release. At Marrowstone Marine Field Station, fish were inventoried for length and weight each month after the initial inventory. Mortalities were recorded daily. Physiological sampling for gill ATPase was conducted June 1, and August 4 and 5, 1998, and all fish in the tanks were weighed and measured. On July 6, fish in all tanks were inventoried for length and weight only. Statistical Analysis Mean gill ATPase, mean skin reflectance, and mean condition factor were compared between treatments and controls using analysis of variance (ANOVA) (Wilkinson 1990). In cases where statistically significant differences were observed, pairwise comparisons were made using the

7

Bonferroni post-hoc procedure to identify which ponds differed. For PIT-tagged steelhead recaptured at Little Goose Dam, mean gill ATPase was compared between the treatment and control groups using a two-sample t-test (Wilkinson 1990). Mean migration rates were compared between treatments and controls using ANOVA (Wilkinson 1990). Interrogation rates were compared between treatments and controls using a chi-square test for differences in probabilities (Conover 1970). For all statistical tests, a P-value of 0.05 was used to designate a significant difference.

8

CHAPTER TWO

AN EVALUATION OF THE EFFECTS OF A MODIFIED FEEDING STRATEGY ON GROWTH AND SMOLTIFICATION OF SUMMER STEELHEAD (ONCORHYNCHUS

MYKISS) AT DWORSHAK NATIONAL FISH HATCHERY

by Robin M. Schrock and Robert E. Reagan

U. S. Geological Survey, Biological Resources Division Columbia River Research Laboratory, Western Fisheries Research Center

Cook, Washington

Ray N. Jones U. S. Fish and Wildlife Service, Idaho Fishery Resource Office

Ahsahka, Idaho

Robert Semple U. S. Fish and Wildlife Service, Dworshak National Fish Hatchery

Ahsahka, Idaho

Nancy Elder U. S. Geological Survey, Biological Resources Division,

Western Fisheries Research Center, Marrowstone Marine Station Nordland, Washington

9

Abstract Growth rates of juvenile summer steelhead at Dworshak National Fish Hatchery were manipulated during winter rearing to promote smoltification in a larger proportion of the production release of summer steelhead (Oncorhynchus mykiss) from Dworshak National Fish Hatchery. By reducing growth rates during winter allowing accelerated growth rates during spring, we attempted to produce a smaller size range of fish at release, an increased number of small fish that undergo parr-smolt transformation, and fewer large fish that potentially residualize. Reduced winter growth was produced by a combined reduced ration/intermittent feeding treatment while accelerated, compensatory growth in the spring was achieved through the return of treatment fish to full hatchery production rations. Growth rates in both the treatment group and control groups were not reduced to the level expected from a review of ten prior years of hatchery records during December and January, when the general production group showed a voluntary reduction in feed intake. Although significant differences in growth rates were seen between the treatment and control groups from December to February, compensatory growth in the treatment group in February and March resulted in the release of fish of the same size and similar size variation. Migration rates of the treatment group were higher than that of the control group, but the difference was not significant. Extended seawater survival and smoltification as measured by gill ATPase did not differ between the groups.

10

Introduction Steelhead (Oncorhynchus mykiss) have been the subject of previous studies designed to alter growth rates to achieve size-at-release goals. Bjornn et al. (1978; 1979) conducted extensive studies with steelhead at Dworshak National Fish Hatchery (NFH) to determine how length at release, date of release, cold water conditioning, fungal infections, saltwater tolerance, and gill sodium, potassium-activated adenosine triphosphatase (Na+, K+-ATPase) activity affected seaward emigration, as measured by recapture at Lower Granite Dam and Little Goose Dam. At that time, the goal at Dworshak was to release fish 200 mm total length (TL), whereas today the target size at release is between 170 and 250 mm. Minimum release sizes of steelhead have been recommended based on the higher recapture rates of tagged fish of larger sizes (> 190 mm) after downstream migration over various distances (Tipping et al. 1995). The assumption drawn from these studies is that large size is critical to smoltification, though the relatively small size of wild emigrating steelhead smolts contradicts this (Beeman et al. 1990, 1991; Maule et al. 1994; Schrock et al. 1999). In our study of steelhead at Dworshak NFH, emphasis on the importance of large size in smoltification is replaced by the hypothesis that manipulation of growth pattern in the months prior to release may stimulate smoltification regardless of final fish size. Some authors consider flow, rather than fish size, the primary predictor variable of migration rate in steelhead (Maule et al. 1994; Giorgi et al. 1997), though the mechanism for the initiation of migration is still unexplained. Differences in smoltification indices between migrating and nonmigrating salmonids have been found, but in steelhead the timing of migration is not tightly coupled with changes in smolt indices. A study comparing migrating and non-migrating rainbow trout found lower condition factor, plasma thyroxine, muscle and liver glycogen levels, and levels of esterified fatty acid, and higher Na+, K+-ATPase levels in migrants (Ewing et al. 1994a). The peak of gill Na+, K+-ATPase in migrants did not correspond to the peak of emigration. In an earlier study, the timing of migration tendency in hatchery winter steelhead did not coincide with indices of smoltification including body silvering and gill Na+, K+-ATPase activity (Ewing et al. 1984). Feeding studies have been conducted to investigate the effects of diet composition, ration rate, feeding frequency, and method of feeding on steelhead growth, development and smoltification. Increased migration rates have been noted in steelhead fed reduced rations during the last month of hatchery rearing, but results differed at two study sites (Tipping and Byrne 1996). The treatment fish were smaller measured by both length and weight, and had lower condition factors than control fish, but migration rates and recapture rates were higher in the treatment fish. Salmonids generally show reduced feeding levels and declining condition factors as they undergo the parr-smolt transformation, but other developmental changes-beyond the reduction in weight that affects condition factor are necessary for smolt development. Only recently has manipulation of growth rate been investigated as a method of accelerating smoltification. Beckman et al. (1998) used temperature to affect growth rate of spring chinook salmon (O. tshawytscha), and found fish with higher spring growth rates emigrated earlier than fish with lower growth rates. Extensive reviews describe the many environmental variables that may be manipulated to

11

modulate the timing and duration of smoltification (Folmar and Dickhoff 1980; Wedemeyer et al. 1980; Zaugg 1981, 1982; Hoar 1988; Maynard et al. 1995), but few methods are practical at a large hatchery. Innovative efforts at hatcheries to increase adult returns, such as exercising fish, have been questioned due to the additional labor and expense required to increase returns by only a small percentage (Evenson and Ewing 1993). Modifications of feeding schedules, rations, or diets are, however, easily achieved at large production facilities. Intermittent feeding schedules have been developed to save labor costs at hatcheries, though rainbow trout fed daily show the best feed conversion efficiency (Kindschi 1988). In a study of intermittent feeding schedules, Kindschi (1988) found that rainbow trout on the longest duration of alternating 4-week fasting and feeding periods showed the least variation in length and weight. After return to full rations, compensatory growth allowed the starved fish to recover weight, although they were smaller at release than control fish. Another study investigated intermittent and reduced feeding schedules, alternating 7 days of feeding with up to 10 days of no feeding, to reduce growth of steelhead reared at 15 oC and ultimately prevent production of large fish (Klontz et al. 1991). Feed reduction resulted in reduced final length and weight, but percent reduction in length and weight did not correspond to the percent reduction in feeding intensity. Fish size may influence the relationship between ration and growth; gross food conversion efficiency or food consumption decreases as ration increases in large fish (Wurtsbaugh and Davis 1977). Demand feeding of steelhead allowed larger fish to feed to satiation, and if enough feed was available, smaller fish then fed. Steelhead from Dworshak NFH are characterized by a wide range of lengths at the time of release. A consequence of the wide size distribution is the production of three groups of fish: small parr that do not migrate, functional smolts that migrate, and large sexually precocious males that residualize and may compete with wild stocks. Efforts to achieve size-at-release requirements, the use of demand feeders, and warm water temperatures may all contribute to an increase in the size range. Results from previous studies strongly suggest that the time of year, holding temperature, and even the stock of fish may influence the outcome of feeding trials (Smith 1987; Kindschi 1988; Klontz et al. 1991). Reductions of feed to 50% of normal rations, regardless if fed continuously or intermittently, were found to reduce weight gain and to control the production of large fish that might residualize (Smith 1987). Klontz et al. (1991) compared the effects of reduced continuous and intermittent feeding regimes on size variation in steelhead and found that neither method attained decreases in size variation. Kindschi (1988) found that when rations were reduced, continuously fed fish varied more in size than fish fed intermittently on a reduced diet. Manipulation of diets, rations, or feeding regimes may negatively affect physiological processes that occur during the parr-smolt transformation. The lipid composition of steelhead is known to change during smoltification, with a decrease in total body lipids and changes in lipid classes found in specific tissues (Sheridan et al. 1983). Both diet and fasting may influence stress response and associated physiological responses, such as hyperglycemia in spring chinook salmon (Barton et al. 1988). Stress and its consequences must be considered when altering feeding regimes of fish that may later be transported or that face a long migration route. Fasting salmonids show negative effects such as increased mortality in as little as 2 weeks. After longer

12

periods of starvation, mortality may continue, and survivors do not reach the size of controls even after a return to feed (Bilton and Robins 1973). However, sockeye salmon (O. nerka) starved for 1 to 3 weeks were able to compensate by utilizing feed more efficiently when feed was presented, and to reach the size of controls after return to full rations. Reduced, intermittent feeding schedules are advantageous in that they control growth without the negative effects of extended fasting. The goal of our study was to determine whether altering growth patterns could enhance smolt development in smaller fish, while preventing production of large fish prone to residualism (Bigelow 1995, 1997). Our objectives were to: 1) increase the number of fish that migrate by promoting smoltification in a wider size range of fish, especially smaller fish; and 2) reduce the final length range at release while shifting the mode of the length frequency distribution toward smaller sizes. Wild steelhead were used as the general model: wild migrants are smaller than hatchery fish, and feed availability in the wild is lower in winter than in spring. Rather than mimic growth rates seen in wild steelhead, we attempted to reproduce growth patterns found in Dworshak steelhead during years in which the stock limited its own feed intake, resulting in release of small fish and later high adult returns. The desired growth rates were determined using known feeding rates from the previous 10 years of monthly inventory summaries (MIS) for Dworshak System II production. Weekly rations were calculated to achieve reduced growth in winter months at expected temperatures, to be followed by accelerated growth in the spring.

Methods Experimental Fish Steelhead from brood year 1997 (spawning take 12) were used for the experiment and were reared under routine hatchery conditions. Eggs were incubated in Heath trays until transfer to nursery tanks in May. In August 1997, fish were stocked at 95 to 100 fish per pound (~28,500 fish per pond) into 4 outdoor Burrows ponds in water reuse System II. The modified feeding schedule treatment group (MFS) and the production control group (CON) were each reared in duplicate ponds for a total of four Burrows ponds in System II. The density index (DI), calculated as [fish weight ⋅ (fish length ⋅ pond volume)-1] was 0.1 on November 1, 1997. All ponds were supplied with ambient river water on a single-pass system until November 19, 1997, when System II ponds were put on reuse water. The following day fish were treated with formalin. Fish were held in the same raceway throughout the experiment. Approximately 75 fish per experimental pond were taken to Marrowstone Marine Field Station on April 24, 1998, for seawater rearing. The remaining fish were released from the hatchery on April 29. Feed Methods and Calculations Fish were hand-fed BioDiet Grower (2.5-mm) from ponding until November 11, 1997. Upon conversion to Babington demand feeders, fish were fed Nelson’s Silver Cup steelhead diet (heat-extruded dry feed). Until the onset of the experiment, all fish were fed unlimited feed, with demand feeders kept full at all times. Demand feeders were located at both ends of each pond. The modified feeding schedule began on November 26, 1997 for treatment fish (Burrows Ponds

13

16 and 18), while control fish (Ponds 20 and 22) continued on full production rations. The feeding rate for the MFS group was determined from hatchery MIS records for System II during the years 1987 to 1996, using the following measurements and calculations: 1) total number of fish; 2) mean weight of fish, calculated from the number of fish per pound; and 3) grams of feed fed per gram of fish, calculated from the total feed fed, mean fish weight, and number of fish. Monthly specific growth rates in length or weight were calculated for the ten-year period and converted to percent per day (% ⋅ day-1) (Tables 2.1 and 2.2). Release years 1988 (BY87) and 1990 (BY89) were used as growth rate reference years based on the release of smaller fish compared to other years, and higher adult returns (Appendix 2.1). Monthly feed consumption rates were calculated for the previous ten years (Table 2.3) , and the lowest growth rate selected for anticipated water temperatures (Table 2.4) in December and January. Based on the small fish size at release and mean temperatures of 51.8 oF (11 oC) for December and January 1993, the feeding rates for BY91 fish were selected as our goal since growth rates were approximately half of those in other years. The two ponds designated for modified feeding (Ponds 16 and 18) were put on rations of 0.25 g feed ⋅ g fish-1 per month based on the weight of fish for the November 1997 MIS (27 fish ⋅lb-1, 16.8 g). Using the estimated number of fish in MFS treatment ponds 16 and 18, the monthly ration for each pond was determined, and weekly rations calculated. Monthly ration was recalculated for January based on the MIS mean weight on January 1. For modified ration ponds, demand feeders were loaded with one-third of the calculated weekly ration on Mondays, Wednesdays, and Fridays. Pond 16 received a total of 528 lb of feed from November 26 through December 31, and 561 lb from January 1 through February 9; Pond 18 received 512 lb and 544 lb during the same periods. On February 10, fish on the modified feed schedule were put back on full production rations. Sampling Methods Three inventory methods were used at the hatchery during the experiment: the monthly inventories, reported in monthly inventory summaries (MIS) by Dworshak NFH; the 100-fish inventories (HFI) conducted by the Idaho Fishery Resource Office (IFRO); and physiological sampling (PS) conducted by the Columbia River Research Laboratory (CRRL). Length and weight data from both the HFI and PS methods were analyzed and compared to determine sample numbers for further production studies. Results are presented based on monthly HFI data until February, when results for both HFI and PS are presented to allow comparison of the two methods.

14

Table 2.1. Mean specific growth rate (SGR, % ⋅ day-1) in length (mm) of summer steelhead at Dworshak National Fish Hatchery, Idaho, based on the Monthly Inventory Summaries (MIS), 1986-1996.

Mean SGR-Length (% × day-1)

Brood Year

August

Sept.

Oct.

Nov.

Dec.

Jan.

Feb.

Mar.

1986

13.67

2.19

1987

19.10

24.53

2.27

7.78

6.19

4.00

1988

16.22

13.95

12.93

7.83

1989

18.30

14.29

13.60

16.20

12.73

7.00

1990

13.40

28.95

17.05

13.90

9.88

7.94

1991

11.22

5.93

6.29

8.55

6.06

1992

11.30

21.34

7.03

18.24

15.43

11.76

1993

22.70

21.74

15.33

6.97

7.69

3.30

1994

23.00

15.89

6.85

7.05

10.18

3.80

1995

20.75

11.52

7.07

1.52

2.00

1996

15.38

13.33

8.82

8.11

9.38

8.00

15

Table 2.2. Mean specific growth rate (SGR, % ⋅ day-1) in weight (g) of summer steelhead at Dworshak National Fish Hatchery, Idaho, based on the Monthly Inventory Summaries (MIS), 1986-1996.

Mean SGR-Length (% × day-1)

Brood Year

August

Sept.

Oct.

Nov.

Dec.

Jan.

Feb.

Mar.

1986

36.36

1987

76.79

92.28

17.13

18.08

14.42

6.04

1988

59.23

46.11

43.87

26.91

1989

64.81

57.03

47.44

56.30

42.98

24.02

1990

47.48

115.73

60.21

48.31

32.42

24.95

1991

39.11

19.25

20.15

27.62

19.05

1992

34.38

80.10

20.86

66.46

52.77

39.65

1993

89.90

80.00

53.46

24.12

24.81

10.10

1994

90.38

54.64

20.18

23.54

32.20

13.26

1995

78.54

37.96

23.12

4.96

6.56

1996

55.22

45.32

28.94

24.90

30.75

27.43

16

Table 2.3. Consumption of grams of feed per grams of fish for Dworshak National Fish Hatchery System II steelhead from 1987 to 19978 calculated from the Monthly Inventory Summaries (MIS). Brood Year

Release Year

December

January

February

March

1987

1988

0.36

0.33

0.20

1988

1989

0.27

0.22

0.19

0.15

1989

1990

0.62

0.57

0.44

0.36

1990

1991

0.48

0.43

0.38

0.33

1991

1992

0.47

0.47

0.38

0.28

1992

1993

0.31

0.21

0.22

0.26

1993

1994

0.29

0.38

0.50

0.47

1994

1995

0.31

0.47

0.20

0.35

1995

1996

0.28

0.38

0.28

0.36

1996

1997

0.50

0.32

0.21

0.08

17

Table 2.4. Mean monthly temperatures at Dworshak National Fish Hatchery during 1986 - 1996. Derived from the Monthly Inventory Summaries (MIS).

Year

Temp.Unit

Aug.

Sept.

Oct.

Nov.

Dec.

Jan.

Feb.

Mar.

Apr.

1987-1988

C F

12.5 54.5

12.4 54.3

10.4 50.7

5.6 42.1

1988-1989

C F

11.5 52.7

11.5 52.7

12.1 52.7

12.2 54.0

9.4 48.9

8.9 48.0

7.8 46.0

5.6 42.1

1989-1990

C F

11.9 53.4

12.3 54.1

12.2 54.0

12.4 54.3

12.8 55.0

11.1 52.0

1990-1991

C F

11.7 53.1

11.1 52.0

11.9 53.4

12.2 54.0

12.2 54.0

12.3 54.1

12.9 55.2

8.4 47.1

1991-1992

C F

8.2 46.8

9.8 49.6

11.9 53.4

11.9 53.4

12.2 54.0

12.3 54.1

12.2 54.0

9.3 48.7

1992-1993

C F

7.6 45.7

11.8 53.2

11.2 52.2

10.6 51.1

9.9 49.8

10.5 50.9

8.2 46.8

1993-1994

C F

12.2 54.0

11.9 53.4

11.7 53.1

12.7 54.9

8.7 47.7

8.7 47.7

12.4 54.3

11.0 51.9

1994-1995

C F

10.0 50.0

11.0 51.9

11.4 52.5

10.3 50.5

12.2 54.0

12.2 54.0

12.5 54.5

7.9 46.2

1995-1996

C F

11.7 53.1

12.2 54.0

10.6 51.1

12.2 54.0

12.3 54.1

12.1 53.8

12.3 54.1

7.3 45.1

1996-1997

C F

12.7 54.9

12.0 53.6

12.1 53.8

12.2 54.0

8.7 47.6

4.4 39.9

4.4 39.9

Dworshak NFH conducted monthly inventories on the entire production, including experimental ponds, and provided us with the MIS records. Sampling consisted of dip-netting a random sample of fish from the pond, taking the total weight of fish in the net, and counting the number of fish to estimate average weight per fish. Mortalities from each pond were recorded daily. The IFRO took monthly inventories of approximately 100 fish per pond from October 1, 1997 through release. Fish were crowded for sampling and were measured for total length ( mm) and weight (g).

