Growth Rate Modulation in Spring ChinookSalmon Supplementation

Annual Report 2004 - 2005 April 2006 DOE/BP-00017450-2

This Document should be cited as follows:

Larsen, Donald, Brian Beckman, Charles Strom, Paul Parkins, Kathleen Cooper, Mark Johnston,David Fast, Walton Dickhoff, "Growth Rate Modulation in Spring Chinook SalmonSupplementation", 2004-2005 Annual Report, Project No. 200203100 (et al.), 73 electronicpages, (BPA Report DOE/BP-00017450-2)

Bonneville Power AdministrationP.O. Box 3621Portland, OR 97208

This report was funded by the Bonneville Power Administration (BPA),U.S. Department of Energy, as part of BPA's program to protect, mitigate,and enhance fish and wildlife affected by the development and operationof hydroelectric facilities on the Columbia River and its tributaries. Theviews in this report are the author's and do not necessarily represent theviews of BPA.

Growth Rate Modulation in Spring Chinook Salmon Supplementation

Annual Report 2005

Project Number 2002-031-00

April 2005

Donald A. Larsen1, Brian R. Beckman1, Charles Strom2, Paul Parkins3, Kathleen A. Cooper3, Mark Johnston4, David E. Fast4 and Walton W. Dickhoff1, 3

1Integrative Fish Biology Progra, Northwest Fisheries Science Center

National Marine Fisheries Service 2725 Montlake Blvd. E. Seattle, Washington 98112, USA

2 Cle Elum Supplementation and Research Facility, Yakama Nation

800 Spring Chinook Way Cle Elum, Washington 98922, USA

3School of Aquatic and Fisheries Science, University of Washington,

Seattle, Washington 98195, USA

4Yakama Nation, Nelson Springs Research Center, 771 Pence Road, Yakima, Washington 98902, USA

Disclaimer: Use of trade names does not imply endorsement by the National Marine Fisheries Service, NOAA.

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SUMMARY

Recommendations of the Columbia River Basin Fish & Wildlife Program (Nov. 14, 2000) for artificial production state: "naturally selected populations should provide the model for successful artificially reared populations, in regard to population structure, mating protocol, behavior, growth, morphology, nutrient cycling, and other biological characteristics." This mirrors guidelines of the NMFS 2000 FCRPS Biological Opinion (9.6.5.3.4, RPA 184). We compared the physiology and development of naturally rearing wild and hatchery-reared spring Chinook salmon in the Yakima River Basin, and found substantial differences. The most serious difference was an approximate 50% incidence of early maturation of Cle Elum Hatchery-reared males (1+ year old minijacks) (Larsen et al. 2004a). This is 2-10 times (depending on year) our estimate of early male maturation in wild spring Chinook salmon in the Yakima River. Apparently, hatchery rearing practices appear to promote early male maturation. Hundreds of thousands of the early maturing hatchery males may residualize in the basin after release and cause negative genetic and ecological impacts. The ecological concerns include competition for space and food, food depletion and predation on emerging salmonids and other species. Furthermore, early male maturation translates into a 25% reduction in anadromous adult production. We have found recently in laboratory studies that modulation of growth rate and/or body energy stores at specific times of the year can reduce the incidence of precocious maturation. Thus, growth rate modulation at the Cle Elum Supplementation Research Facility may reduce early male maturation to levels similar to natural wild fish. This project has four central objectives: 1) estimate the incidence of precocious maturation and characterize the related maturational physiology in wild Yakima spring Chinook salmon for comparison to the hatchery fish, 2) monitor the incidence of yearling precocious maturation in the hatchery population, and 3) conduct a series of growth modulation experiments to control precocious maturation in the Cle Elum hatchery population, 4) collaborate with tribal and state biologists in developing and implementing production scale growth modulation studies designed to reduce precocious male maturation while producing a successful smolt. Our ultimate goal is to develop rearing protocols to produce fish with morphological, physiological, and life-history attributes similar to naturally reared cohorts.

Results, to date, are as follows: Precocious male maturation rates of wild Yakima Spring Chinook salmon sampled at Roza dam in mid-winter 2005 (BY 2003) were 6%. However, validation tests conducted this year indicated that this and previous mid-winter estimates of precocious male maturation rates based on GSI are relatively inaccurate, but these collections do provide useful information on gender ratios of wild fish. In 2005 the female:male ratio of 300 wild fish (BY 2003) sampled at Roza Dam in January was 56:44. In brood year 2003 production fish were grown for a second year on either a High or Low Growth treatment and the precocious male maturation rate was 27% and 13% in these two groups, respectively. Furthermore, physiological comparisons between the two treatments showed that the Low growth fish were smaller, as expected, but did not differ from the High Growth treatment with regard to smolt development. Relative comparisons of precocious male maturation rates between wild and hatchery fish were obtained from collections of migrating smolts (and minijacks) at Prosser Dam in the spring. Minijack rates in 2005 (brood year 2003) were 1.6% wild, 10.7% Low Growth

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treatment, and 20% High Growth treatment. Gender ratios among wild fish favored females on 1 of 2 sampling dates, but were not significantly different from 50:50 for both the Low and High growth hatchery treatments. A second laboratory scale growth modulation experiment investigating the combined effect of alterations in fry pond timing and dietary lipid content on age-1 and age-2 precocious male maturation rates was successfully completed in 2005. Age-2 maturation rates in all treatments ranged from 12-18% of males and were not significantly different. Furthermore, with the exception of size differences during the early stages of the study, the treatments did no differ with regard to condition factor, plasma insulin-like growth factor-I (a growth regulating hormone), and gill Na+/K+-ATPase activity. However, earlier pond timing produced 1-4% age-1 precociously maturing males. Thus, future investigations will be required to determine what effects planned production scale alterations in pond timing at CESRF (to increase smolt release size) will have on both age-1 and age-2 precocious male maturation rates.

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TABLE OF CONTENTS Summary…………………………………………………………………………………..ii Table of Contents…………………………………………………………………………iv List of Figures…………………………………………………………………….….……v List of Tables……………………………………………………………………..…..…...x Introduction………………………………………………………………………………..1 Objective 1………………………………………………………………………………...2 Objective 2……………………………………………………………………………….12 Objective 3…………………………………………..…………………………….……..18 Objective 4……………………………………………………………………………….49 Acknowledgements………………………………………………………………...…….56 Literature Cited…………………………………………………………………………..57

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LIST OF FIGURES Figure 1. Frequency distributions of Log10 plasma 11-Ketotestosternone (11-KT) and gonadosomatic index (GSI) from Cle Elum Hatchery, CESRF spring Chinook salmon sampled in January and February 2005. Minijacks were designated by 11-KT using a threshold of 0.8 (ng/ml) according to the method of Larsen et al (2004a) and GSI using a threshold of 0.1%……………………….…………………………………….....3 Figure 2. Regression relationship between Log10 gonadosomatic index (GSI) and plasma 11-Ketotestosterone (11-KT) levels in male Cle Elum Hatchery (CESRF) spring Chinook salmon sampled in January (a) and February (b) 2005. Minijacks were designated by 11-KT using a threshold of 0.8 (ng/ml) according to the method of Larsen et al (2004a) and GSI using a threshold of 0.1%…………………..…………………...…4 Figure 3. Regression relationship between Log10 gonadosomatic index (GSI) and plasma 11-Ketotestosterone (11-KT) levels in male Cle Elum Hatchery (CESRF) spring Chinook salmon sampled on 7 March 2005. Minijacks were designated by 11-KT using a threshold of 0.8 (ng/ml) according to the method of Larsen et al (2004a) and GSI using a threshold of 0.1%. Precocious male maturation rates were 37% of males using 11-KT and 31% using GSI………………………………………………………………..………5 Figure. 4 Frequency distribution of log10transformed gonadasomatic index (GSI) of wild male fish collected at Roza Dam on the Yakima River on 26 January 2005. Male fish were classified as minijacks if their GSI exceeded 0.1% (log -1.0) according to a modification of the method of Larsen et al. (2004a). The total number of male and female fish sampled (N) and female:male ratios are presented………………………..… 6 Figure. 5 Frequency distribution of log10transformed gonadasomatic index (GSI) of male wild fish collected at Prosser Dam on the Yakima River on 22 April and 5 May 2003. Male fish were classified as minijacks if their GSI exceeded 0.1 (log -1.) according to a modification of the method of Larsen et al. (2004a). The total number of male and female fish sampled (N) and female:male ratios are presented for each date………….....9 Figure. 6 Frequency distribution of log10transformed gonadasomatic index (GSI) of male hatchery fish collected at Prosser Dam on the Yakima River on 22 April and 5 May 2003. Male fish were classified as minijacks if their GSI exceeded 0.1 (log -1.) according to a modification of the method of Larsen et al. (2004a). The total number of male and female fish sampled (N) and female:male ratios are presented for each date………..….10 Figure. 7 Frequency distribution of log10transformed plasma 11-ketotestosterone (11-KT) levels of male High Growth and Low growth treated hatchery spring Chinook salmon collected at the Clark Flat Acclimation site on the Yakima River in March 2005. Male fish were classified as minijacks if their plasma 11-KT level exceeded 0.8 ng/ml (Log -0.09) according to the method of Larsen et al. (2004a). The total number of male and female fish sampled on each date (N) and female:male ratios are presented for each acclimation site……………………………………………………………………….….14