18

Physiological sampling (PS) of 20 fish per pond was initiated at the time MFS fish were returned to full rations in February 1998, at 1-month intervals until the time of release in April, and at 1 and 3 months after transfer to seawater. The sample size was limited by the time necessary to collect physiological measurements. Fish were non-lethally anesthetized using tricaine methanesulfonate (MS-222) at 80 mg ⋅ L-1 and were measured for fork length (mm), weight (g), skin reflectance, and short-wave near-infrared total body fat. Samples of mucus for lysozyme and gill filaments for Na+, K+-ATPase activity were also collected. In order to compare the IFRO length samples with those taken by CRRL staff, it was necessary to convert total length to fork length based on a calculated regression equation (r2 = 0.9926, n = 78). Physiological Analyses Physiological assessment of the 20-fish samples included fork length (mm); weight (g); skin reflectance; mucus lysozyme (ng ⋅ mL-1, hen egg white lysozyme standard HEWL); gill Na+, K+-ATPase (μmol Pi ⋅ mg protein-1 ⋅ hr-1); short-wave near-infrared spectroscopy for lipids and protein; total body fat; and tissue moisture analysis. Length and weight data were used to calculate condition factor (K) as [105 ⋅(weight ⋅ length-3)]. Skin reflectance was measured with a modification of the technique described by Haner et al. (1995), and is described in Section Five of this report. Gill Na+, K+-ATPase was assayed by the method of Schrock et al. (1994). Short-wave, near-infrared readings and analysis for lipids and protein were performed by Eastern Oregon University (EOU) researchers by the method of Lee et al. (1992). Body lipid analysis was completed by an acid hydrolysis method (method 948.15, Cunniff 1997). Five fish from each experimental group were frozen and shipped to the Washington State University for the analysis. A core sample for analysis was taken from each fish in an area below the lateral line, centered just above the vent, corresponding to the area where reflectance was measured (see Section Five). Moisture content was determined by drying a pre-weighed sample in a drying oven at 100 to 105 oC for 17 to 18 hr. All lipid data were reported on a dry weight basis. Tissue samples were analyzed in triplicate if the sample size permitted, or in duplicate if inadequate sample was available. Drs. Cavinato (EOU) and Rasco (WSU) will report the combined results. Transport to Marrowstone Marine Field Station Approximately seventy-five fish from each experimental pond were transported in individual 45-gal totes to Marrowstone Marine Field Station on April 24, 1998 for extended seawater rearing trials. Fish were netted from the raceways, transferred to 15-gal totes and poured into the 3 x 4 x 2.5 ft transport totes. Each transport tote was equipped with an air stone which provided oxygen during the 12-hr trip. Water temperature (maintained at 11 to 13 oC) and oxygen levels were monitored during transport. Four fish died during transport when they were either washed out of the covered totes or suffocated in the plastic tote liners. At Marrowstone, the fish were netted and transferred by bucket to separate circular tanks supplied with 13 oC freshwater. Seawater Rearing

19

Fish at the seawater facility were allowed to acclimate in freshwater for 1 week before conversion to seawater was initiated on May 3. Escalating salinities were reached by controlling the flow of both freshwater and seawater to the experimental tanks. Fish were held at 1/3-strength seawater (9.5 ppt salinity) for two days, 2/3-strength seawater (19.0 ppt salinity) for two days, and were converted to full-strength seawater (27.9 to 29.5 ppt salinity) on May 7. Fish were maintained at an average flow of 3 gpm (11 Lpm) with a temperature range of 50.0 to 54.5 oF (10.0 to 12.5 oC) throughout the rearing period (April 25 to August 5, 1998). After conversion to seawater was completed, the temperature and salinity in the tanks were ambient ocean conditions and were monitored daily. Fish were fed to satiation once daily. Mortalities were removed and recorded daily. Tanks were cleaned every two weeks or as needed. Each tank had its own net and brush for cleaning, to prevent the spread of disease. An initial length and weight inventory was made of all fish on April 25, 1998. Fish were then inventoried for length and weight each month at the time of physiological sampling for gill ATPase (June 1, July 6, August 4, and August 5). In-River Migration and Survival To measure migration rate, estimate survival during migration, and assess smolt development after release, 400 fish from one replicate treatment pond and one control pond were marked with passive integrated transponder (PIT) tags (800 total). To determine the effect of fish size, PIT-tags were injected into 200 small fish (< 200 mm TL) and 200 large fish (>200 mm) from each pond. Tagging was conducted on April 22, 1998. Migration time was measured to Lower Granite, Little Goose, and Lower Monumental dams on the Snake River and McNary Dam on the Columbia River (Figure 1.1). Survival to Lower Granite Dam during migration was estimated using the cumulative number of unique interrogations at each of the four dams. To compare smolt development before and after release, PIT-tagged fish from the study were recaptured at the NMFS interrogation-by-code facilities at Little Goose Dam, and gill Na+, K+-ATPase activity was measured for comparison with activity levels prior to release. Statistical analysis Statistical analysis was performed using SAS (version 6.0). General linear model (GLM) tests and two sample t-tests were performed between replicate MFS groups (ponds 16 and 18) and replicate control groups (ponds 20 and 22). Tests were conducted by month comparing fork length (mm), weight (g), and condition factor. Gill Na+, K+-ATPase activities were analyzed for the 20 fish physiological samples. GLM analysis was used rather than analysis of variance (ANOVA) because of unequal sample sizes. It was determined that the replicate MFS groups and replicate control groups were not significantly different (P < 0.05); therefore the results were pooled to increase the sample sizes. Pooled MFS and pooled control samples were then compared to each other, again using two sample t-test and GLM by month evaluating fork length (mm), weight (g), condition factor, and gill Na+, K+-ATPase activities (twenty fish physiology samples). A significance level of P = 0.05 was used for all statistical tests.

20

For statistical analysis of PIT-tag information, pre-release mean gill ATPase and mean condition factor from treatment and control groups were compared using the Bonferroni post-hoc procedure. For PIT-tagged steelhead recaptured at Little Goose Dam, mean gill ATPase was compared between the treatment and control groups using a two-sample t-test (Wilkinson 1990). Mean migration rates were compared between treatment and control groups using a chi-square test for differences in probabilities (Conover 1970). A P-value of 0.05 was used to test for significant difference.

Results

Growth, Condition Factor, and ATPase A statistically significant difference was seen between the mean fork lengths of treatment (MFS) and control (CON) fish one month after the modified feeding schedule began (P < 0.05), as measured by the HFI in January (Figure 2.1). The significant difference persisted and was measured in both the HFI and PS samples in February and March (Figures 2.1, 2.2, and 2.3). Significant differences were also seen between weights of the two groups during this same period measured in both the HFI and PS samples (Figures 2.4, 2.5, and 2.6). During the first month after conversion back to production rations (March), the mean specific growth rate was much higher in the MFS group than the CON group as calculated by both length (Figure 2.7) and weight (Figure 2.8). At the time of release, mean lengths and weights did not differ between the two groups (Figures 2.1 - 2.6).

21

Sample Date (1997-1998)Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep

Mea

n Fo

rk L

engt

h (m

m)

80

100

120

140

160

180

200

220

240

260

Modified Feeding Seawater

*

*

**

Seaw

ater

Tra

nsfe

r

TreatmentControl

* Significant Difference (P < 0.05)

22

Figure 2.1. Mean fork length (mm) of summer steelhead in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 100-fish inventories (HFI).

23

Sample Date (1997-1998)Feb Mar Apr May Jun Jul Aug Sep

Mea

n Fo

rk L

engt

h (m

m)

150

180

210

240

Seawater

*

Sea

wat

er T

rans

fer

* Significant Difference (P < 0.05)

TreatmentControl

24

Figure 2.2. Mean fork length (mm) of summer steelhead in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 20-fish physiology samples (PS).

25

Sample Date (1997-1998)Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep

Mea

n Fo

rk L

engt

h (m

m)

80

100

120

140

160

180

200

220

240

260Modified Feeding Seawater

Sea

wat

er T

rans

fer

Treatment (HFI)Control (HFI)Treatment (PS)Control (PS)

26

Figure 2.3. Mean fork length (mm) of summer steelhead in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 100-fish inventories (HFI) and 20-fish physiology samples (PS).

Experimental Fork LengthControl Fork Length

* Significant Difference (P < 0.05)

Sample Date (1997 - 1998)Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep

Mea

n W

eigh

t (g)

0

20

40

60

80

100

120

140

160

180Modified Feeding Seawater

*

*

**

*

Sea

wat

er T

rans

fer

* Significant Difference (P < 0.05)

TreatmentControl

27

Figure 2.4. Mean weight (g) of summer steelhead in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 100-fish inventories (HFI).

28

Sample Date (1997-1998)

Feb Mar Apr May Jun Jul Aug Sep

Mea

n W

eigh

t (g)

30

60

90

120

150

180

*

Seawater

Sal

twat

er T

rans

fer

* Significant Difference (P < 0.05)

TreatmentControl

29

Figure 2.5. Mean weight (g) of summer steelhead in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 20-fish physiology samples (PS).

Sample Date (1997-1998)Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep

Mea

n W

eigh

t (g)

0

20

40

60

80

100

120

140

160

180Modified Feeding Seawater

Sea

wat

er T

rans

fer

Treatment (HFI)Control (HFI)Treatment (PS)Control (PS)

30

Figure 2.6. Mean weight (g) of summer steelhead in the modified feeding schedule treatment (BP16 and BP 18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 100-fish inventories (HFI) and 20-fish physiology samples (PS).

31

Growth Period (1997-1998)Sept30 - Oct 30

Oct 30 - Dec 2

Dec 2 - Jan 7

Jan 7 - Feb 17

Feb 17 - Mar 4

Mar 4 - Apr 25

Apr 25 - June 1

June 1 - July 6

July 6 - Aug 4

Mea

n S

GR

-Len

gth

(% %

day

-1)

0.0

0.2

0.4

0.6

0.8SeawaterModified Feeding

Sea

wat

er T

rans

fer

TreatmentControl

32

Figure 2.7. Mean specific growth rate (SGR, % ⋅ day-1) in fork length (mm) of summer steelhead

in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 100-fish inventories (HFI).

Growth Period (1997-1998)Sept30 - Oct 30

Oct 30 - Dec 2

Dec 2 - Jan 7

Jan 7 - Feb 17

Feb 17 - Mar 4

Mar 4 - Apr 25

Apr 25 - June 1

June 1 - July 6

July 6 - Aug 40.0

0.5

1.0

1.5

2.0

2.5

3.0SeawaterModified Feeding

Mea

n S

GR

-Wei

ght (

% %

day

-1)

Seaw

ater

Tra

nsfe

r

TreatmentControl

33

Figure 2.8. Mean specific growth rate (SGR, % ⋅ day-1) in body weight (g) of summer steelhead in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 100-fish inventories (HFI).

34

Initially a significant difference in condition factor (Figure 2.9) was found between the MFS and CON groups at the time of ponding in August, but the difference was not apparent in the November inventory, and was attributed to measurement error with small fish. After treatment fish were placed on the modified feeding schedule in late November, a significant difference in condition was seen between the treatment and control groups (P < 0.05) at the next inventory, and the difference persisted until March. At the time of release, the difference in condition factor between the MFS and CON groups, as measured by the HFI, was negligible. However, there was a significant difference in condition factor based on the PS method (Figure 2.10). Between March and the time of release in April, both MFS and CON fish showed the decline in condition factor indicative of smoltification, although the timing of the decrease differed between the two groups. Discrepancies were apparent between results calculated from HFI and PS methods (Figure 2.11). Gill Na+, K+-ATPase was monitored beginning February 12, 1998 during the physiology sampling (n = 20). No significant differences in ATPase activity between the MFS and CON groups were found throughout the study (Figure 2.12). In both groups, mean ATPase values remained low in freshwater (< 5 μmol Pi ⋅ mg protein-1 ⋅ hr-1), but increased dramatically after transfer to seawater (~23 μmol Pi ⋅mg protein-1 ⋅ hr-1 on June 1). Feed Consumption Feed consumption (g feed ⋅ g fish-1 ) by System II production fish, as determined from the MIS (excluding data from MFS ponds 16 and 18), was low compared to prior years (Appendix 2.3 and Table 2.3), with the exception of the 1988 release (BY87). In December 1997, feed consumption by System II fish was 0.27 g feed ⋅ g fish-1 per day, decreasing to 0.22 g feed ⋅ g fish-1 in January. Feed consumption for experimental fish was calculated using both the HFI and MIS data (Appendix 2.3). In December, MFS fish consumed an estimated 0.26 or 0.19 g feed ⋅ g fish-1 based on weights from the HFI and MIS, respectively, and in January consumed 0.19 g feed ⋅ g fish-1 according to both methods. Control fish consumed 0.39 (HFI) or 0.24 (MIS) g feed ⋅ g fish-1 in December, and 0.28 or 0.24 g feed ⋅ g fish-1 in January. Feed consumption by MFS fish was 68 to 79% of consumption by CON fish during the same period. Seawater Performance Specific growth rates (% ⋅ day-1) for the MFS fish were higher than for CON fish for the first two months in seawater, but the difference was not significant. A significant difference in condition factor in seawater was seen only in the July inventory results, after a disease episode resulted in high mortality in one of the MFS tanks. A diagnosis of Pseudomonas infection in fish from that tank was made by the IFHC (K. Clemens, personal communication).

35

Experimental Fork LengthControl Fork Length

* Significant Difference (P < 0.05)

Sample Date (1997 - 1998)Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep

Mea

n C

ondi

tion

Fact

or (K

)

0.92

0.94

0.96

0.98

1.00

1.02

1.04

1.06

1.08

1.10

1.12Modified Feeding Seawater

**

*

*

*

Sea

wat

er T

rans

fer

* Significant Difference (P < 0.05)

TreatmentControl

36

Figure 2.9. Mean condition factor (K) of summer steelhead in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 100-fish inventories (HFI).

37

Sample Date (1997-1998)Feb Mar Apr May Jun Jul Aug Sep

Mea

n C

ondi

tion

Fact

or (K

)

0.90

0.93

0.96

0.99

1.02

1.05

*

*

Seawater

Sea

wat

er T

rans

fer

* Significant Difference (P < 0.05)

TreatmentControl

38

Figure 2.10. Mean condition factor (K) of summer steelhead in the modified feeding schedule

treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 20-fish physiology samples (PS).

Sample Date (1997-1998)Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep

Mea

n C

ondi

tion

Fact

or (K

)

0.92

0.94

0.96

0.98

1.00

1.02

1.04

1.06

1.08

1.10

1.12Modified Feeding Seawater

Seaw

ater

Tra

nsfe

rTreatment (HFI)Control (HFI)Treatment (PS)Control (PS)

39

Figure 2.11. Mean condition factor (K) of summer steelhead in the modified feeding schedule treatment and control groups at Dworshak National Fish Hatchery, Idaho, based on the 100-fish inventories (HFI) and 20-fish physiological samples (PS)

40

Sample Date ( 1997 - 1998)Feb Mar Apr May Jun Jul Aug Sep

Na+

, K+

- ATP

ase

Act

ivity

(:

mol

Pi %

mg

prot

ein-

1 %

hr-1

)

0

5

10

15

20

25Seawater

Sea

wat

er T

rans

fer

TreatmentControl

41

Figure 2.12. Mean Na+, K+-ATPase activity (µmol Pi ⋅ mg protein-1 ⋅ hr-1) of summer steelhead in the modified feeding schedule treatment (BP16 and BP 18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho.

42

In-river Migration and Survival PIT-Tag Interrogation Rates. Of the 800 steelhead smolts marked with PIT-tags and released from Dworshak NFH for this experiment, 632 unique interrogations were made, for a recovery rate of 79.0% (Table 2.5). This interrogation rate is among the highest recorded for Dworshak steelhead, second only to the 85.2% rate recorded for the 1993 release (Table 2.6). The CON smolts were interrogated at a slightly higher rate at downriver dams than the MFS group, with 81% versus 77%, respectively (Table 2.5). Large smolts (> 200 mm) had a higher interrogation rate than small smolts (< 200 mm) in both the CON and MFS groups . The percent of PIT-tags detected for small and large smolts (MFS and CON groups combined) was 78% and 80%, respectively (Table 2.5). Small smolts were detected at a rate of 81% for the CON group and 75% for the MFS group (Table 2.5). A comparison of the number of PIT-tag recoveries by 10-mm length groups showed little difference between the MFS and CON ponds (Table 2.7). Generally, those length groups nearest the center of the distribution had the highest rates of interrogation. However, chi-square analysis did not detect a significant difference between the distributions of the two groups. Migration Time. Steelhead smolts in this experiment traveled relatively quickly through the Lower Snake River after release. Mean migration time to Lower Granite Dam was less than six days and mean migration time to Lower Monumental Dam was less than 14 days. Only 11 fish from the experiment were interrogated at McNary Dam (Table 2.8). Smolts in the MFS treatment group traveled significantly (P < 0.05) faster to Lower Granite Dam than smolts in the control group. In almost every case, large steelhead smolts in the MFS group migrated more rapidly than large smolts in the CON group. Small smolts in the MFS group also traveled more quickly to Lower Granite Dam than those from the CON group; however, the difference in mean migration time was not significant.

43

Table 2.5. Number of small and large steelhead smolts from modified feeding treatment (MFS 16) and control (CON 20) groups at Dworshak National Fish Hatchery, Idaho, that were PIT-tagged, released, and detected at lower Snake and Columbia River dams in 1998. Treatment

Small Smolts1

Large Smolts2

Total

Group

Marked

Detected

Percent

Marked

Detected

Percent

Marked

Detected

Percent

Modified Feeding

199

149

74.9

201

159

79.1

400

308

77.0

Control

200

162

81.0

200

162

81.0

400

324

81.0

Totals

399

311

77.9

401

321

80.0

800

632

79.0

1 Smolts < 200 mm Fork length. 2 Smolts > 200 mm Fork length.

44

Table 2.6. Summary of PIT-tag release and interrogation data for steelhead released from Dworshak National Fish Hatchery, Idaho, and interrogated at lower Snake and Columbia River dams from 1992 through 1997.

Year

Number Released Number Interrogated

Percent

1992

2,969

2,119

71.4

1993

1,471

1,254

85.2

1994

1,468

985

67.0

1995

5,118

3,798

74.2

1996

5,088

3,321

65.3

1997

5,589

4,215

75.4

45

Table 2.7. Number of steelhead that were PIT-tagged and released at Dworshak National Fish Hatchery, Idaho, and interrogated at lower Snake and Columbia River dams, presented for each 10-mm length group released from MFS 16 and CON 20 in 1998.

Treatment Pond 16

Control Pond 20

Length Group

Release

s

Interrogations

Interrogation

Rate

Release

s

Interrogations

Interrogati

on Rate

130

0

0

0

1

0

0

140

4

1

.25

1

1

1.00

150

3

1

.33

3

3

1.00

160

7

5

.71

7

3

.43

170

8

5

.62

4

3

.75

180

29

24

.83

22

17

.77

190

62

50

.81

51

44

.86

200

86

63

.73

111

90

.82

210

95

77

.81

100

79

.79

220

63

47

.74

55

43

.78

230

31

26

.84

36

31

.86

240

10

8

.80

8

8

1.00

250

1

1

1.00

1

1

1.00

260

1

0

0

0

0

0

46

Table 2.8. Mean migration time of small (<200 mm) and large (>200 mm) steelhead smolts from MFS 16 and CON 20 at Dworshak National Fish Hatchery, Idaho, that were PIT-tagged, released, and interrogated at lower Snake and Columbia River dams in 1998.

Mean Migration Time (Days)

Dam Site

Experimental Group

Small Smolts1

Large

Smolts2

Combined

Lower Granite

MFS

4.6

5.2

4.9

CON

6.1

5.1

5.6

Little Goose

MFS

9.1

7.7

8.4

CON

11.2

8.1

9.1

Lower Monumental

MFS

11.8

11.4

11.6

CON

14.1

12.0

13.2

McNary

MFS

13.9

12.5

13.0

CON

13.0

21.0

17.0

1 Smolts < 200 mm fork length. 2 Smolts > 200 mm fork length

Discussion This study is unique in its application of hatchery-specific rearing specifications and stock performance history to develop a modified feeding schedule for manipulating growth in summer steelhead. Using the monthly inventory summaries, we examined feed consumption and temperature data from the 10 years prior to the experiment. Feed consumption rates for fish similar in size to the experimental fish, and held at similar temperatures, were calculated and applied in the study. Additionally, years when smaller fish were released were referenced to establish target growth rates for the winter months. Feed for the modified feeding schedule treatment was decreased to the lowest level the stock had limited itself to in the presence of constantly full demand feeders. Results indicate that the production group as a whole, including control ponds, voluntarily reduced their feed intake during December and January. Therefore, differences between the experimental ponds and control ponds were not as great as expected based on feed consumption and temperature data from previous years. Growth A reduction in growth was seen after one month in the fish fed reduced rations, as evidenced by significantly lower mean lengths and weights in MFS fish than controls from January through March (Figures 2.1 - 2.6). Slower growth during periods of reduced, intermittent feeding has

47

been demonstrated in other studies (Klontz et al. 1991). With conversion back to production rations in February, fish that had been on the reduced, intermittent rations demonstrated compensatory growth and achieved the same mean length and weight in April as control fish (Figures 2.7 and 2.8). Dobson and Holmes (1984) found that compensatory growth in rainbow trout after undergoing 3-week periods of alternating fasting and feeding was manifested as an increase in length rather than gut fat deposits or water uptake. During the reduced ration treatment, MFS fish emptied the demand feeders on the day they were filled, resulting in 1-day fasting periods on Tuesdays and Thursdays and 2-day fasts on the weekends. These periods of fasting were short compared to a previous study where fish reached a non-feeding mode after extended periods of fasting (Smith 1987). Klontz et al. (1991) found no difference in rates of growth between fish fed on reduced continuous or intermittent schedules. Therefore, the growth pattern we achieved demonstrates that relatively precise control of growth can be achieved by using past growth performance in combination with feed consumption and temperature data. Condition Factor Changes in condition factor during the experiment could be explained as seasonal changes seen in both experimental groups, as a reflection of ration amount, or as a developmental change associated with smoltification. Mean condition factor was significantly lower in the MFS fish than in controls from December through February (Figure 2.9). The simultaneous reduction in condition factor from early January in both the MFS and CON groups (Figures 2.9 - 2.11) has several possible explanations. Reduced feeding was seen in the treatment groups, control groups, and the entire System II production. Though this voluntary reduction in feeding might be explained by a disease episode, none was documented during routine health screening (K. Clemens, IFHC, personal communication). Alternatively, a reduction in condition factor and feeding is often associated with smoltification (Wedemeyer et al. 1980; Ewing et al. 1984). In previous years, growth rates of Dworshak steelhead decreased in January or February, although in some cases the decrease followed an increase in length or weight in December (Figure 2.1 and 2.2). Mean condition factor in the treatment group was significantly lower than in controls from December through February, but increased for a short period in March after the treatment fish were returned to full rations (Figures 2.9, 2.10, and 2.11). This initial increase in condition factor due to compensatory growth as weight gain was followed by a decrease that would more likely be attributable to smoltification. The decrease in condition factor was seen in both the MFS and CON groups between March and the time of release in April, when gill Na+, K+-ATPase levels were also similar (Figures 2.11 and 2.12). Condition factor is also influenced by the timing of measurement, whether it followed periods of starvation or after fish returned to feeding (Klontz et al. 1991). The fasting periods in our study did not exceed 2 days, therefore, condition factor measurements should accurately reflect the length-weight relationship. In both our study and that of Klontz et al. (1991), significant reductions in growth of steelhead occurred with no negative effects from reductions in feeding intensity. Size Variation and Smoltification

48

A population of relatively smaller treatment fish upon which to test the use of growth modification for enhancement of smoltification was not produced. Furthermore, the voluntary reduction in feed consumption in controls during the winter, coupled with the high compensatory growth in MFS fish, resulted in treatment and control fish of similar final size at release. Control and MFS fish had similar migration, seawater performance, and seawater survival, indicating no difference in smolt condition. Feed Consumption When the feed rate of the entire System II production was recalculated for the study period, it was found to be 0.27 and 0.23 g feed ⋅ g fish-1 during December and January, respectively. These rates were low compared to other years (Table 2.3), and were near the minimum levels we had selected (release years 1988 and 1990) for the MFS fish. Feed consumption rates in the MFS fish were found to be between 21 and 32% below that of the CON fish. Dworshak NFH steelhead show high variability in feed consumption rates among months and years (Table 2.3) which is not always explained by differences in temperatures (Table 2.4). Another factor to consider was that fish from spawning take 12 used in the experiment were on average larger than the System II production fish, based on the Dworshak NFH monthly inventory summaries. It has been shown that maintenance requirements may decrease in larger fish (Brown 1946), but it was not within the scope of this study to determine if the relatively small difference in size among spawning takes at Dworshak NFH influenced feed consumption. We did not achieve the reduction in ration of 50% compared to the production fish that would be necessary to produce a significant reduction in growth (Smith 1987). The estimation of the target feeding rate from the previous 10 years’ records did not anticipate the low feeding rate of the production group as a whole. The small difference in feeding rates and therefore growth between the experimental groups was evidenced by few differences in physiological or migration performance. Seawater Performance Controls and fish from the MFS treatment demonstrated similar seawater performance. After transfer to seawater, no differences in length, weight, condition factor, or mortality were found between the two groups (Figures 2.1 - 2.6 and 2.9 - 2.11, Appendix 2.2). Both groups had reached the same level of smoltification as measured by gill Na+, K+-ATPase (23 μmol Pi ⋅ mg protein-1 ⋅ hr-1) after 1 month in seawater, and ATPase levels remained similar after 3 months (Figure 2.12). Fast pre-smolt growth in the hatchery does not necessarily predict fast seawater growth, although in steelhead, hatchery growth explained approximately one-third of the variation in seawater growth (Johnsson et al. 1997). Though growth patterns at the hatchery differed between MFS and CON fish, our results indicated no difference in seawater growth. A limitation of the study was the direct transfer of experimental fish to the seawater facility, eliminating the opportunity for smolt development during in-river migration. Survival of all fish after the abrupt transition to seawater, in effect a seawater challenge, indicates the MFS fish were at the same level of smolt development as control fish.

49

In-river Migration and Survival Fish from the MFS treatment arrived approximately 1 to 2 days sooner at the first 3 detection sites (Lower Granite, Little Goose, and Lower Monumental dams) than control fish, and 4 days sooner at the fourth site (McNary Dam) (Table 2.8). Larger fish also appeared to migrate more slowly as the distance from the hatchery increased, as indicated by the longer migration time to McNary Dam. Fängstam (1993) found that smolts of Baltic salmon (Salmo salar) spent more time actively swimming at higher rates than non-migrants, although non-migrants were capable of the same swimming speeds. Faster migration rates for MFS fish may therefore be interpreted as an indication of smoltification when smolt indices such as gill ATPase are too low to distinguish between groups. Previous studies at Dworshak NFH have investigated holding conditions and characteristics of production steelhead that appear to affect seaward emigration (Bjornn et al. 1978, 1979). Size has been found to be an important factor determining emigration in other steelhead stocks, with fish of large sizes (> 190 mm) migrating at higher rates than small fish (Tipping et al. 1995). However, a review of hatchery records at Dworshak NFH revealed that high adult returns were realized during years when smaller fish were released from the hatchery (Appendix 2.1). Tipping and Byrne (1996) found that by limiting feed in the final month before release, a reduction in condition factor was effected, and fish migrated more quickly and were recaptured in greater numbers than control fish. The causal mechanism for an increased migration rate being induced by a reduction in condition factor was not explained, but in that study, smaller fish were recaptured a higher rates than larger fish downstream. Bjornn et al. (1979) determined that length at release was the single most important factor determining recapture at dams, yet even small fish conditioned in cold water prior to an early release, as in our study, were recaptured in large numbers. A careful review of data from that study shows that among fish released in May, smaller fish were recaptured at higher rates than larger fish. Tipping and Byrne (1996) also found that smaller fish were recaptured at higher rates than larger fish in a feed reduction study, but a minimum release size was predetermined by reaching a target size before the feed reduction. Recapture rates for fish may depend on several, possibly interacting, factors that have not been considered, such as the size selectivity of dam recapture systems, river conditions such as flow, and physiological or behavioral differences between fish of the same size on different release dates. During substantially decreased flows in the Snake River, only 5 to 15% of Dworshak steelhead were recaptured at Lower Granite Dam, and it was suggested that the fish did not migrate successfully (Bjornn et al. 1979). Comparison of Inventory Methods Results of the HFI for controls, used to monitor production growth, did not alert us to the reduced feeding rate of the System II production group as a whole. New guidelines for inventory procedures for the hatchery fish should be incorporated into future production studies (Ewing et al. 1998a). Discrepancies between results calculated from inventory or physiology samples may be explained by differences in samples size and the level of variation among individuals. By all inventory methods, reduced ration fish were the same size at release as the control fish, and showed the same acclimation, growth rate, and smolt development after seawater transfer.

50

Comparison of the 3 sources of measurements (MIS, HFI, PS) for length, weight, condition factor, and specific growth rate reveal that evaluations of production fish must provide appropriately large sample sizes. Ewing et al. (1994b) found that variability in fish size affected the minimum error associated with estimates of fish size, and errors of 2 to 5% are to be expected. In that study, 7 to 9 random samples from a raceway were recommended to reduce error. The results of our study indicate that samples sizes for physiological indices should be increased. Furthermore, HFI results did not duplicate MIS-calculated estimates of length and weight during the study. We will continue to evaluated discrepancies between different sampling methods and sample sizes to determine appropriate methods for future production studies. Conclusions Hatchery production records provided the information necessary to achieve a growth pattern similar to wild fish by reducing feed amounts in winter, and to produce fish physiologically comparable to those on full production rations at the time of release. Compensatory growth allowed the modified feeding schedule group to reach the same size at release as control fish. Migration rates were enhanced in smaller fish, and performance during seawater rearing, as measured by growth rates and gill ATPase was similar in both treatment and control groups. Future trials will reduce feeding rates substantially below what the stock records suggest, and will include longer periods of reduced ration and intermittent feeding, with the goal of producing smaller fish in the treatment group. Calculation of rations based on known feeding rates and holding conditions of the specific stock provides additional benefits such as prevention of overfeeding and reduction in feed costs.

51

Appendices Appendix 2.1 Mean total length (mm) and weight (g) at release and adult returns for Dworshak National Fish Hatchery summer steelhead (Oncorhynchus mykiss) from coded wire tag rack returns for the Modified Feeding Schedules Project at Dworshak National Fish Hatchery. Brood Year

Release Year

Release Total Length (mm)

Release Weight (g)

Recovery Year

Return Rates

1985

1986

1989

.283

1986

1987

187

64.8

1990

.393

1987

1988

213

91.3

1991

3.18

1988

1989

179

56.0

1992

.160

1989

1990

199

76.8

1993

.530

1990

1991

204

82.5

1994

.144

1991

1992

175

51.5

1995

1992

1993

209

87.8

1996

.043

52

Apr May Jun Jul Aug Sep

Num

ber o

f Mor

talit

ies

0

5

10

15

Treatment Tank 7Treatment Tank 9Control Tank 3Control Tank 8

53

Appendix 2.2 Number of mortalities of summer steelhead in the modified feeding schedule treatment (MFS 16/Tank 7 and 18/Tank 9) and control (CON 20 / Tank 3 and 22 / Tank 8) groups at Marrowstone Marine Station, Washington.

54

Appendix 2.3. Food consumption by summer steelhead in the modified feeding schedule treatment (BP16 and BP18) and control (BP20 and BP22) groups at Dworshak National Fish Hatchery, Idaho, based on 100-fish inventories (HFI) and monthly inventory summaries (MIS).

Month

Pond

Pounds of Feed

Number of Fish

100-Fish Inventory g feed g fish-1

100-Fish Inventory Weight of Fish (g)

MIS g feed g fish

MIS Weight of fish (g)

December

16

363

24,057

0.26

26.0

0.19

36.0

18

352

23,126

0.26

26.7

0.19

37.1

20

520

25,801

0.35

26.4

0.35

40.5

22

380

26,487

0.23

27.7

0.43

44.0

January

16

429

23,920

0.23

35.5

0.20

41.6

18

416

22,980

0.21

39.4

0.19

42.7

20

600

25,656

0.25

43.2

0.24

49.1

22

800

26,371

0.32

42.7

0.32

53.2

March

16

958

23,249

0.32

57.9

0.32

58.2

18

958

22,050

0.32

61.9

0.32

61.3

20

958

24,904

0.27

65.5

0.27

64.8

22

958

25,885

0.26

65.7

0.23

72.0

April

16

672

22,946

0.17

79.7

0.18

73.2

18

672

21,625

0.18

80.1

0.17

82.5

20

672

24,506

0.16

78.7

0.16

75.6

22

672

26,388

0.14

83.5

0.14

80.9

55

CHAPTER THREE

EVALUATION OF AN EXTRUDED ABERNATHY DIET ENHANCED WITH (SCHIZOCHYTRIUM) AS A MEANS OF PROMOTING SMOLTIFICATION IN

SUMMER STEELHEAD (ONCORHYNCHUS MYKISS) AT DWORSHAK NATIONAL FISH HATCHERY, IDAHO

by

Ann Gannam and Carl Burger U. S. Fish and Wildlife Service, Abernathy Salmon Culture Technology Center

Longview, Washington

Ray N. Jones U. S. Fish and Wildlife Service, Idaho Fishery Resource Office

Ahsahka, Idaho

Corie Samson and Marilyn Blair U. S. Fish and Wildlife Service, Dworshak Fish Health Center

Ahsahka, Idaho

Robert Semple U. S. Fish and Wildlife Service, Dworshak National Fish Hatchery

Ahsahka, Idaho

Robin M. Schrock and Alec G. Maule U. S. Geological Survey, Biological Resources Division

Columbia River Research Laboratory, Western Fisheries Research Center Cook, Washington

Nancy Elder

U. S. Geological Survey, Biological Resources Division Western Fisheries Research Center, Marrowstone Marine Station

Nordland, Washington

56

Abstract

Dworshak summer steelhead were fed an enhanced extruded Abernathy Dry diet during the later part of their rearing period to improve health and survival of the released smolts. The control diet was the extruded Hagerman steelhead diet. No differences in growth rate were observed between the treatment and the control fed fish in freshwater. Condition factor, skin reflectance and gill ATPase also showed no significant differences. Percent weight gain of the treatment fish in saltwater was higher than that of the control fish.

57

Introduction Wide length frequency distributions are a common characteristic of juvenile steelhead (Oncorhynchus mykiss) at Dworshak National Fish Hatchery (NFH). Lengths often range from 80 to 240 mm (total length, TL) by the time the fish are released as smolts. Recent research conducted by the Idaho Fishery Resource Office and Dworshak NFH indicates that a high proportion of the steelhead that are smaller than 170 mm at the time of release may not be migrating down river to the ocean (Bigelow 1997). Based on hatchery pre-release inventory results, as much as 25% of the smolts released may be less than 170 mm. This situation conflicts with recommendations in the Draft Snake River Salmon Recovery Plan (NMFS 1995) and the National Marine Fisheries Service (NMFS) Biological Opinion for Hatchery Operations in the Columbia River Basin (NMFS 1999). The documents recommend that hatchery steelhead be released at sizes between 180 and 240 mm (TL) in order to minimize residualization. Steelhead within the range of 180 to 220 mm exhibit more complete parr-smolt transformation and are therefore more likely to actively migrate; but fish larger than 220 mm are more prone to residualize (Partridge 1985, 1986; Cannamela 1992). In addition, steelhead larger than 250 mm may be more capable of predation (Cannamela 1993). One particular concern is that steelhead, both large and small, that fail to emigrate may negatively impact sensitive fish species such as the endangered Snake River fall chinook salmon (O. tshawytscha), spring/summer chinook salmon, or the proposed threatened wild Snake River steelhead through displacement, competition for food, and/or behavioral influences (Viola and Schuck 1995). Juvenile hatchery steelhead have been collected in several tributary streams below Dworshak NFH after smolts were released in 1994, 1995, and 1996. However, data have not been collected to determine effects on wild steelhead in those streams. The primary objective of the SRSRP recommendation is to reduce the potential impacts of various state and federal hatchery programs on listed fish species by attempting to minimize the number of non-migrating hatchery summer steelhead smolts released into the Snake River basin. This study addressed the issue of size at release from a different perspective. The purpose of this study was to determine if diet could produce healthy, high-quality, actively migrating steelhead smolts, regardless of size. Wild juvenile steelhead typically spend 2 to 3 years in freshwater, whereas hatchery fish spend only 1 year (Pauley et al. 1986). While subordinate individuals may have a more limited access to food than dominant individuals because of their social position, providing enhanced proportions of specific dietary components in the food they do receive may help offset that disadvantage and actually help stimulate the parr-smolt transformation that may be otherwise suppressed. Presently, there are no known diets that have been formulated specifically for conservation production programs that meet the physiological requirements of anadromous fish during smoltification. Omega-3 polyunsaturated fatty acids found in fish feeds are proposed to increase disease resistance in fish (Blazer 1992; Rowley et al. 1995). Cellular membranes become stronger and more resistant to lysis. Fluidity of the cell membrane depends on the fatty acid composition. Therefore, leukocyte functions may also be affected. The ratio of fatty acids is also important for disease resistance. Results of a study of Atlantic salmon (18 to 20 g) fed diets with either

58

high levels of n-3 or n-6 fatty acids suggest that fish fed the low ratio n-3/n-6 polyunsaturated fatty acids were less resistant to infection (Thompson et al. 1996). Additionally, lipid metabolism and changes in fatty acid composition are known to occur during smoltification in salmonids (Love 1974; Sheridan et al. 1985; Hoar 1988; Sheridan 1988). The lipid composition of the smolts becomes more like sea-run than freshwater fish. In selective breeding programs, to increase disease resistance, a significant positive correlation has been found between growth rate and survival (Fjalestad et al. 1993). The increase in growth rate seen with glucan immunostimulation may be acting on general fish condition and on the immune system. Substances administered as immunostimulants in feed may have a separate, positive effect on fish condition by increasing growth rates, as was demonstrated in sea bream (Sparus aurata) (Mulero et al. 1998). In a dietary study of the effect of levamisole administered to 100-g gilthead bream, increased disease resistance against Vibrio anguillarum and increased growth were demonstrated. Phagocytosis and respiratory burst activity increased and a delayed rise in serum complement occurred. The levamisole-treated group showed increased phagocytosis of V. anguillarum by head-kidney leucocytes and lower mortality after injection challenge with Vibrio. We proposed to approach the issue of enhancing steelhead smoltification by providing an enhanced Abernathy Diet formulated to facilitate or stimulate the physiological processes that occur during smoltification. The objectives of this project were: 1) to increase the proportion of smaller-sized juvenile steelhead that exhibit characteristics of smoltification prior to release from the hatchery, during seaward emigration, and during extended seawater rearing; 2) to increase the proportion of smaller sized steelhead smolts that successfully emigrate to down river dams on the Snake and Columbia Rivers; and 3) to increase the proportion of smaller-sized steelhead smolts that survive during extended seawater rearing.