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Figure. 8 Frequency distribution of log10transformed plasma 11-ketotestosterone (11-KT) levels of male High Growth and Low growth treated hatchery spring Chinook salmon collected at the Easton Acclimation site on the Yakima River in March 2005. Male fish were classified as minijacks if their plasma 11-KT level exceeded 0.8 ng/ml (Log -0.09) according to the method of Larsen et al. (2004a). The total number of male and female fish sampled on each date (N) and female:male ratios are presented……….15 Figure. 9 Frequency distribution of log10transformed plasma 11-ketotestosterone (11-KT) levels of male High Growth and Low Growth treated hatchery spring Chinook salmon collected at the Jack Creek Acclimation site on the Yakima River in March 2005. Male fish were classified as minijacks if their plasma 11-KT level exceeded 0.8 ng/ml (Log -0.09) according to the method of Larsen et al. (2004a). The total number of male and female fish sampled on each date (N) and female:male ratios are presented……….16 Figure 10. Weight (g) of treatment groups in growth modulation experiment examining early and late ponding date and high and low fat diet: (Early/High, Early/Low, Late/High, and Late/Low). Magnification of data February to July is included to aid in viewing early treatment differences. Dashed lines fit by eye…………………………...22 Figure 11. Weight (g) of treatment groups in growth modulation experiment examining early and late ponding date and high and low fat diet: (Early/High, Early/Low, Late/High, and Late/Low) and Cle Elum hatchery Production fish reared under High and Low Growth treatments (Prod./ High and Prod./Low). Magnification of data from February to July is included to aid in viewing early treatment differences. Dashed lines fit by eye.………...………………………………………………………………………23 Figure 12. Fork length (mm) of treatment groups in growth modulation experiment examining early and late ponding date and high and low fat diet: (Early/High, Early/Low, Late/High, and Late/Low). Dashed lines fit by eye………………………………...…...24 Figure 13. Fork length (mm) of treatment groups in growth modulation experiment examining early and late ponding date and high and low fat diet: (Early/High, Early/Low, Late/High, and Late/Low) and Cle Elum hatchery Production fish reared under High and Low Growth treatments (Prod./ High and Prod./Low). Dashed lines fit by eye………...25 Figure 14. Condition Factor (%) of treatment groups in growth modulation experiment examining early and late ponding date and high and low fat diet: (Early/High, Early/Low, Late/High, and Late/Low)…………………………………………………...26 Figure 15. Condition factor (%) of treatment groups in growth modulation experiment examining early and late ponding date and high and low fat diet: (Early/High, Early/Low, Late/High, and Late/Low) and Cle Elum hatchery Production fish reared under High and Low Growth treatments (Prod./ High and Prod./Low)……………...….27