Methods Experimental Design The experiment was initiated after juvenile steelhead were transferred from inside nursery tanks to Burrows ponds outside. The experiment consisted of 2 Burrows ponds (Ponds 28 and 30) that received the enhanced Abernathy Diet prior to release (treatment), and 2 Burrows ponds (Ponds 24 and 26) that received the standard production diet throughout the rearing period (control). Smoltification was evaluated by examining 3 different indices: gill sodium, potassium-activated adenosine triphosphatase (Na+, K+-ATPase) activity, skin reflectance and condition factor (K). Emigration was evaluated by examining migration time and survival of smolts after they were released from the hatchery. Survival during initial entry into seawater and during early seawater rearing was evaluated by transferring representative samples from each pond to circular tanks at the U. S. Geological Survey (USGS) Marrowstone Marine Field Station on Puget Sound, Washington. Fish Culture

59

Experimental fish were from brood year 1997, the progeny of steelhead that were spawned during Take 11 in 1997. After being raised in the nursery, the fish were transferred to outside Burrows ponds in System II on August 21, 1997, at an average size of 96 fish per pound. Approximately 28,500 fish were stocked into each of the Burrows ponds. A summary of the monthly production inventories (number, mortality, size, and weight) is provided in Appendix Table 4.1. All ponds were supplied with ambient river water on a single-pass system until November 11, 1997, when the reuse system was started. All fish were hand-fed BioMoist 2.5-mm feed from the time of ponding until November 11, 1997. At that time, the fish were converted from hand feeding to Babington demand feeders. At the same time fish were converted from BioMoist feed to the control diet (Hagerman floating steelhead diet) or the extruded Abernathy Dry diet. Fish size at conversion was about 25 fish per pound. The enhanced Abernathy Diet was started on March 30, 1998, and was administered until release on April 29, 1998. Prior to release on April 24, 75 fish from each of the treatment and control ponds were transferred from Dworshak NFH to the seawater rearing facilities at Marrowstone Marine Field Station. The fish were transferred in 170-L totes supplied with oxygen. Temperature was kept 12 + 1 oC using ice. At Marrowstone, the fish were transferred to freshwater circular rearing tanks. Fish from each pond were randomly stocked into the circular tanks so that each replicate was represented. On April 25, weights and lengths were taken. After a week, the fish were gradually acclimated to full seawater over a period of seven days. The fish were fed a BioOregon moist diet ad libitum, and the amount of food consumed each day was recorded. Feed Methods Silver Cup Feed Company made both extruded Abernathy Diets and the control diet used in this study (Table 3.1). Schizochytrium (a dried algae) was added at 1.0% of the diet in the enhanced feed. Table 3.2 shows the proximate analysis and fatty acid composition of the added algae. The proximate composition of the Abernathy Diet with and without the additive, as well as the control diet (the Hagerman floating steelhead diet) can be found in Table 3.3. Data Collection The hatchery conducted monthly sample counts on representative ponds in System II and used that data to project growth for all ponds in the system. Mortalities were collected and reported daily. Starting in September, random samples of about 100 fish were collected from each pond to measure length and weight. In February, March and April 1998, random samples of 20 fish were taken from each pond for physiological assessments. Fish were non-lethally anesthetized using tricaine methanesulfonate (MS-222) and were measured for fork length (mm) and weight (g). Gill ATPase activity, skin reflectance and condition factor were measured to assess the degree of smoltification. Gill ATPase activity was measured using the method described by Schrock et al. (1994). Skin reflectance was measured using a modification of the technique described by Haner et al. (1995). Condition factor was calculated as K (Anderson and Gutreuter 1983).

60

Table 3.1. Composition of the Abernathy Dry and enhanced Abernathy Dry extruded feeds used in the diet trial at Dworshak NFH, 1997-98.

Abernathy Extruded Feed

Diet proportion (%)

Diet proportion (%)

Fish meal

45.5

45.5

Feather meal

10.0

10.0

Blood meal

2.5

2.5

Wheat germ

5.0

5.0

Wheat flour

25.495

24.495

Schizochytrium

-

1.0

Vitamins

1.5

1.5

Choline

0.58

0.58

Stay-C

0.2

0.2

Minerals1

0.1

0.1

Ca propionate

0.125

0.125

Fish oil2

9.0

9.0

Total

100

100 1 The vitamin and mineral premixes to supply the following concentrations (mg/kg of diet unless indicated otherwise): choline chloride, 3500; crystalline ascorbic acid, 2000; biotin, 0.6; B12, 0.06; folic acid, 16.5; inositol, 132; vitamin K, 9.2; niacin, 220.6; pantothenic acid, 106; pyridoxine, 30.9; riboflavin, 52.9; thiamin, 43.0; vitamin E, 503 IU; vitamin A palmitate, 6614 IU; vitamin D, 441 IU; zinc, 75; manganese, 20; copper, 1.5; and iodine, 10. 2 Fish oil stabilized with 500 mg of liquid ethoxyquin per kg of oil (National Research Council, 1981).

61

Table 3.2. Proximate analysis and fatty acid profile of the Schizochytrium used in the enhanced Abernathy diet.

Proximate Analysis

Fatty Acid Profile

Protein 39% Fatty Acid Content (%w/w) 29.5% Fat 32%

Fatty Acid (% of total Fatty Acids)

Carbohydrate 13% 14:00 11.0

Ash 12% 16:00 38.5

Moisture 3% 16:1 7.3

Cholesterol (mg/g) 2.6 18:00 1.1

Brassicasterol (mg/g) 0.5 18:1 4.1

Stigmasterol (mg/g) 1.5 20:3ω6 0.4

Lecithin (mg/100g) 2860.0 20:5ω3 0.6

Carotene (mg/lb) 22.5 22:5ω6 12.9

Xanthophyll (mg/lb) 6.3 22:6ω3 24.0

62

Table 3.3. Proximate analyses of the diets used in the Dworshak NFH diet trial, 1997-98. Proximate analysis(%)

Abernathy Diet

Abernathy + Schizochytrium

Hagerman floating steelhead Diet

Nutra Transfer FW (Moore-Clark)

Protein

45.6

43.7

47.6

49.8

Fat

16.9

19.9

16.7

22.8

Ash

9.1

8.6

8.2

9.4

Moisture

4.8

4.5

7.1

4.8

63

To measure migration rate, estimate survival during migration and assess smolt development during seaward emigration, 400 fish each from treatment pond 28 and control pond 24 were marked with passive integrated transponder (PIT) tags. To assess differences between smaller and larger fish, 200 fish < 200 mm (TL) and 200 fish > 200 mm (TL) were tagged in each pond. Tagging was conducted on April 22, 1998. Migration time was measured to Lower Granite, Little Goose, Lower Monumental and McNary dams on the lower Snake and Columbia rivers (Figure 1.1). Survival during migration was estimated to Lower Granite Dam by using the cumulative number of unique interrogations at each of the dams listed above. To assess smolt development during seaward emigration, arrangements were made with NMFS to use the interrogation-by-code facilities at Little Goose Dam. These facilities allowed recapture of the PIT-tagged fish so that gill clips could be collected for ATPase activity measurements. Data Analysis Replicate treatment ponds and replicate control ponds were combined. The pooled data were used to test for significant differences in mean gill ATPase, mean skin reflectance and mean condition factor between the treatment and control groups prior to release using a two-sample t-test (Wilkinson 1990). A P-value of 0.05 was used to designate a significant difference. Small (< 200 mm) fish in the treatment and control ponds were tested for significant differences in mean gill ATPase, mean skin reflectance and mean condition factor in April prior to release using the same procedure. Mean gill ATPase was compared between treatment and control groups that were recaptured at Little Goose Dam during seaward emigration using a two-sample t-test. The same procedure was used to compare mean gill ATPase for small fish in those groups. Mean migration rates were compared between treatments and controls using a two-sample t-test (Wilkinson 1990). A P-value of 0.05 was used to designate a significant difference. Small (< 200 mm) fish from the treatment and control ponds were compared for significant differences in mean migration rates using the same procedure. Treatment and control ponds were tested for significant differences in interrogation rates using a chi-square test for differences in probabilities (Conover 1971). A P-value of 0.05 was used to designate a significant difference. The same procedure was used to test for differences between small smolts from the treatment and control ponds. At Marrowstone, mean monthly mortality rates were compared between the control and treatment group using analysis of variance (ANOVA) (Wilkinson 1990). The number of mortalities for each pond was summed for each month, divided by the number of fish in the pond at the beginning of the month, and multiplied by 100 to obtain a monthly percent mortality. The percentages were transformed using a square root arcsine procedure (Sokal and Rohlf 1981). Data for replicate ponds were combined to form one treatment group and one control group for comparison. A P-value of 0.05 was used to designate a significant difference.

64

Results Smoltification Pre-release. Mean gill ATPase was relatively low and exhibited little variability during the later period of hatchery rearing. However, levels did increase slightly in April prior to release. Levels ranged from a low of 2.9 in Treatment Pond 30 in February to a high of 5.5 in Control Pond 24 in April (Table 3.4). Mean gill ATPase was higher for the control group prior to release. The only statistically significant difference (P < 0.04) in gill ATPase observed between treatment and control ponds was in February. The difference between the means was 0.39. In April, small fish in the control ponds had a higher mean gill ATPase level than the small fish in the treatment ponds but the difference was not significant. Mean skin reflectance exhibited very little variability during the later period of hatchery rearing but did increase over time. Levels ranged from 3.8 for Treatment Pond 30 in February to 5.7 for Control Pond 24 in April (Table 3.5). Mean skin reflectance was consistently higher for the control group prior to release. However, the only significant difference (P < 0.04) was in February. In April, small fish in the treatment group had a higher mean skin reflectance than the control group, but the difference was not significant. Mean condition factor was quite variable prior to release but did exhibit a decreasing trend from March to April in both the treatment and control ponds (Table 3.6). Mean condition factor was consistently lower for the treatment group prior to release. However, the only statistically significant difference (P < 0.03) was in February. In April, small fish in the treatment group had a lower mean condition factor than the small fish in the control group, but the difference was not significant. Downstream Migration. Gill ATPase increased considerably between the time of release from the hatchery and the time of recapture at Little Goose Dam. Mean gill ATPase (Control Pond 24) increased from 5.1 in April at Dworshak NFH to 12.3 in May at Little Goose Dam). Mean Gill ATPase was higher for the control group than for the treatment group, 12.0 versus 11.4, respectively, but the difference was not significant. For small fish, the control group again had a higher mean gill ATPase level than the treatment group, 12.5 versus 12.1, and again the difference was not significant. Extended Seawater Rearing. In seawater, the fish that had been fed the Abernathy Diet in fresh water grew faster than the fish fed the control diet (70.4% weight gain versus 47.5% weight gain). A disease outbreak in some tanks reduced the number of tanks that could be compared. The tanks that had no mortality were used. Downstream Migration Survival PIT-tag Interrogation Rates. Of the 800 smolts that were tagged, 799 were released. A total of 619 unique interrogations, or 77.5 %, were made for the treatment and control ponds combined (Table 3.7). These results fall within the upper 50% of the historical range of 65.3% to 85.2% for other groups of steelhead released from Dworshak NFH (Table 3.8). The differences in

65

Table 3.4. Mean monthly gill ATPase levels for summer steelhead in the treatment (BP28 and BP30) and control (BP24 and BP26) ponds in the Enhanced Abernathy Diet Evaluation Trial at Dworshak NFH, Idaho.

Gill ATPase Levels

Month

Pond

Sample

Size Mean

SD

Minimum

Maximum

February

24

20

3.3

0.7

2.0

5.3

26

19

3.7

1.1

2.5

6.7

28

20

3.3

0.5

2.7

4.5

30

20

2.9

0.7

1.3

4.1

March

24

20

3.6

0.8

2.3

5.4

26

20

3.8

1.3

1.3

5.8

28

19

3.9

1.0

2.4

6.4

30

19

2.8

0.7

1.7

4.0

April

24

20

5.5

1.7

3.1

9.1

26

20

4.9

1.5

1.9

8.2

28

20

4.8

1.2

2.9

6.7

30

19

4.5

1.5

1.7

8.9

66

Table 3.5. Mean monthly skin reflectance levels for summer steelhead in the treatment (BP28 and BP30) and control (BP24 and BP26) ponds in the Enhanced Abernathy Diet Evaluation Trial at Dworshak NFH, Idaho.

Skin Reflectance Levels

Month

Pond

Sample

Size Mean

SD

Minimum

Maximum

February

24

20

4.1

1.4

0.4

6.1

26

20

4.9

1.0

2.2

6.8

28

20

4.4

1.1

1.6

6.4

30

20

3.8

1.2

1.3

5.8

March

24

20

4.8

0.9

2.0

6.1

26

20

4.7

1.6

1.4

7.3

28

20

5.1

1.3

1.5

6.7

30

20

4.9

0.8

3.2

6.4

April

24

20

5.7

1.1

3.9

7.4

26

20

5.0

1.0

3.2

6.7

28

20

5.5

0.9

3.7

6.9

30

20

5.3

1.0

3.3

6.8

67

Table 3.6. Mean monthly condition factor (K) for summer steelhead in the treatment (BP28 and BP30) and control (BP24 and BP26) ponds in the Enhanced Abernathy Diet Evaluation Trial at Dworshak NFH, Idaho.

Condition Factor (K)

Month

Pond

Sample

Size

Mean

SD Minimum

Maximum

February

24

20

0.97

0.06

0.77

1.05

26

20

0.99

0.04

0.89

1.05

28

20

0.95

0.12

0.56

1.10

30

20

0.93

0.09

0.67

1.05

March

24

20

1.01

0.08

0.87

1.01

26

20

0.97

0.09

0.77

1.11

28

20

0.94

0.11

0.65

1.11

30

20

0.96

0.05

0.86

1.03

April

24

20

0.96

0.06

0.81

1.05

26

20

0.94

0.05

0.86

1.06

28

20

0.93

0.04

0.86

1.00

30

20

0.94

0.07

0.73

1.03

68

Table 3.7. Number of small and large summer steelhead smolts in the treatment pond 28 and control pond 24 that were PIT-tagged, released, and detected at down river dams as part of the Enhanced Abernathy Diet Evaluation Trial at Dworshak NFH in 1998.

Treatment

Small Smolts1

Large Smolts2

Total

Marked

Detected

Percent

Marked

Detected

Percent

Marked

Detected

Percent

Diet Control

198

145

73.2

201

168

83.6

399

313

78.4

Abernathy Diet

201

141

70.1

199

165

82.9

400

306

76.5

Totals

399

286

71.7

400

333

83.3

799

619

77.5

1 Smolts < 200 mm fork length. 2 Smolts > 200 mm fork length.

69

Table 3.8. Summary of PIT-tag release and interrogation data for steelhead released from Dworshak NFH from 1992 through 1997.

Year

Number Released Number Interrogated

Percent

1992

2,969

2,119

71.4

1993

1,471

1,254

85.2

1994

1,468

985

67.0

1995

5,118

3,798

74.2

1996

5,088

3,321

65.3

1997

5,589

4,215

75.4

70

performance between the treatment and control groups were so small that statistical testing was not performed to detect significant differences (Figure 3.1). The percentage of marked smolts interrogated were 76.5% for the Abernathy Diet treatment group and 78.4% for the controls. For small and large smolts, detection rates were 70.1% for small treatment smolts, 73.2% for small control smolts, 82.9% for large treatment smolts, and 83.6% for large control smolts (Figure 3.2 and Table 3.7). The number of interrogations obtained was within the range of historical observations (Table 3.8). Figure 3.3 compares the number of releases and interrogations for the Abernathy Diet treatment and control ponds by 10-mm length intervals. Again, very little difference was observed between the two groups. Those length groups closest to the center of the distributions had the lowest rates of interrogation. A comparison of the percentage of smolts interrogated for both ponds revealed that the treatment group had a higher percent interrogation for smolts between 190 and 220 mm, but not for the smaller size groups 170 mm and below (Figure 3.4). Even though there was a treatment effect for part of the population, a chi-square test for differences between the two groups (Conover 1971) in that range did not result in significance (P > 0.05). Migration Time. The mean migration time for steelhead released from Dworshak NFH to various down river dams is listed for each of the experimental and control groups in Table 3.9. Mean migration times from Dworshak NFH to Lower Granite Dam ranged from 6.0 days for the small smolts to 6.5 days for the large smolts on the Abernathy Diet. Migration times for the control fish ranged from 6.5 days for the small smolts to 5.6 days for the large smolts. Means were tested for significant differences between treatments and controls using a t-test. No significant differences were observed between the diet treatment and the control. No large differences were consistently observed in the mean migration times of small and large smolts. Small smolts tended to travel more slowly than large smolts, but this trend was also not consistent. Extended Seawater Rearing Survival Seawater survival is shown in Table 3.10. The results are confounded by a disease outbreak. High mortality was seen in one control tank and one treatment tank. The other two tanks, one control and one treatment tank, had no mortalities.

71

Figure 3.1. Total number of smolts marked with PIT-tags and subsequently interrogated at downriver dams for treatment pond 28 and control pond 24 in the Abernathy Diet experiment conducted at Dworshak NFH in May1998.

Abernathy Diet Diet Control0

100

200

300

400

500

Num

ber

Marked Interrogated

72

Figure 3.2. Percent of small (<200 mm) and large (>200 mm) smolts marked with PIT-tags and subsequently interrogated at downriver dams for treatment pond 28 and control pond 24 in the Abernathy Diet experiment conducted at Dworshak NFH in May1998.

Abernathy Diet Diet Control0

10

20

30

40

50

60

70

80

90

100

Perc

ent

Small Smolts Large Smolts

73

130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280

Length (mm)

0

20

40

60

80

100

120N

umbe

rReleases Interrogations Pond 28

Abernathy Diet

74

Figure 3.3. Number of PIT-tagged steelhead smolts released and interrogated at downriver dams, by 10 mm length group, for the treatment pond 28 and control pond 24 in the Abernathy Diet experiment conducted at Dworshak NFH in 1998.

130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280

Length (mm)

0

20

40

60

80

100

120

Num

ber

Releases Interrogations Pond 24Control Diet

75

130 140 150 160 170 180 190 200 210 220 230 240 250

Length (mm)

0

20

40

60

80

100

Perc

ent

Treatment Control

76

Figure 3.4. Percentage of smolts released that were interrogated at downriver dams for the treatment and control ponds, by length group, in the Abernathy Diet experiment conducted at Dworshak NFH in 1998.

77

Table 3.9. Mean migration time of small (< 200 mm) and large (> 200 mm) steelhead smolts that were PIT-tagged, released, and detected at down river dams as part of the Enhanced Abernathy Diet Evaluation Project at Dworshak NFH in 1998.

Mean Migration Time (Days)

Dam Site

Treatment Small

Smolts1

Large

Smolts2

Combined

Lower Granite

Abernathy Diet

6.0

6.5

6.3

Diet Control

6.5

5.6

6.0

Little Goose

Abernathy Diet

9.3

8.1

8.7

Diet Control

7.3

7.6

7.4

Lower Monumental

Abernathy Diet

11.6

9.3

10.5

Diet Control

10.6

10.3

10.5

McNary

Abernathy Diet

24.2

-

24.2

Diet Control

11.3

10.1

10.7

1 Smolts < 200 mm fork length. 2 Smolts > 200 mm fork length

78

Table 3.10. Mortalities that occurred during extended seawater rearing at Marrowstone Field Station for summer steelhead in the Enhanced Abernathy Diet Evaluation Project at Dworshak NFH in 1998.

Group

Pond Number

Mortalities

Percent

Abernathy Diet

28

75

37

49

30

75

0

0

Diet Control

24

75

23

31

26

75

0

0

79

Discussion

Algae, Spirulina platensis and Tetraselmis suecica, have been used as an immunostmulants with some success (Duncan and Klesius 1996; Austin and Day 1990). In this study, Schizochytrium sp. was used. The algal preparation was used not only as a possible immunostimulant but as a feed enhancer because of its levels of polyunsaturated fatty acids. Sargent et. al (1999) reported that Atlantic salmon, when fed a vegetable oil compared to a fish oil diet, were better able to osmoregulate in a salt water challenge. The vegetable oil provided fatty acids the fish could use to form docosahexaenoic acid (DHA, 22:6n-3) eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (AA, 20:4n-6) used in cell membranes and precursors of eicosanoids. Ogata and Murai (1989) and Ashton et al. (1993) also suggest that the ratios of fatty acids, linoleic (18:2n-6)/linolenic (18:3n-3) are very important for the facilitation of smolting. Bergstrom (1989) stated that smolts may have different fatty acid requirements from parr in that they may need a good supply of the fatty acid DHA which would be beneficial for the smolting process. The results of the present study did not provide enough information to make conclusions. However the increased survival of the fish in salt water may be an indication that the enhanced Abernathy was better.

80

Appendix Table 3.1 Monthly mortality, weight, and size of steelhead in each treatment and control pond in the Moore-Clark Nutra-Transfer Diet evaluation project at Dworshak National Fish Hatchery, Idaho. Month1

Pond

Mortalities

Number

Weight

No./lb

Length (mm)

8/97

24

0

28,550

288

99

79

26

0

28,500

291

98

78

28

30

9/97

24

299

28,251

307

92.1

80

26

519

27,981

310

90.4

80

28

472

28,028

323

86.9

81

30

231

28,269

329

86.0

82

10/97

24

337

27,914

709

39.8

106

26

361

27,670

696

39.7

106

28

238

27,790

749

37.1

108

30

322

27,947

692

40.4

105

11/97

24

199

27,715

1100

25.2

123

26

123

27,497

1034

26.6

121

28

108

27,682

1045

26.5

121

30

102

27,845

1071

26.0

122

12/97

24

104

27,611

1793

15.4

145

26

108

27,389

1680

16.3

142

28

70

27,612

1578

17.5

138

30

88

27,757

1551

17.9

137

1/98

24

102

27,509

2571

10.7

164

26

34

27,355

2464

11.1

162

28

35

27,577

1978

13.9

150

30

9

27,748

1893

14.7

147

81

Appendix Table 3.1 Continued Month

Pond

Mortalitie

s

Number

Weight

No./lb

Length (mm)

2/98

24

192

27,317

3056

8.9

174

26

222

27,133

2893

9.4

171

28

267

27,310

2350

11.6

159

30

497

27,251

2568

10.6

164

3/98

24

629

26,688

3983

6.7

191

26

766

26,367

3465

7.6

183

28

2040

25,270

2865

8.8

174

30

2935

24,316

2947

8.3

178

4/98

24

300

26,388

4712

5.6

203

26

402

25,965

4555

5.7

202

28

865

24,405

3813

6.4

194

30

1124

23,192

3865

6.0

198

Release

24

81

26,307

5597

4.7

215

26

101

25,864

4781

5.4

205

28

84

24,321

3994

6.1

197

30

80

23,112

4026

5.7

201

1Except for August, data is recorded for the beginning of the month.

82

CHAPTER FOUR

AN EVALUATION OF FEEDING THE MOORE-CLARK NUTRA TRANSFER FW DIET AS A MEANS OF PROMOTING SMOLTIFICATION IN SUMMER STEELHEAD (ONCORHYNCHYS MYKISS) AT DWORSHAK NATIONAL FISH HATCHERY, IDAHO

by

Ray N. Jones U. S. Fish and Wildlife Service, Idaho Fishery Resource Office

Ahsahka, Idaho

Steve Boggio Moore-Clark USA, Inc. La Conner, Washington

Robin M. Schrock and Robert E. Reagan

U. S. Geological Survey, Biological Resources Division, Columbia River Research Laboratory, Western Fisheries Research Center

Cook, Washington

Corie Samson U. S. Fish and Wildlife Service, Idaho Fish Health Center

Ahsahka, Idaho

Robert Semple U. S. Fish and Wildlife Service, Dworshak National Fish Hatchery

Ahsahka, Idaho

83

Abstract The Moore-Clark Nutra-Transfer diet was evaluated at Dworshak National Fish Hatchery to determine if smoltification in summer steelhead could be enhanced. The diet was fed for about six weeks prior to smolts being released. Smoltification was evaluated at several different life stages. Gill ATPase activity was monitored to evaluate the effect on physiology. Skin reflectance and condition factor were monitored to evaluate the effect on morphology. Migration rate and survival during downstream migration were monitored to evaluate emigration. Representative groups were reared at Marrowstone Marine Field Station in seawater to simulate and evaluate initial ocean entry. Comparisons were made with control groups that were fed the standard hatchery diet. Although growth was slightly better in the treatment groups, no significant differences were observed between treatments and controls for any of the parameters measured.

84

Introduction At Dworshak National Fish Hatchery (NFH), when juvenile steelhead (Oncorhynchus mykiss) reach a size of about 20 fish per pound, they are converted from BioMoist (hand-fed) to a heat-extruded pellet (steelhead diet) administered using Babington Response Feeders. No changes in the diet are made during the remainder of the production cycle except for an increase in the size of the pellet to accommodate increased fish size. Presently, no known diets have been formulated specifically for conservation production programs to meet the physiological requirements of anadromous fish during smoltification. Moore-Clark USA, Inc. presently manufactures Nutra-Transfer FW, a diet that was specifically designed to facilitate the transfer of Atlantic salmon (Salmo salar) parr from freshwater to saltwater rearing pens for commercial production. Although good success has been experienced by the commercial fish industry, the effectiveness of Nutra-Transfer FW has never been evaluated at a conservation or a mitigation hatchery. Anadromous salmonids go through a series of physiological changes in preparation for entry into the ocean from freshwater, including changes in associated hormonal processes that modify behavior (Iwata 1995). However, some physiological parameters used as indices of the smoltification process are not always positively correlated with behavior, depending on the species (Iwata 1995; Pirhonen and Forsman 1998). While the Nutra-Transfer FW diet does enhance the physiological processes involving saltwater tolerance and osmoregulation in seawater, an evaluation of its effects on behavioral changes associated with smoltification has never been conducted. The ability of the Nutra-Transfer FW diet to enhance emigration behavior (i.e., migration rate) associated with smoltification in steelhead was of particular interest in this study. The primary goal of the study was to enhance the smoltification of summer steelhead at Dworshak NFH, to increase the proportion of smaller steelhead that smolt.

Methods Experimental Design The experiment was initiated after juvenile steelhead were transferred from inside nursery tanks to outside Burrows ponds. The treatment group consisted of 2 Burrows ponds that received the Moore-Clark Nutra-Transfer FW diet prior to release, while the control group consisted of 2 Burrows ponds that received the standard production diet throughout the rearing period. Smoltification was evaluated by examining 3 different indices: gill sodium, potassium-activated adenosine triphosphatase (Na+, K+-ATPase) activity, skin reflectance, and condition factor (K). Emigration behavior was evaluated by examining migration time and survival after the smolts were released from the hatchery. Survival during initial entry into seawater and during early seawater rearing was evaluated by transferring representative groups from each pond to circular tanks at the Marrowstone Marine Field Station (U. S. Geological Survey) on Puget Sound, Washington. Fish Culture

85

Experimental fish were from brood year 1997, the progeny of steelhead spawned during Take 11. After nursery rearing, the fish were transferred to outside Burrows ponds on August 21, 1997, at an average size of 93.5 fish per pound. About 28,500 fish were stocked into each Burrows pond. A summary of the monthly production inventories (number, mortality, size, and weight) is provided in Appendix Table 4.1. All ponds were supplied with ambient river water on a single-pass system until November 11, 1997, when the reuse system was started. All fish were hand-fed BioMoist 2.5-mm feed from the time of ponding until November 11, 1997. At that time, the fish were converted from hand feeding to Babington demand feeders, and were converted from BioMoist feed to Rangen’s heat-extruded dry feed. Fish size at conversion was about 25 fish per pound. The Nutra-Transfer FW diet was started on March 16, 1998 and was fed until release. A proximate analysis of the Nutra-Transfer FW diet is listed in Appendix Table 4.2. The daily feeding record is listed in Appendix Table 4.3. The fish were released on April 29, 1998. On April 24, about 75 fish from each treatment and control pond were transferred from Dworshak NFH to the seawater rearing facilities at Marrowstone Marine Field Station. The fish were transferred in 100-gal totes supplied with oxygen. Temperatures were kept near 13 °C using ice. At Marrowstone, the fish were measured for fork length, weighed, and transferred to freshwater circular rearing tanks. After 2 days, the fish were gradually acclimated to full seawater. The acclimation process took about 7 days. The fish were fed the same diet at the same rate that was used at Dworshak NFH. Data Collection Dworshak NFH conducted monthly sample counts on representative ponds and used that data to project growth for all ponds in the system. Mortalities were collected and reported daily. Starting in September, random samples of about 100 fish were collected from each treatment and control pond to measure mean length and weight. In February, March, and April 1998, random samples of 20 fish were taken from each treatment and control pond to measure gill ATPase activity and skin reflectance. Fish were non-lethally anesthetized using tricaine methanesulfonate (MS-222) and were measured for fork length (mm) and weight (g). Gill ATPase activity was measured using the method described by Schrock et al. (1994). Skin reflectance was measured using a modification of the technique described by Haner et al. (1995). Condition factor was calculated as K (Anderson and Gutreuter 1983). To measure migration rate, estimate survival during migration, and assess smolt development after release, 400 fish each from treatment pond 32 and control pond 24 were marked with passive integrated transponder (PIT) tags. To assess differences between smaller and larger fish, 200 fish smaller than 200 mm (TL) and 200 fish larger than 200 mm (TL) were tagged in each pond. Tagging was conducted on April 22, 1998. Migration time was measured to Lower Granite, Little Goose, Lower Monumental, and McNary dams on the lower Snake and Columbia rivers (see Figure 1.1). Survival during migration was estimated to Lower Granite Dam by using the cumulative number of unique interrogations at each of the dams listed above. To assess smolt development after release, arrangements were made with the National Marine Fisheries Service to use the interrogation-by-code facilities at Little Goose Dam. These facilities allowed

86

us to collect our PIT-tagged fish so that gill ATPase activity could be measured for comparison with activity levels prior to release. Data Analysis Mean gill ATPase, mean skin reflectance, and mean condition factor were compared between treatment and control groups using analysis of variance (ANOVA) (Wilkinson 1990). For those cases where statistically significant differences were observed, pairwise comparisons were made using the Bonferroni post-hoc procedure to identify which ponds differed. For those PIT-tagged steelhead that were recaptured at Little Goose Dam, mean gill ATPase was compared between the treatment and control groups using a two-sample t-test (Wilkinson 1990). Mean migration rates were compared between treatments and controls using ANOVA (Wilkinson 1990). Interrogation rates were compared between treatments and controls using a chi-square test for differences in probabilities (Conover 1970). For the statistical analysis of gill ATPase at Marrowstone Marine Field Station, the median fork length was calculated and used to differentiate small and large smolts. For all statistical tests, a P-value of 0.05 was used to designate a significant difference.

Results Indices of Smoltification Pre-release. Feeding the Moore-Clark Nutra-Transfer FW diet did not appear to have any significant affect on smoltification of steelhead prior to their release from the hatchery. Gill ATPase levels were low for both treatment and control ponds from February through April, and exhibited no distinct increasing trend that would characterize rapidly developing smoltification (Table 4.1). The only statistically significant difference observed was for the April sample. Treatment Pond 32 had a significantly (P < 0.05) higher level of ATPase activity than Control Ponds 24 and 26. However, the absolute differences between these ponds was less than 4.0 micromoles, the minimum amount of difference required for a biologically significant difference (Schrock et al. 1994). Skin reflectance did not exhibit any distinct increasing trend that would indicate rapidly developing smoltification (Table 4.2). The only statistically significant (P < 0.05) difference observed was between Control Pond 26 and Treatment Pond 34 in February. However, the difference, only 1.1 reflectance units, is not considered to be biologically significant. Condition factor did not exhibit any decreasing trend from February through April, and no significant differences between any of the ponds were observed (Table 4.3). The Moore-Clark Nutra-Transfer FW diet did not appear to significantly increase the proportion of smaller-sized juvenile steelhead that exhibited characteristics of smoltification. In April, there were no significant differences in gill ATPase activity between small (< 200 mm) and large (> 200 mm) juvenile steelhead in either the treatment or control groups. No significant differences in gill ATPase were observed between small juvenile steelhead from control ponds compared to small juvenile steelhead in treatment ponds. Larger juveniles had significantly (P < 0.01) higher skin reflectance values than smaller juveniles in both the treatment and control groups. However, no significant differences were observed between small juveniles from treatment ponds compared to control ponds.

87

Emigration. The Moore-Clark Nutra-Transfer FW diet did not appear to have a significant affect on smolt development during seaward emigration. Mean gill ATPase increased greatly for both the treatment and control groups between the time of release and the time of recapture at Little Goose Dam. Mean gill ATPase increased from 7.3 to 13.3 for the treatment group and increased from 5.5 to 12.5 for the control group. However, there was no significant difference in ATPase between the treatment and control groups collected at Little Goose Dam. Similarly, for small smolts (< 200 mm), there was no significant difference in mean gill ATPase. Extended Seawater Rearing. The treatment did not appear to have a significant affect on gill ATPase levels once fish were transferred to seawater. Mean gill ATPase levels of summer steelhead increased sharply from a range of 4.9 to 7.3 in April at Dworshak NFH (Table 4.1) to a range of 19.3 to 21.8 (Table 4.4) in June after being transferred to Marrowstone Marine Field Station. After being held at Marrowstone for two months, gill ATPase levels decreased somewhat in August. However, there were no significant differences between treatments and controls for either June or August (Table 4.4). We compared the gill ATPase of smaller smolts in the treatment and control groups and found no significant differences. Emigration PIT-tag Interrogation Rates. Smolt survival to Lower Granite Dam was very good compared to previous years. A total of 797 steelhead smolts were marked with PIT-tags and released from Dworshak NFH in May 1998 for this experiment. Of those, 626 unique interrogations were made, or 78.5% (Table 4.5). These results fall within the upper 50% of the historical range of 65.3 to 85.2 % for other groups of steelhead released from Dworshak NFH (Table 4.6). The Moore-Clark Nutra-Transfer FW diet did not appear to have a significant affect on smolt survival during seaward emigration. Both the treatment and control groups had very similar survival rates to Lower Granite Dam. The percentages of PIT-tagged fish detected from the control and treatment groups were 78.4 and 78.6%, respectively, a difference so small that statistical testing was not performed (Figure 4.1). Large smolts had a higher survival rate than smaller smolts in both the control and treatment groups (Figure 4.2). The percent of PIT-tags detected for small and large smolts (treatment and control groups combined) was 73.6 and 83.5%, respectively (Table 4.5). There was no significant difference in survival rates for small smolts in the treatment and control groups (Table 4.5 and Figure 4.2). For the small smolts, the percentages of PIT-tags detected for the control and treatment groups was 73.2 and 74.0%, respectively (Table 4.5), a difference so small that statistical testing was not performed. The number of releases and interrogations for the treatment and control groups is presented in Figure 4.3 by 10-mm length intervals. There was very little difference between the two groups. Those length groups nearest the center of the distribution had the lowest rates of interrogation. A comparison of the percentage of smolts interrogated for both ponds revealed that the treatment group had a higher percent interrogation for those smolts between 170 and 210 mm, but not for the smaller size groups below that (Figure 4.4). Even though there was a treatment effect for part of the population, a chi-square test for differences between the two groups in that range did not result in a significant difference.

88

Migration Time. Steelhead smolts traveled relatively quickly through the Lower Snake River after release. Mean migration time to Lower Granite Dam was less than 8 days, and mean migration time to Lower Monumental Dam was less than 12 days. Only 4 fish were interrogated at McNary Dam. In almost every case, steelhead smolts in the control group traveled faster to each of the dams than smolts in the treatment group (Table 4.7), although the differences were not statistically significant. The same pattern held true for small smolts: those in the control group traveled faster than the treatment group but the difference was not statistically significant. Extended Seawater Rearing Survival Overall, mortality during the period of seawater rearing was low. Mortality was 8% in the treatment ponds and 31% in the control ponds (Table 4.8). However, most of the mortality in the control ponds was the result of a disease episode in Control Pond 24 during mid-June involving Pseudomonas. Twenty of the 23 mortalities that occurred in Pond 24 were the result of Pseudomonas during the period June 6 to June 13, 1998. Control Pond 26 had no mortalities during the rearing period.