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Figure 16. Plasma IGF-I (ng/ml) of treatment groups in growth modulation experiment examining early and late ponding date and high and low fat diet: (Early/High, Early/Low, Late/High, and Late/Low)……………………………………………..…….28 Figure 17. Plasma IGF-I (ng/ml) of treatment groups in growth modulation experiment examining early and late ponding date and high and low fat diet: (Early/High, Early/Low, Late/High, and Late/Low) and Cle Elum hatchery Production fish reared under High and Low Growth treatments (Prod./ High and Prod./Low)…………………29 Figure 18. Gill Na+/K+-ATPase activity (μmoles PO4 / mg protein x hr) of treatment groups in growth modulation experiment examining early and late ponding date and high and low fat diet: (Early/High, Early/Low, Late/High, and Late/Low)……….………….30 Figure 19. Gill Na+/K+-ATPase activity (μmoles PO4 / mg protein x hr) of treatment groups in growth modulation experiment examining early and late ponding date and high and low fat diet: (Early/High, Early/Low, Late/High, and Late/Low) and Cle Elum hatchery Production fish reared under High and Low Growth treatments (Prod./ High and Prod./Low)……………………………………………………………………………….31 Figure 20. Frequency distribution of log10-GSI of male Yakima spring Chinook salmon reared under (Early/High, Early/Low, Late/High, Late/Low) experimental growth treatments. Fish were sampled on 6 June, 2005. Percentages of precociously mature males (both age-1 and age-2) are designated for each treatment………………….……..35 Figure 21. Comparison of frequency distribution of log10GSI for immature and age-2 (left graph) and immature and age-1 (right graph) precociously mature males from Yakima spring Chinook salmon reared in the growth modulation experiment. Fish were sampled on 6 June 2005. Arrow designates the 0.1% GSI threshold below and above which fish were designated as immature or precociously mature, respectively. Note1, Immature males are the same fish plotted on each graph. Note 2, to aid in viewing, the y-axis for the right plot is on a log10 scale……………………………………...…………..36 Figure 22. Percentages of immature, and age-1 and age-2 precociously mature males among all males in the Early/High, Early/Low, Late/High, Late/Low) treatment groups from Cle Elum Hatchery spring Chinook salmon growth modulation experiment (averaged from 4 replicate tanks per treatment). Percentages ± SE are presented above each category………………………………………………………………...…………..37 Figure 23. Percentages of female, immature, and age-1 and age-2 precociously mature males among all fish in the Early/High, Early/Low, Late/High, Late/Low treatment groups from Cle Elum Hatchery spring Chinook growth modulation experiment (averaged from 4 replicate tanks per treatment). Percentages ± SE are presented above each category……………………………………………………………………….……38

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Figure 24. Scatter-plot of plasma 11-ketotestosterone (11-KT) levels in al male spring Chinook salmon sampled in the growth modulation experiment from January to June 2005 (open circles. 11-KT levels (mean±se) for maturing and immature males were determined by designating fish with levels above and below 0.8 ng/ml as maturing or immature, respectively according to the method of Larsen et al. (2004a). The dashed line indicates the 0.8ng/ml….…………………………………………………………...……42 Figure 25. Plasma 11-ketotestosterone levels from all immature, age-1 and age-2 precociously maturing spring Chinook salmon from the growth modulation experiment sampled from January to May, 2005. Different letters denote significant differences between male maturation types………………...……………………………….………..43 Figure 26. Weight of immature and precociously maturing male spring Chinook salmon from the growth modulation experiment in spring. Precociously maturing males were designated based on having plasma 11-KT levels above 0.8 ng/ml according to the method of Larsen et al. (2004a). Asterisks indicate significant differences between immature and maturing fish. Significance level = 0.05……………………...….……44 Figure 27. Fork length of immature and precociously maturing male spring Chinook salmon from the growth modulation experiment in spring. Precociously maturing males were designated based on having plasma 11-KT levels above 0.8 ng/ml according to the method of Larsen et al. (2004a). Asterisks indicate significant differences between immature and maturing fish. Significance level = 0.05………………...…………….45 Figure 28. Plasma IGF-I of immature and precociously maturing male spring Chinook salmon from the growth modulation experiment in spring. Precociously maturing males were designated based on having plasma 11-KT levels above 0.8 ng/ml according to the method of Larsen et al. (2004a). Asterisks indicate significant differences between immature and maturing fish. Significance level = 0.05……...…46 Figure 29. Condition factor of immature male and precociously maturing male spring Chinook salmon from the growth modulation experiment in spring. Precociously maturing males were designated based on having plasma 11-KT levels above 0.8 ng/ml according to the method of Larsen et al. (2004a). Asterisks indicate significant differences between immature and maturing fish. Significance level = 0.05…….…..47 Figure 30. Gill Na+/K+-ATPase activity of immature male and precociously maturing male spring Chinook salmon from the growth modulation experiment in spring. Precociously maturing males were designated based on having plasma 11-KT levels above 0.8 ng/ml according to the method of Larsen et al. (2004a). Asterisks indicate significant differences between immature and maturing fish. Significance level