Discussion In addition to the standard components making up most fish diets, Nutra-Transfer FW has several additional ingredients that help facilitate the transition of anadromous fish from freshwater to saltwater. Inorganic salt (NaCl) is the primary ingredient added to Nutra-Transfer FW which has been demonstrated to increase survival in Pacific salmon (Zaugg and McLain 1969; Zaugg 1982; Zaugg et al. 1983). Addition of salt to the diet apparently activates the physiological process involved with extrarenal salt excretion, evidenced by distinct increases in gill Na+, K+-ATPase activity. We did not observe any increases in gill ATPase activity in steelhead at Dworshak NFH prior to their release, and no significant differences were observed between treatment and control groups. According to the literature, caution needs to be taken when feeding diets with higher than normal percentages of inorganic salts. Westgate et al. (1976) observed that spring chinook salmon (O. tshawytscha) fed diets with as much as 3 to 4% salt (NaCl) ate less readily than the controls, resulting in significantly reduced weight gain and feed conversion compared to controls. Small decreases in weight gain and food utilization have been observed in coho (O. kisutch) and chinook salmon fed salt-enhanced diets (Zaugg and McLain 1969; Westgate et al. 1976) although Shaw et al. (1975) reported no effect in Atlantic salmon and Zaugg et al. (1983) reported no significant differences in weight gain between treatment and control groups at Spring Creek NFH and Garrion Springs Hatchery. We observed that steelhead fed the Nutra-Transfer FW diet were slightly larger than the control groups and appeared to be in generally better condition, although we did not calculate growth and feed conversion rates. Although this was only a single-year pilot study, the need for assessing smoltification based on both a physiological and behavioral perspective appears relevant. Though the importance of life-stage associated behavioral characteristics to salmonids is well known, as are the physiological changes associated with freshwater-saltwater adaptation, better assessment and understanding of smoltification in the future will likely come with the more advanced ability to assess

89

physiological and behavioral attributes in context with each other and their cues. Studies on growth rate prior to smoltification (Dickhoff et al. 1995; Beckman et al. 1998), visual pigment dominance (Alexander et al. 1998), and phototaxis (Iwata et al. 1989) are examples of important steps in this direction. Studies like these suggest that a broad range of smolt condition values are possible for a given salmonid stock in a given production program situation. Our study attempted to relate physiology with behavior, to eventually gain the ability to produce smolts of optimal condition within the context of program goals for an enhancement program. Commercial smoltification diets are proving beneficial and successful in improving survival and optimizing growth during many freshwater-to-saltwater transfer situations. Success of these diets are based on the ability of dietary salt to lower post-transfer plasma chloride levels, and the ability of high-energy formulations to impart rapid growth during this time. Other proprietary ingredients are added to enhance osmoregulatory ability, palatability, and health. As yet, the nature of behavioral effects of these diets to smolting salmonids has not drawn much attention, though schooling behavior associated with feeding salt has been observed. Based on the innate association between physiological and behavioral parameters, however, it is logical to assume that behavioral effects from these commercial smoltification diets exist. These effects are likely of little practical importance to direct freshwater-saltwater transfer situations, but for inland enhancement hatcheries releases, understanding the nature of these effects on the juvenile migration may be instrumental to maximizing adult returns. Enhancement efforts should be aware that these diets and many more are being made available via the research-and-development-driven commercial salmonid industry.

90

Table 4.1. Mean monthly gill ATPase levels for summer steelhead in the treatment (BP32 and BP34) and control (BP24 and BP26) ponds in the Moore-Clark Nutra-Transfer Diet Evaluation Project at Dworshak NFH, Idaho.

Gill ATPase Levels

Month

Pond

Sample

Size Mean

SD

Minimum

Maximum

February

24

20

3.3

0.7

2.0

5.3

26

19

3.7

1.1

2.5

6.7

32

20

3.3

0.6

2.0

4.1

34

19

3.3

0.6

2.4

4.4

March

24

20

3.6

0.8

2.3

5.4

26

20

3.8

1.3

1.3

5.8

32

20

3.3

0.7

2.2

5.0

34

19

3.5

0.8

1.2

4.7

April

24

20

5.5

1.7

3.1

9.1

26

20

4.9

1.5

1.9

8.2

32

20

7.3

2.5

4.2

14.0

34

20

6.4

2.1

3.1

10.4

91

Table 4.2. Mean monthly skin reflectance levels for summer steelhead in the treatment (BP32 and BP34) and control (BP24 and BP26) ponds in the Moore-Clark Nutra-Transfer Diet Evaluation Project at Dworshak NFH, Idaho.

Skin Reflectance Levels

Month

Pond

Sample

Size Mean

SD

Minimum

Maximum

February

24

20

4.1

1.4

0.4

6.1

26

20

4.9

1.0

2.2

6.8

32

20

4.4

1.1

1.6

6.4

34

20

3.8

1.2

1.3

5.8

March

24

20

4.8

0.9

2.0

6.1

26

20

4.7

1.6

1.4

7.3

32

20

5.1

1.3

1.5

6.7

34

20

4.9

0.8

3.2

6.4

April

24

20

5.7

1.1

3.9

7.4

26

20

5.0

1.0

3.2

6.7

32

20

5.5

0.9

3.7

6.9

34

20

5.3

1.0

3.3

6.8

92

Table 4.3. Mean monthly condition factor (K) for summer steelhead in the treatment (BP32 and BP34) and control (BP24 and BP26) ponds in the Moore-Clark Nutra-Transfer Diet Evaluation Project at Dworshak NFH, Idaho.

Condition Factor (K)

Month

Pond

Sample

Size Mean

SD

Minimum

Maximum

February

24

20

0.97

0.06

0.77

1.05

26

20

0.99

0.04

0.89

1.05

32

20

0.94

0.18

0.54

1.10

34

20

0.96

0.08

0.68

1.05

March

24

20

1.01

0.08

0.87

1.01

26

20

0.97

0.09

0.77

1.11

32

20

0.95

0.08

0.71

1.07

34

20

0.97

0.05

0.88

1.09

April

24

20

0.96

0.06

0.81

1.05

26

20

0.94

0.05

0.86

1.06

32

20

0.95

0.03

0.91

1.00

34

20

0.97

0.05

0.90

1.05

93

Table 4.4. Mean monthly gill ATPase levels for summer steelhead from treatment (BP32 and BP34) and control (BP24 and BP26) ponds in the Moore-Clark Nutra-Transfer Diet Evaluation Project during seawater rearing trials at Marrowstone Marine Station, Washington.

Gill ATPase Levels

Month

Pond

Sample

Size Mean

SD

Minimum

Maximum

June

24

20

20.9

3.2

15.4

28.5

26

20

21.8

5.5

10.9

32.2

32

20

19.3

5.2

6.3

29.8

34

19

21.3

6.0

8.5

29.6

August

241

-

-

-

-

-

26

18

19.2

7.7

8.7

35.2

32

20

18.0

4.5

11.7

27.6

34

19

19.7

6.2

5.8

30.2

1 Because of the disease outbreak in Pond 24, these fish were not sampled in August.

94

Table 4.5. Number of small and large steelhead smolts that were PIT-tagged, released, and detected at down river dams for treatment pond 32 and control pond 24 in the Moore-Clark Nutra-Transfer Diet Evaluation Project at Dworshak NFH, Idaho in 1998. Treatment

Small Smolts1

Large Smolts2

Total

Marked

Detected

Percent

Marked

Detected

Percent

Marked

Detected

Diet Control

198

145

73.2

201

168

83.6

399

313

Moore-Clarke Diet

200

148

74.0

198

165

83.3

398

313

Totals

398

293

73.6

399

333

83.5

797

626

1 Smolts < 200 mm fork length. 2 Smolts > 200 mm fork length.

95

Table 4.6. Summary of PIT-tag release and interrogation data for steelhead released from Dworshak NFH from 1992 through 1997.

Year

Number Released Number Interrogated

Percent

1992

2,969

2,119

71.4

1993

1,471

1,254

85.2

1994

1,468

985

67.0

1995

5,118

3,798

74.2

1996

5,088

3,321

65.3

1997

5,589

4,215

75.4

96

Table 4.7. Mean migration time of small and large steelhead smolts that were PIT-tagged, released, and detected at down river dams for treatment pond 32 and control pond 24 in the Moore-Clark Nutra-Transfer Diet Evaluation Project at Dworshak NFH in 1998.

Small Smolts1

Large Smolts2

Dam Site

Treatment N

Mean Migration Time (Days)

N

Mean Migration Time (Days)

Lower Granite

Moore-Clark Diet

100

7.4

118

6.8

Diet Control

96

6.0

124

6.5

Little Goose

Moore-Clark Diet

26

10.5

31

10.6

Diet Control

29

9.3

26

8.1

Lower Monumental

Moore-Clark Diet

21

9.8

14

11.9

Diet Control

19

11.6

18

9.3

McNary

Moore-Clark Diet

1

28.1

2

15.6

Diet Control

1

24.2

0

-

1 Smolts < 200 mm fork length. 2 Smolts > 200 mm fork length

97

Table 4.8. Mortalities that occurred during extended seawater rearing at Marrowstone Field Station for summer steelhead in the Moore-Clark Nutra-Transfer Diet Evaluation Project at Dworshak NFH in 1998.

Group

Pond

Number

Mortalities

Percent

Moore-Clark Diet

32

75

6

8

34

75

6

8

Diet Control

24

75

23

31

26

75

0

0

98

Moore-Clark Diet Diet Control0

100

200

300

400

500

Num

ber

Marked Interrogated

99

Figure 4.1. Total number of smolts marked with PIT-tags and subsequently interrogated at down river dams for the control and treatment groups in the Moore-Clark Nutra-Transfer Diet Evaluation Project at Dworshak NFH in May 1998.

100

Figure 4.2. Percent of small (<200 mm) and large (>200 mm) steelhead smolts marked with PIT-tags and subsequently interrogated at downriver dams for the control and treatment groups in the Moore-Clark Nutra-Transfer Diet Evaluation Project at Dworshak NFH in May

Moore-Clark Diet Diet Control0

10

20

30

40

50

60

70

80

90

100

Perc

ent

Small Smolts Large Smolts

101

Figure 4.3. Number of PIT-tagged steelhead smolts released and interrogated at downriver dams, by 10 mm length group, for the treatment and control ponds in the Moore-Clark Nutra-Transfer Diet Evaluation project conducted at Dworshak NFH in 1998.

130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280

Length (mm)

0

20

40

60

80

100

120

Num

ber

Releases Interrogations Pond32Moore-Clark Diet

130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280

Length (mm)

0

20

40

60

80

100

120

Num

ber

Releases InterrogationsPond 24Diet Control

102

Figure 4.4. Percentage of smolt released that were interrogated at downriver dams for the treatment and control ponds, by length group, in the Moore-Clark Nutra-Transfer Diet Evaluation project conducted at Dworshak NFH in 1998.

130 140 150 160 170 180 190 200 210 220 230 240 250 260

Length (mm)

0

20

40

60

80

100Pe

rcen

tN<10 Treatment N<10 Control Treatment Control

103

Appendix Table 4.1. Monthly mortality, weight, and size of steelhead in each treatment pond (Ponds 32 and 34) and control pond (Ponds 24 and 26) in the Moore-Clark Nutra-Transfer Diet evaluation project at Dworshak National Fish Hatchery, Idaho. Month1

Pond

Mortalities

Number

Weight

No./lb

Length (mm)

8/21/97

24

0

28,550

288

99

79

26

0

28,500

291

98

78

32

0

28,500

312

91

80

34

0

27,500

320

86

81

9/97

24

299

28,251

307

92.1

80

26

519

27,981

310

90.4

80

32

536

27,964

331

84.6

82

34

98

27,402

339

80.9

83

10/97

24

337

27,914

709

39.8

106

26

361

27,670

696

39.7

106

32

322

27,642

660

41.9

104

34

134

27,268

775

35.2

110

11/97

24

199

27,715

1100

25.2

123

26

123

27,497

1034

26.6

121

32

427

27,215

993

27.4

120

34

28

27,240

1044

26.1

122

12/97

24

104

27,611

1793

15.4

145

26

108

27,389

1680

16.3

142

32

280

26,935

1480

18.2

137

34

52

27,188

1536

17.7

138

1/98

24

102

27,509

2571

10.7

164

26

34

27,355

2464

11.1

162

32

98

26,837

1938

13.8

150

34

19

27,169

1964

13.8

150

104

Appendix Table 4.1. Cont. Month

Pond

Mortalities

Number

Weight

No./lb

Length (mm)

2/98

24

192

27,317

3056

8.9

174

26

222

27,133

2893

9.4

171

32

318

26,519

2424

10.9

162

34

328

26,841

2449

10.6

162

3/98

24

629

26,688

3983

6.7

191

26

766

26,367

3465

7.6

183

32

1255

25,264

2938

8.6

176

34

1261

25,580

2933

8.7

175

4/98

24

300

26,388

4712

5.6

203

26

402

25,965

4555

5.7

202

32

551

24713

4413

5.6

203

34

718

24,862

4200

5.9

199

Release

24

81

26,307

5597

4.7

215

26

101

25,864

4781

5.4

205

32

91

24,622

4628

5.3

206

34

75

24,787

4426

5.6

203

1 Except for August, data is recorded for the beginning of the month.

105

Appendix Table 4.2. Proximate analyses of the diets used in the Dworshak NFH diet trial, 1997-98.

Proximate analysis(%)

Abernathy

Diet

Abernathy +

Schizochytrium

Hagerman

floating steelhead

Diet

Nutra Transfer

FW (Moore-Clark)

Protein

45.6

43.7

47.6

49.8

Fat

16.9

19.9

16.7

22.8

Ash

9.1

8.6

8.2

9.4

Moisture

4.8

4.5

7.1

4.8

106

Appendix Table 4.3. Daily feeding records for the treatment (BP32 and BP34) and control (BP24 and BP26) ponds for the Moore-Clark Nutra-Transfer Diet Evaluation Project for summer steelhead at Dworshak NFH, Idaho. Month

Day

BP24

BP26

BP32

BP34

November

1

18

18

18

18

2

18

18

0

0

3

0

0

0

0

4

0

0

18

18

5

0

0

0

0

6

20

19

20

21

7

18

18

18

18

8

18

18

18

18

9

18

18

18

18

10

18

18

18

18

11

18

18

18

18

12

18

18

18

18

13

23

23

23

23

14

30

30

30

30

15

30

30

29

29

16

27

27

27

27

17

25

25

25

25

18

0

0

0

0

19

80

80

80

80

20

0

0

0

0

21

120

120

80

80

22

0

0

0

0

23

0

0

0

0

24

0

0

0

0

25

69

40

40

80

26

41

40

40

40

27

0

0

0

0

28

0

0

80

40

29

0

0

0

0

30

40

40

40

0 December

1

0

0

0

0

2

0

0

0

0

3

40

40

40

40

107

4 0 0 0 0

5

80

80

40

40 Month

Day

BP24

BP26

BP32

BP34

6

0

0

0

0

7

0

0

0

0

8

120

40

120

80

9

0

0

0

0

10

40

80

40

40

11

0

0

0

0

12

40

40

40

40

13

0

0

0

0

14

0

0

0

0

15

40

40

40

40

16

0

0

0

0

17

40

0

0

40

18

0

0

0

0

19

80

80

80

80

20

0

0

0

0

21

80

80

80

40

22

0

0

0

0

23

0

0

0

0

24

80

80

40

40

25

0

0

0

0

26

0

40

80

80

27

0

0

0

0

28

80

80

40

40

29

40

0

0

0

30

0

0

0

0

31

0

0

0

0 January

1

0

0

0

0

2

0

40

80

40

3

0

0

0

0

4

0

0

0

0

5

40

40

40

40

6

0

0

0

0

7

80

40

40

40

8

0

0

0

0

108

9 40 40 0 80

10

0

0

0

0

11

0

0

0

0

12

80

80

120

40 Month

Day

BP24

BP26

BP32

BP34

13

0

0

0

0

14

40

40

40

40

15

0

0

0

0

16

80

40

40

80

17

0

0

0

0

18

0

0

0

0

19

80

80

80

40

20

0

0

0

0

21

0

0

40

80

22

0

0

0

0

23

120

80

40

80

24

0

0

0

0

25

0

0

0

0

26

120

120

120

80

27

0

0

0

0

28

0

0

40

40

29

0

0

0

0

30

80

80

80

40

31

0

0

0

0 February

1

80

80

40

120

2

0

0

0

0

3

0

0

0

0

4

40

40

40

40

5

0

0

0

0

6

80

40

40

40

7

0

0

0

0

8

80

80

120

80

9

0

0

0

0

10

80

80

80

80

11

40

0

0

0

12

0

0

40

40

13

40

80

0

40

14

0

0

0

0

109

15

40

80

80

40

16

0

0

0

0

17

40

0

0

0

18

0

0

0

0

19

0

40

0

0 Month

Day

BP24

BP26

BP32

BP34

20

40

0

40

0

21

0

0

0

0

22

80

120

80

80

23

0

40

40

0

24

0

0

0

0

25

80

40

0

40

26

40

0

40

0

27

80

80

80

40

28

0

0

0

0 March

1

80

80

40

40

2

0

40

40

0

3

0

40

40

40

4

80

40

80

40

5

0

40

0

0

6

40

0

40

80

7

0

0

0

0

8

0

0

0

0

9

80

80

120

40

10

80

80

80

80

11

0

0

0

0

12

0

0

0

0

13

0

0

0

0

14

0

0

0

0

15

0

0

0

0

16

160

160

220

220

17

0

0

0

0

18

80

80

128

73

19

0

0

0

0

20

120

160

55

180

21

0

0

0

0

22

0

0

0

0

110

23 80 80 110 55

24

0

0

0

0

25

0

0

0

0

26

40

40

40

95

27

120

50

95

40

28

0

0

0

0

29

0

0

0

0 Month

Day

BP24

BP26

BP32

BP34

30

80

80

95

95

31

0

0

0

0 April

1

0

0

0

0

2

0

0

0

0

3

80

80

40

165

4

0

0

0

0

5

0

0

0

0

6

0

0

0

0

7

40

0

55

55

8

0

80

55

55

9

0

0

0

0

10

80

0

0

0

11

0

0

0

0

12

0

0

0

0

13

72

80

110

110

14

0

0

0

0

15

0

0

0

0

16

0

0

0

0

17

0

0

0

0

18

0

0

0

0

19

0

0

0

0

20

40

30

15

0

21

0

0

0

0

22

10

0

0

0

23

0

0

0

0

24

40

40

31

0 Monthly Totals

Month

BP24

BP26

BP32

BP34

111

November 649 618 658 619 December

760

680

640

600

January

760

680

760

720 February

840

800

720

640

March

1040

1050

1183

1078 April

362

310

306

385

112

CHAPTER FIVE

SKIN REFLECTANCE AS A NON-LETHAL MEASURE OF SMOLTIFICATION FOR JUVENILE STEELHEAD AT DWORSHAK NATIONAL FISH HATCHERY,

IDAHO

by Ray N. Jones

U. S. Fish and Wildlife Service, Idaho Fishery Resource Office Ahsahka, Idaho

Corie Samson

U. S. Fish and Wildlife Service, Idaho Fish Health Center Ahsahka, Idaho

and

Robin M. Schrock and Robert E. Reagan U. S. Geological Survey, Biological Resources Division

Western Fisheries Research Center, Columbia River Research Laboratory Cook, Washington

Abstract The goal of this study was to determine if skin reflectance could be used as a non-lethal, low-maintenance, low-cost method of monitoring juvenile steelhead smoltification at Dworshak NFH during the 2 to 3-month period prior to release. Changes in gill ATPase, skin reflectance, and condition factor were measured for five separate experimental groups of summer steelhead at Dworshak NFH once monthly during February, March, and April of 1998. Mean skin reflectance increased from February to April for all five experimental groups. However, the rate of increase varied considerably. Similar to mean skin reflectance, mean gill ATPase activity increased each month for all five experimental groups as well. Except for one experimental group, mean condition factor did not exhibit the decreasing trend characteristic of smoltification. Very strong positive correlations were found between mean skin reflectance and mean gill ATPase activity. Strong negative correlations between gill ATPase and condition factor, and between skin reflectance and condition factor, were exhibited for most of the groups examined. However, none of the correlations were statistically significant. The strong correlation between skin reflectance and gill ATPase activity in our study, and previous demonstrations of significant increases in skin reflectance of steelhead during the juvenile migration, strongly support development of skin reflectance as a non-lethal quantitative measurement of development during the parr-smolt transformation.

113

Introduction The Idaho Fish Health Center (IFHC) has responsibility for conducting smolt assessments of summer steelhead (Oncorhynchus mykiss) at Dworshak National Fish Hatchery (NFH) prior to their release in the spring. The assessment includes a wide array of fish health and condition parameters and the use of the Goede’s Fish Health/Condition Assessment Procedures (Goede 1989). However, the IFHC assessment does not include an index which measures the smoltification process as it develops throughout the spring. A non-lethal index of smoltification could be very useful in assessing the readiness of summer steelhead for release from the hatchery and as an evaluation tool for recommending changes in rearing and production practices. Juvenile salmonids go through a series of physiological, morphological, and behavioral changes while they are rearing in freshwater that prepare them for migration to the ocean. The biological changes that occur are collectively referred to as the parr-smolt transformation process, or smoltification (Wedemeyer et al. 1980; Folmar and Dickhoff 1981; Wedemeyer 1996). A common index of smoltification is the condition factor, the relation between fish length and fish weight (Wedemeyer 1996). The condition factor typically decreases during smoltification and has been used successfully as a criterion of smoltification in steelhead (Wagner 1974b). The change in the length-weight relationship is also associated with decreases in total body lipids (Fessler and Wagner 1969). Another change that occurs during smoltification is the development of saltwater tolerance and an increase in hypo-osmoregulatory ability. This change is associated with an increase in levels of gill sodium, potassium-activated adenosine triphosphatase (Na+, K+-ATPase), an enzyme system that provides the energy necessary for chloride cells in the gills to actively transport ions absorbed from seawater back into the ocean (Wedemeyer 1996). Gill ATPase levels have been used extensively in research to evaluate the success of various rearing programs at many Columbia River hatcheries (Zaugg et al. 1991). However, measuring gill ATPase levels is not practical for routine monitoring at the production level. Collecting and analyzing the tissue samples is labor intensive, and the turn around time for receiving the data after it has been summarized is often so long that the information is not available for making timely management decisions. Another change that smolting fish exhibit is an increase in body silvering due to the reflection of light from guanine and other purines deposited in the scales and deep skin tissues (Hoar 1976; Folmar and Dickhoff 1980). Guanine and hypoxanthine are produced as a result of seasonal thyroid hormone-induced increases in protein catabolism (Wedemeyer 1996). Guanine levels have been used as an assessment tool in the past, but the sampling method is lethal and the process is very tedious and time consuming (Johnson and Eales 1968). Haner et al. (1995) developed a non-lethal method for quantitatively measuring skin silvering using a photo reflectance video analysis system (PRVAS). The authors found that mean skin reflectance of steelhead and spring chinook salmon (O. tshawytscha) released from Dworshak and Kooskia National Fish Hatcheries was significantly correlated with mean gill ATPase activity and mean skin guanine concentrations. The authors concluded that skin reflectance could be used as a non-lethal and cost effective indicator of smolt development. Haner et al.

114

(1995) monitored skin reflectance immediately prior to release and during the outmigration process, but did not collect data for any extended period leading up to release. Use of their method as a means of pre-release monitoring and assessment has never before been tested. The goal of this study was to determine if skin reflectance could be used as a non-lethal, low-maintenance, low-cost method of monitoring juvenile steelhead smoltification at Dworshak NFH during the 2 to 3-month period prior to release. The objectives of the study were: 1) to measure the quantitative changes in gill ATPase activity, skin reflectance, and condition factor of steelhead during the 3-month period before release from the hatchery; and 2) to determine the relationships between gill ATPase activity, skin reflectance, and condition factor of steelhead during the 3-month period before release from the hatchery.

Methods Fish Collection and Processing To measure changes in gill ATPase, skin reflectance, and condition factor over time, steelhead from brood year 1997 were sampled at Dworshak NFH once monthly during February, March, and April of 1998. Random samples of 20 fish were taken from each of 10 Burrows ponds representing the 3 separate experiments presented in this report. The first experiment consisted of 2 treatment ponds that underwent a reduced, intermittent diet during December 1997 and January 1998 (Growth Treatment) and 2 control ponds (Growth Control). The second and third experiments consisted of 2 treatment ponds receiving an Abernathy Diet and 2 treatment ponds receiving a Moore-Clark Diet, respectively; these experiments shared 2 control ponds that were fed the standard hatchery diet (Diet Control). Fish were anesthetized using tricaine methanesulfonate (MS-222) at a concentration of 100 mg ⋅ L-1. Data for skin reflectance was obtained by placing each fish into a 24 x 13 x 10 cm plexiglass box equipped with 2 photo bulbs that produced diffuse light (Haner et al. 1995). The fish was placed into a small plexiglass trough that could be easily inserted and removed from the box. The fish, one black and one gray calibration tab, a label identifying the fish, and enough water to just cover the fish were placed in the trough. An image of the fish was taken using an Epson Photo PC 600 XGA color digital camera. After the photo image was obtained, each fish was measured for fork length (mm) and weight (g), and a gill tissue sample was taken to determine gill ATPase activity. Gill ATPase was measured using the microassay technique developed by Schrock et al. (1994), and is reported in units of micromoles inorganic phosphate per milligrams protein per hour (μmol Pi ⋅ mg protein-1 ⋅ hr-1). Condition factor (K) was calculated as (105 ⋅ [weight ⋅ length-3]) (Anderson and Gutreuter 1983). Converting Photos to Quantitative Values of Skin Reflectance The images were downloaded from the camera to a computer using Epson Image Expert 1.1.2 software. Sigma Scan Pro Image Analysis (Version 4.01.003) was used to calculate reflectance. Area measurement was used for skin reflectance and was defined as the sum of calibrated pixel units in a defined area. The black and gray tabs were used to calibrate the

115

pixels. The area was chosen by first drawing a line from the posterior end of the pectoral fin up to the lateral line. The second line was drawn along the lateral line just to the anterior portion of the dorsal fin. A line was then drawn directly down to the ventral side of the fish. The final line was drawn back to the initial starting point (Figure 5.1). The reflectance values were saved into Lotus for further evaluation. Data Analysis and Statistical Comparisons Data from replicate ponds were combined for statistical analyses. Mean values for skin reflectance, gill ATPase activity, and condition factor were calculated monthly. Pearson’s correlation coefficients were calculated for comparisons between mean monthly values of skin reflectance, condition, and gill ATPase activity. Statistical analyses were performed using Systat (Wilkinson 1990) and differences were considered significant at P < 0.05.

116

Figure 5.1. Photo of steelhead smolt inside a plexiglass case for measuring skin reflectance. The area used for assessing skin reflectance is outlined by the black lines. The black and gray calibration tabs are located in the upper left.

117

Results Skin Reflectance Mean skin reflectance increased from February to April for all five experimental groups (Table 5.1). However, the rate of increase varied considerably. For example, mean skin reflectance for the Growth Treatment group increased by 1.3 units from February to March but by only 0.1 unit from March to April. In contrast, mean skin reflectance for the Growth Control group increased by 0.5 unit from February to March and from March to April. The Abernathy Diet and the Diet Control were the only groups that exhibited an accelerating rate of increase in mean skin reflectance from February to April. Gill ATPase Similar to mean skin reflectance, mean gill ATPase activity increased each month for all five experimental groups (Table 5.1). Unlike skin reflectance, however, mean gill ATPase exhibited a consistent rate of increase from February to April for all five groups, with the exception of the Growth Control group, which did not exhibit an increase in mean gill ATPase from February to March. Condition Factor Mean condition factor did not exhibit the decreasing trend characteristic of smoltification except in the Growth Control fish (Table 5.1). This group exhibited an accelerated rate of decrease in mean condition factor from February to April. The other four experimental groups exhibited a slight increase in mean condition factor from February to March, followed by a decrease from March to April; however, the decrease did not always result in an April mean condition factor which was lower than the February mean. Correlations Between Indices Very strong positive correlations were found between mean skin reflectance and mean gill ATPase activity (Table 5.2). Strong negative correlations between gill ATPase and condition factor, and between skin reflectance and condition factor, were exhibited for most of the groups examined. However, the results were not consistent. None of the correlations were statistically significant.

Discussion Gill Na+, K+-ATPase activity for steelhead at Dworshak NFH normally shows little change during the 2 to 3-month period prior to release from the hatchery (Rondorf et al. 1989; Beeman et al. 1990, 1991) and mean values usually fall below 10 μmol Pi ⋅ mg protein-1 ⋅ hr-1. It is therefore not surprising to see such a slow rate of increase in skin reflectance prior to release. However, the results are encouraging, since there appears to be a strong relationship between skin reflectance and gill ATPase activity.

118

The reflectance measurements recorded in our study were higher than those reported by Haner et al. (1995). Our use of grey and black reference tabs, rather than the white and black tabs used by Haner et al. (1995), would explain our higher reflectance values because the contrast between silvering and gray is more distinct than in comparison with white. Therefore our results are not directly comparable to those of Haner et al. (1995). We were unable to confirm the significant differences in skin reflectance between samples taken pre-release and during migration reported in Haner et al. (1995), because all of our samples were taken prior to release. Differences in skin reflectance and ATPase activities between years in other steelhead stocks (Haner et al. 1995) suggest that annual differences in baseline reflectance levels and ATPase activity exist, but the increase in both measurements seen between release and during emigration appears to be common in different species and stocks. Further work with skin reflectance needs to be conducted to validate its reliability as a pre-release measure of smolt development in steelhead. The magnitude of annual differences of baseline levels and the potential magnitude of change during the migration need to be established for individual hatchery stocks. The strong correlation between skin reflectance and gill ATPase activity in our study, and previous demonstrations of significant increases in skin reflectance of steelhead during the juvenile migration, strongly support development of skin reflectance as a non-lethal quantitative measurement of development during the parr-smolt transformation. The pre-release smolt assessment on brood year 1997 spring chinook salmon, conducted in April 1999, provides a good example of how this technique could have been useful. The IFHC examined 60 fish and determined that they were probably not quite ready for release because they did not appear to be as silvery as in previous years. Parr marks were clearly visible. However, because the visual assessment was purely qualitative, it was not easily compared with assessments from previous years. Had skin reflectance data been available, it would have provided a quantitative assessment for comparison between years.

119

Table 5.1. Statistical summary of skin reflectance, gill ATPase, and condition factor for each of the experimental groups sampled at Dworshak NFH, February to April 1998.

Skin Reflectance

Gill ATPase

Condition Factor

Treatment

Group Month

N

Mean SD Mean SD

Mean SD

February

39

4.3 1.2 3.4 1.2

0.95 0.12

March

39

5.6 0.9 3.7 0.9

1.01 0.07

Growth

April 40

5.7 0.8 5.5 1.6

0.95 0.05

February

38

4.5 1.3 3.6 0.7

0.97 0.05

March

39

5.0 1.3 3.6 1.1

0.95 0.11

Growth Control

April

40

5.5 1.1 5.0 1.4

0.92 0.04

February

40

4.0 0.9 3.1 0.7

0.94 0.11

March

38

4.5 1.0 3.4 1.0

0.95 0.09

Abernathy

Diet

April

39

5.1 0.9 4.6 1.3

0.94 0.06

February

39

4.1 1.2 3.3 0.6

0.95 0.14

March

38

5.0 0.9 3.4 0.8

0.97 0.05

Moore-Clark Diet

April 40

5.4 0.9 6.8 2.3

0.96 0.04

February

39

4.3 1.3 3.5 0.9

0.98 0.05

March

40

4.7 1.3 3.7 1.1

0.99 0.09

Diet

Control

April

40

5.3 1.1 5.2 1.7

0.95 0.05

120

Table 5.2. Pearson correlation coefficients calculated for comparisons of skin reflectance, condition factor, and gill ATPase for experimental groups sampled at Dworshak NFH, February to April 1998. Treatment Group

Reflectance x

Condition

Reflectance x

ATPase

Condition x

ATPase

Growth

0.44

0.66

-0.38

Growth Control

-0.99

0.87

-0.92

Abernathy Diet

-0.05

0.96

-0.33 Moore-Clark Diet

-0.80

0.75

0.03

Diet Control

-0.80

0.96

-0.94

121

CONCLUSIONS This report details three approaches to producing a higher proportion of summer of steelhead from Dworshak National Fish Hatchery that emigrate successfully, quickly, and survive the transition to seawater. Results show that small gains in growth, emigration rates, and seawater survival may be achieved, but the cost of modifying routine hatchery practices should be considered in evaluating a transition to new feeding methods. All treatment groups, including the controls, that were reared under routine hatchery procedures outmigrated in similar numbers, and acclimated to seawater. All were at similar levels of smoltification as measured by gill ATPase at the time of release, and during seawater residence. Further, reduced ration, intermittent feeding trials may be refined from this study for further reductions in feed and to test different phasing of the modified feeding schedule prior to release. Results of the reduced ration, intermittent feeding modification suggest that feeding studies based on the specific stock and holding conditions offer control of ration rates and expected growth rates.

122

LIST OF REFERENCES Abbott, J. C., and L. M. Dill. 1989. The relative growth of dominant and subordinate

juvenile steelhead trout (Salmo gairdneri) fed equal rations. Behavior 108(2): 104-111.

Adams, B.L., W.S. Zaugg, and L.R. McLain. 1973. Temperature effect on parr-smolt

transformation in steelhead trout (Salmo gairdneri) as measured by gill sodium-potassium stimulated adenosine triphosphatase. Comp. Biochem. Physiol. 44A:1333-1339.

Adams, B.L., W.S. Zaugg, and L.R. McLain. 1975. Inhibition of salt water survival and Na-

K-ATPase elevation in steelhead trout (Salmo gairdneri) by moderate water temperatures. Transactions of the American Fisheries Society 104:766-769.

Alexander, G., R. Sweeting, and B. McKeown. 1998. The effect of thyroid hormone and

thyroid hormone blocker on visual pigment shifting in juvenile coho salmon (Oncorhynchus kisutch). Aquaculture 168(1-4): 157-168.

Anderson, R.O. and S.J. Gutreuter. 1983. Length, weight, and associated structural indices.

Pages 283-300 in L.A. Nielsen and D.L. Johnson, editors. Fisheries Techniques. American Fisheries Society, Bethesda, Maryland.

Ashton, H. J., D. O. Farkvam, and B. E. March. 1993. Fatty acid composition of lipids in the

eggs and alevins from wild and cultured chinook salmon (Oncorhynchus tshawytscha). Canadian Journal of Fisheries and Aquatic Sciences 50: 648-655.

Austin, B and J. G. Day. 1990. Inhibition of prawn pathogenic Vibrio spp. by a commercial

spray-dried preparation of Tetraselmis suecica. Aquaculture 90:389-392. Banks, J. L. 1992. Effects of density and loading on coho salmon during hatchery rearing and

after release. Progressive Fish Culturist 54: 137-147. Banks, J. L. 1994. Raceway density and water flow as factors affecting spring chinook

salmon (Oncorhynchus tshawytscha) during rearing and after release. Aquaculture 119: 201-217.

Barron, M. G. 1986. Endocrine control of smoltification in anadromous salmonids. Journal

of Endocinology 108: 313-319. Barton, B. A., C. B. Schreck, and L. Fowler. 1988. Fasting and diet content affect stress-

induced changes in plasma glucose and cortisol in juvenile chinook salmon. Progressive Fish-Culturist 50: 16-22.

123

Beckman, B. R., D. A. Larsen, B. Lee-Pawlak, and W. W. Dickhoff. 1998. Relation of fish size and growth rate to migration of spring chinook salmon smolts. North American Journal of Fisheries Management 18(3): 537-546.

Beeman, J. W., D. W. Rondorf, J. C. Faler, M. E. Free, and P. V. Haner. 1990. Assessment

of smolt condition for travel time analysis. Annual report 1989 (Contract DE-A179-87BP35245) to Bonneville Power Administration, Portland, Oregon.

Beeman, J. W., D. W. Rondorf, J. C. Faler, M. E. Free, P. V. Haner, S. T. Sauter, and D. A.

Venditti. 1991. Assessment of smolt condition for travel time analysis. Annual report 1990 (Contract DE-A179-87BP35245) to Bonneville Power Administration, Portland, Oregon.

Bergström, E. 1989. Effect of natural and artificial diets on seasonal changes in fatty acid

composition and total body lipid content of wild and hatchery-reared Atlantic salmon (Salmo salar L.) parr-smolt. Aquaculture 82: 205-217.

Bigelow, P. E. 1995. Migration to Lower Granite Dam of Dworshak National Fish Hatchery

Steelhead. Pages 42-58 in Interactions of hatchery and wild steelhead in the Clearwater River of Idaho. Fisheries Stewardship Project, 1994 Progress Report. U. S. Fish and Wildlife Service, Ahsahka, Idaho.

Bigelow, P. E. 1997. Emigration to Lower Granite Dam of Dworshak National Fish

Hatchery steelhead. Pages III-1 to III-22 in Interactions of hatchery and wild steelhead in the Clearwater River of Idaho. Fisheries Stewardship Project, 1995 Progress Report. U. S. Fish and Wildlife Service, Ahsahka, Idaho.

Bilton, H. T., D. F. Alderdice, and J. T. Schnute. 1982. Influence of time and size at release

of juvenile coho salmon (Oncorhynchus kisutch) on returns at maturity. Canadian Journal of Fisheries and Aquatic Sciences 39: 426-447.

Bilton, H. T., and G. L. Robins. 1973. The effects of starvation and subsequent feeding on

survival and growth of Fulton Channel sockeye salmon fry. Journal of the Fisheries Research Board of Canada 30(1): 1-5.

Bjornn, T. C., R. R. Ringe, and P. Hiebert. 1978. Seaward migration of Dworshak hatchery

steelhead trout in 1976. U. S. Fish and Wildlife Service, Idaho Cooperative Fishery Research Unit. Contract No. 14-16-0001-6314FH. University of Idaho, Moscow, Idaho.

Bjornn, T. C., R. R. Ringe, and P. Hiebert. 1979. Seaward migration of Dworshak hatchery

steelhead trout in 1977. U. S. Fish and Wildlife Service, Idaho Cooperative Fishery Research Unit. Contract No. 14-16-0008-2144. University of Idaho, Moscow, Idaho.

Blazer, V. S. 1992. Nutrition and disease resistance in fish. Annual Rev. of Fish Diseases.

pp 309-323.

124

Brown, M. 1946. The growth of brown trout (Salmo trutta L.) III. The effect of temperature

on the growth of two-year-old trout. Journal of Experimental Biology 22: 145-155. Cannamela, D. A. 1992. Potential impacts of releases of hatchery steelhead trout “smolts” on

wild and natural juvenile chinook and sockeye salmon. Idaho Department of Fish and Game, Boise, Idaho. 36 pp.

Cannamela, D. A. 1993. Hatchery steelhead smolt predation of wild and natural juvenile

chinook salmon fry in the upper Salmon River, Idaho. Idaho Department of Fish and Game, Boise, Idaho. 23 pp.

Cavinato, A. G., M. M. Clark, D. M. Mayes, and B. A. Rasco. 1996. Non-invasive

spectrophotometric method for determining fat content in live fish. 51st Northwest Regional Meeting of the American Chemical Society, Corvallis, Oregon.

Conover, W. J. 1971. Practical non-parametric statistics. John Wiley and Sons, New York,

New York. Conte, F. P., and H. H. Wagner. 1965. Development of osmotic and ionic regulation in

juvenile steelhead trout Salmo gairdneri. Comparative Biochemistry and Physiology 14: 603-620.

Cunniff, P. (ed.). 1997. Official Methods of Analysis of AOAC International, 16th Edition

(3rd Revision). American Organization of Analytical Chemists (AOAC) International, Gaithersburg, Maryland.

Dickhoff, W. W., B. R. Beckman, D. A. Larsen, C. Duan, and S. Moriyama. 1997. The role

of growth in endocrine regulation of salmon smoltification. Fish Physiology and Biochemistry 17: 231-236.

Dickhoff, W. W., B. R. Beckman, D. A. Larsen, C. V. W. Mahnken, C. B. Schreck, C. Sharpe,

and W. S. Zaugg. 1995. Quality assessment of hatchery-reared spring chinook salmon smolts in the Columbia River basin. American Fisheries Society Symposium 15: 292-302.

Dobson, S. H., and R. M. Holmes. 1984. Compensatory growth in the rainbow trout, Salmo

gairdneri Richardson. Journal of Fish Biology 25: 649-656. Duncan, P. L. and P. H. Klesius. 1996. Effects of feeding Spirulina on specific and

nonspecific immune responses of channel catfish. Journal of Aquatic Animal Health 8:308-313.

Evenson, M. D., and R. D. Ewing. 1993. Effect of exercise of juvenile winter steelhead on

adult returns to Cole Rivers Hatchery, Oregon. Progressive Fish-Culturist 55(3): 180-183.

125

Ewing, R. D., D. Barratt, and D. Garlock. 1994. Physiological changes related to migration

tendency in rainbow trout (Oncorhynchus mykiss). Aquaculture 121(1-3): 277-289. Ewing, R. D., M. D. Evenson, E. K. Birks, and A. R. Hemmingsen. 1984. Indices of parr-

smolt transformation in juvenile steelhead trout (Salmo gairdneri) undergoing volitional release at Cole Rivers Hatchery, Oregon. Aquaculture 40: 209-221.

Ewing, R. D. And S. K. Ewing. 1995. Review of the effects of rearing density on survival to

adulthood for Pacific salmon. The Progressive Fish-Culturist 57(1):1-25. Ewing, R. D., M. A. Lewis, J. E. Sheahan, and S. K. Ewing. 1998. Evaluation of inventory

procedures for hatchery fish. III. Nonrandom distributions of chinook salmon in raceways. Progressive Fish-Culturist 60(3): 159-166.

Ewing, R. D., J. E. Sheahan, M. A. Lewis, and A. N. Palmisano. 1998. Effects of rearing

density and raceway conformation on growth, food conversion, and survival of juvenile spring chinook salmon. Progressive Fish-Culturist 60: 167-178.

Ewing, R. D., T. R. Walters, M. A. Lewis, and J. E. Sheahan. 1994. Evaluation of inventory

procedures for hatchery fish. I. Estimating weights of fish in raceways and transport trucks. Progressive Fish-Culturist 56(3): 153-159.

Fängstam, H. 1993. Individual downstream swimming speed during the natural smolting

period among young of Baltic salmon (Salmo salar). Canadian Journal of Zoology 71: 1782-1786.

Fessler, J.L. and H.H. Wagner. 1969. Some morphological and biochemical changes in

steelhead trout during parr-smolt transformation. Journal of Fisheries Research Board of Canada 26:2823-2841.

Fjalestad K. T., T. Gjedrem, and B. Gjerde. 1993. Genetic improvement of disease resistance

in fish: an overview. Aquaculture 111: 65-74. Folmar, L. C., and W. W. Dickhoff. 1980. The parr-smolt transformation (smoltification) and

seawater adaptation in salmonids: a review of selected literature. Aquaculture 21: 1-37.

Folmar, L. C., and W. W. Dickhoff. 1981. Evaluation of some physiological parameters as

predictive indices of smoltification. Aquaculture 23: 309-324. Giorgi, A. E., T. W. Hillman, J. R. Stevenson, S. G. Hays, and C. M. Peven. 1997. Factors

that influence the downstream migration rates of juvenile salmon and steelhead through the hydroelectric system in the mid-Columbia River Basin. North American Journal of Fisheries Management 17: 268-282.

126

Goede, R. W. 1989. Fish health/condition assessment procedures. Utah Division of Wildlife Resources, Logan, Utah.

Haner, P. V., J. C. Faler, R. M. Schrock, D. W. Rondorf, and A. G. Maule. 1995. Skin reflectance as a nonlethal measure of smoltification for juvenile salmonids. North American Journal of Fisheries Management 15: 814-822.

Hansen, L. P., W. C. Clarke, R. L. Saunders, and J. E. Thorpe. 1989. Salmonid

Smoltification III. Proceedings of a workshop sponsored by the Directorate for Nature Management. Aquaculture 82(1-4):93-102.

Hoar, W. S. 1976. Smolt transformation: evolution, behavior, and physiology. Journal of the

Fisheries Research Board of Canada 33:1234-1252. Hoar, W. S. 1988. The physiology of smolting salmonids. Pages 275-343 in W. S. Hoar and

D. J. Randall (eds.), Fish Physiology Vol. XI, The physiology of developing fish, Part B. Academic Press, New York, New York.

Hvidsten, N. A., A. J. Jensen, H. Vivås, Ø. Bakke, and T. G. Heggberget. 1995. Downstream

migration of Atlantic salmon smolts in relation to water flow, water temperature, moon phase and social interaction. Nordic Journal of Freshwater Research 70: 38-48.

Hosmer, M. J., J. G. Stanley, and R. W. Hatch. 1979. Effects of hatchery procedures on later

return of Atlantic salmon to rivers in Maine. The Progressive Fish Culturist 41(3):115-119.

Iwata, M. 1995. Downstream migratory behavior of salmonids and its relationship with

cortisol and thyroid hormones: a review. Aquaculture 135: 131-139. Iwata, M., T. Yamanome, M.. Tagawa, H. Ida, and T. Hirano. 1989. Effects of thyroid

hormones on phototaxis of chum and coho salmon juveniles. Aquaculture 82:329-338. Johnson, C.E. and J.G. Eales. 1968. Influence of temperature and photoperiod on guanine

and hypoxanthine levels in skin and scales of Atlantic salmon (Salmo salar) at parr-smolt transformation. Journal of the Fisheries Research Board of Canada 25:1901-1909.

Johnsson, J. I., J. Blackburn, W. C. Clarke, and R. E. Withler. 1997. Does pre-smolt growth

rate in steelhead trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch) predict growth rate in seawater? Canadian Journal of Fisheries and Aquatic Sciences 54: 430-433.

Johnston, C. E., and R. L. Saunders. 1981. Parr-smolt transformation of yearling Atlantic

salmon (Salmo salar) at several rearing temperatures. Canadian Journal of Fisheries and 38: 1189-1198.

127

Jonsson, N. 1991. Influence of water flow, water temperature and light on fish migration in rivers. Nordic Journal of Freshwater Research 66: 20-35.

Kindschi, G. A. 1988. Effect of intermittent feeding on growth of rainbow trout, Salmo

gairdneri Richardson. Aquaculture and Fisheries Management 19: 213-215. Klontz, G. W., M. G. Maskill, and H. Kaiser. 1991. Effects of reduced continuous versus

intermittent feeding of steelhead. Progressive Fish-Culturist 53: 229-235. Lee, M. H., A. G. Cavinato, D. M. Mayes, and B. A. Rasco. 1992. Non-invasive SW-NIR

(short wavelength near infrared) spectrophotometric method to estimate the crude lipid content in the muscle of intact rainbow trout. Journal of Agricultural Food Chemistry 40: 2176-2181.

Love, R. M. 1974. The Chemical Biology of Fishes. Academic Press, New York, New York. Maule, A. G., J. W. Beeman, R. M. Schrock, and P. V. Haner. 1994. Assessment of smolt

condition for travel time analysis. Annual report 1991-1992 (Contract DE-A179-87BP35245) to Bonneville Power Administration, Portland, Oregon.

Maynard, D. J., T. A. Flagg, and C. V. W. Mahnken. 1995. A review of seminatural culture

strategies for enhancing the postrelease survival of anadromous salmonids. American Fisheries Society Symposium 15: 307-314.

Muir, W. D., W. S. Zaugg, A. E. Giorgi, and S. McCutcheon. 1994. Accelerating smolt

development and downstream movement in yearling chinook salmon with advanced photoperiod and increased temperature. Aquaculture 123: 387-399.

Mulero, V., M. A. Estaban, J. Muñoz, and J. Meseguer. 1998. Dietary intake of levamisole

enhances the immune response and disease resistance of the marine teleost gilthead seabream (Sparus aurata L.). Fish & Shellfish Immunology 8: 49-62.

National Research Council. 1981. Nutrient requirements of coldwater fishes. National

Academy Press, Washington, D. C. 63 pp. NMFS (National Marine Fisheries Service). 1995. Proposed recovery plan for Snake river

salmon. U.S., Department of commerce. National Oceanic and Atmospheric Administration.

NMFS (National Marine Fisheries Service). 1999. Biological opinion for hatchery

operations in the Columbia River Basin. U.S. Department of Commerce, National Marine Fisheries Service, Silver Spring, Maryland.

128

Ogata, H. and T. Murai. 1989. Effects of dietary fatty acid composition on growth and smolting of underyearling Masu salmon, Oncorhynchus masou. Aquaculture 82: 181-189.

Partridge, F. E. 1985. Effects of steelhead smolt size on residualism and adult return rates.

U.S. Fish and Wildlife Service, Lower Snake River Compensation Plan. Contract No. 14-16-001-83605. Idaho Department of Fish and Game, Boise, Idaho.

Partridge, F. E. 1986. Effects of steelhead smolt size on residualism and adult return rates. U.S. Fish and Wildlife Service, Lower Snake River Compensation Plan. Contract No. 14-16-001-83605. Idaho Department of Fish and Game, Boise, Idaho.

Pauley, G., B. Bortz, and M. Shepard. 1986. Species profiles: Life histories and

environmental requirements of coastal fishes and invertebrates (Pacific Northwest)–Steelhead Trout. Biological Report 82(11.62), National Wetlands Research Center, Fish and Wildlife Service, U. S. Dept. of Interior, Washington, DC.

Peven, C. M., R. R. Whitney, and K. R. Williams. 1994. Age and length of steelhead smolts

from the mid-Columbia River basin, Washington. North American Journal of Fisheries Management 14: 77-86.

Pirhonen, J. and L. Forsman. 1998. Relationship between Na+,K+-ATPase activity and

migration behaviour of brown trout and sea trout (Salmo trutta L.) during the smolting period. Aquaculture 168:41-47.

Rondorf, D.W., J.W. Beeman, J.C. Faler, M.E. Free, and R.J. Wagner. 1989. Assessment of

smolt condition for travel time analysis. Annual report 1988. Prepared by U.S. Fish and Wildlife Service for Bonneville Power Administration, Portland, Oregon.

Rowley, A. F., J. Knight, P. Lloyd-Evans, J. W. Holland, and P. J. Vickers. 1995. Eicosanoids

and their role in immune modulation in fishBa brief overview. Fish & Shellfish Immunology 5: 549-567.

Sargent, J., G. Bell, L. McEvoy, D. Tocher, and A. Estevez. 1999. Recent developments in

the essential fatty acid nutrition of fish. Aquaculture 177: 191-199. Schrock, R. M., J. W. Beeman, D. W. Rondorf, and P. V. Haner. 1994. A microassay for gill

sodium, potassium-activated ATPase in juvenile Pacific salmonids. Transactions of the American Fisheries Society 123: 223-229.

Schrock, R. M., P. V. Haner, K. M. Hans, J. W. Beeman, S. P. VanderKooi, J. D. Hotchkiss,

P. A. Petrusso, S. G. Smith, and A. G. Maule. 1999. Assessment of smolt condition for travel time analysis. Annual report 1993-1994 (Contract DE-A179-87BP35245) to Bonneville Power Administration, Portland, Oregon. Internet publication at http://www.efw.bpa.gov/Environment/EW/EWP/DOCS/REPORTS/DOWNSTRM/ withpdf.htm.

129

Sigholt, T., T. Åsgård, and M. Staurnes. 1998. Timing of parr-smolt tranformation in Atlantic salmon (Salmo salar): effects of changes in temperature and photoperiod. Aquaculture 160: 129-144.

Shaw, H.M., R.L. Saunders, H.C. Hall, and E. B. Henderson. 1975. Effect of dietary sodium

chloride on growth of Atlantic salmon (Salmo salar). Journal of Fisheries Research Board of Canada 32:1813-1819.

Sheridan, M. A. 1989. Alterations in lipid metabolism accompanying smoltification and

seawater adaptation of salmonid fish. Aquaculture 82:191-203 Sheridan, M. A., W. V. Allen, and T. H. Kerstetter. 1983. Seasonal variations in the lipid

composition of the steelhead trout, Salmo gairdneri Richardson, associated with the parr-smolt transformation. Journal of Fish Biology 23(2): 125-134.

Sheridan, M. A., W. V. Allen, and T. H. Kerstetter. 1985. Changes in the fatty acid

composition of steelhead trout, Salmo gairdneri Richardson, associated with parr-smolt transformation. Comparative Biochemistry and Physiology 80B(4): 671-676.

Shrimpton, J. M., N. J. Bernier, G. K. Iwama, and D. J. Randall. 1994. Differences in

measurements of smolt development between wild and hatchery-reared juvenile coho salmon (Oncorhynchus kisutch) before and after saltwater exposure. Canadian Journal of Fisheries and Aquatic Sciences 51(10): 2170-2178,

Smith, R. R. 1987. Methods of controlling growth of steelhead. Progressive Fish-Culturist

49(4): 248-252. Solbakken, V. A., T. Hansen, and S. O. Stefansson. 1994. Effects of photoperiod and

temperature on growth and parr-smolt transformation in Atlantic salmon (Salmo salar L.) and subsequent performance in seawater. Aquaculture 121: 13-27.

Sokal and Rohlf 1981. Biometry: the Principals and Practices of Statistic in Biological

Research (second edition). W.H. Freeman and Company, New York. Symons, P.E. 1970. The possible role of social and territorial behavior of Atlantic salmon

parr in the production of smolts. Technical Report No. 2. Fisheries Research Board of Canada. 25p.

Thompson, K. D., M. F. Tatner, and R. J. Henderson. 1996. Effects of dietary (n-3) and (n-6)

polyunsaturated fatty acid ratio on the immune response of Atlantic salmon, Salmo salar L. Aquaculture Nutrition 2: 21-31

Thorpe, J.E., N.B. Metcalfe, and F.A. Huntingford. 1992. Behavioural influences on life-

history variation in juvenile Atlantic salmon, Salmo salar. Environmental Biology of Fishes 33:331-340.

130

Tipping, J. M., and J. B. Byrne. 1996. Reducing feed levels during the last month of rearing enhances emigration rates of hatchery-reared steelhead smolts. Progressive Fish-Culturist 58:128-130.

Tipping, J. M., R. V. Cooper, J. B. Byrne, and T. H. Johnson. 1995. Length and condition

factor of migrating and nonmigrating hatchery-reared winter steelhead smolts. Progressive Fish-Culturist 57: 120-123.

Viola, A. E., and M. L. Schuck. 1995. A method to reduce the abundance of residual

hatchery steelhead in rivers. North American Journal of Fisheries Management 15(2): 488-493.

Wagner, H. H. 1974a. Seawater adaptation independent of photoperiod in steelhead trout

(Salmo gairdneri). Canadian Journal of Zoology 52: 805-812. Wagner, H. H. 1974b. Photoperiod and temperature regulation of smolting in steelhead trout

(Salmo gairdneri.) Canadian Journal of Zoology 52:219-234. Wedemeyer, G.A. 1996. Physiology of Fish in Intensive Culture Systems. Chapman and

Hall, New York, New York. Wedemeyer, G. A., R. L. Saunders, and W. C. Clarke. 1980. Environmental factors affecting

smoltification and early marine survival of anadromous salmonids. Marine Fisheries Review 42: 1-4.

Westgate, J.W. , T.B. McKee, D.L. Crawford, and D.K. Law. 1976. Nutritional effects of

using salty herring meal in Oregon Moist Pellets. Progressive Fish Culturist 38:118-119.

Whitesel, T. A. 1991. Performance of juvenile Atlantic salmon (Salmo salar) introduced into a stream: smolt development and thyroid hormones. Dissertation Abstracts International, Part B: Science and Engineering 51(11): 156 pp.

Wilkinson, L. 1990. SYSTAT: the system for statistics. Evanston, Illinois. SYSTAT, Inc.,

1990. Wurtsbaugh, W. A., and G. E. Davis. 1977. Effects of fish size and ration level on the

growth and food conversion efficiency of rainbow trout, Salmo gairdneri Richardson. Journal of Fish Biology 11(2): 99-104.

Zaporozhec, O. M., and G. V. Zaporozhec. 1993. Preparation of hatchery-reared chum fry

for life at sea: osmoregulatory dynamics. Fisheries Oceanography 2(2): 91-96. Zaugg, W. S. 1981. Advanced photoperiod and water temperature effects on Na+, K+-

adenosine triphosphatase activity and migration of juvenile steelhead (Salmo gairdneri). Can. J. Fish. Aquat. Sci. 38: 758-764.

131

Zaugg, W. S. 1982. Some changes in smoltification and seawater adaptability of salmonids resulting from environmental and other factors. Aquaculture 28: 143-151.

Zaugg, W. S., B. L. Adams, and L. R. McLain. 1972. Steelhead migration: potential

temperature effects as indicated by gill adenosine triphosphatase activities. Science 176: 415-416.

Zaugg, W. S., J. E. Bodle, J. E. Manning, and E. Wold. 1986. Smolt transforation and

seaward migration in 0-age progeny of adult chinook salmon (Oncorhynchus tshawytscha) matured early with photoperiod control. Can. J. Fish. Aquat. Sci. 43: 885-888.

Zaugg, W.S. and L.R. McLain. 1969. Inorganic salt effects on growth, salt water adaption,

and gill ATPase of Pacific salmonids. Pages 293-305. In Neuhaus and Halver (eds) Fish in Research. Academic Press, New York.

Zaugg, W. S., and H. H. Wagner. 1973. Gill ATPase activity related to parr-smolt

transformation and migration in steelhead trout (Salmo gairdneri): influence of photoperiod and temperature. Comparative Biochemistry and Physiology 45B: 955-965.

Zaugg, W.S., D.D. Roley, E.F. Prentice, K.X. Gores, and F.W. Waknitz. 1983. Increased

seawater survival and contribution to the fishery of chinook salmon (Oncorhynchus tshawytscha) by supplemental dietary salt. Aquaculture 32:183-188.

Zaugg, W.S., W.W. Dickhoff, B.R. Beckman, C.V.W. Mahnken, G.A. Winans, T.W.

Newcomb, C.B. Schrock, A.N. Palmisano, R.M.Schrock, G.A. Wedemeyer, R.D. Ewing, and C.W. Hopley. 1991. Smolt quality assessment of spring chinook salmon. Annual Report prepared for Bonneville Power Administration, Portland, Oregon.