= 0.05……………………………………………………………………………….…48

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Figure 31. Weight of Conventional (High) growth and Modified (Low) growth CESRF spring Chinook salmon from production scale growth modulation experiment. To aid in viewing insert shows magnification of early growth period. Asterisk indicates significant differences between treatments at a given date. Dashed line fit by eye. Significance level =0.05………………….……………………………….………….50 Figure 32. Fork length of Conventional (High) growth and Modified (Low) growth CESRF spring Chinook salmon from production scale growth modulation experiment. Dashed line fit by eye. Significance level =0.05…………………………....……….51 Figure 33. Plasma IGF-I levels of Conventional (High) growth and Modified (Low) growth CESRF spring Chinook salmon from production scale growth modulation experiment. Asterisk indicates significant differences between treatments at a given date. Significance level =0.05…………………………………………………….………..52 Figure 34. Condition factor of Conventional (High) growth and Modified (Low) growth CESRF spring Chinook salmon from production scale growth modulation experiment. Asterisk indicates significant differences between treatments at a given date. Significance level =0.05…………………………………………………..………….53 Figure 35. Gill Na+/K+-ATPase activity of Conventional (High) growth and Modified (Low) growth CESRF spring Chinook salmon from production scale growth modulation experiment. Significance level =0.05………………………………………..………54 Figure 36. Plasma thyroxine (T4) levels of Conventional (High) growth and Modified (Low) growth CESRF spring Chinook salmon from production scale growth modulation experiment. Significance level =0.05………………………………..………….…...55

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LIST OF TABLES

Table 1. Fork length, weight, total number of fish sampled (N), number of females and males, female:male ratio, X2 value for F:M ratio comparison to a 50:50 ratio, and percent of all males categorized by GSI as precociously maturing among wild Yakima Spring Chinook salmon captured at Roza Dam during the mid-winter re-distribution 2003-2005. NS = not significantly different from a 50:50 gender ratio…………….……6 Table 2. Fork length, weight, total number of fish sampled (N), number of females and males, female:male ratio, X2 value for F:M ratio comparison to a 50:50 ratio, and percent of all males categorized by GSI as precociously maturing among hatchery (Conventional and Modified) and Wild Yakima Spring Chinook salmon captured at Prosser Dam during the spring smolt migration 2003-2005. NS = not significantly different from a 50:50 gender ratio…………………………………………………..……8 Table 3. Fork length, weight, total number of fish sampled (N), number of females (F) and males (M), female:male ratio, X2 value for F:M ratio comparison to a 50:50 ratio, and percent of all males categorized by plasma 11-KT levels as precociously maturing Cle Elum Hatchery spring Chinook salmon sampled prior to volitional release form Clark Flat, Easton, and Jack Creek acclimation sites in 2003- 2005 (brood years 2001 - 2003). Brood year 2002 and 2003 were reared under either Low or High Growth treatments to control precocious male maturation rates. NS = not significantly different from a 50:50 gender ratio…………………………………………………………..…………………..17 Table 4. Final demographic data (fork length, weight, # males and females, female:male ratio and precocious male maturation rates) from fish sampled on the final sampling date of 6 July, 2005 from the growth modulation experiment examining the effect of early and late fry ponding date and high and low lipid diet on age-1 and age-2 male maturation in Cle Elum Hatchery spring Chinook salmon………...………………32 Table 5 series. ANOVA, mean, and multiple range test tables for mean weights of fish from treatments (Early/High, Early/Low, Late/High, Late/Low) in the growth modulation experiment sampled at the end of the experiment on 6 June 2005. Significance level = 0.05…………...…………………...……………………………..33 Table 6 series. ANOVA, mean, and multiple range test tables for mean fork lengths of fish from treatments (Early/High, Early/Low, Late/High, Late/Low) in growth modulation experiment sampled at the end of the experiment on 6 June 2005. Significance level = 0.05 indicated by S on third table…………………..……………34 Table 7 series. ANOVA, mean, and multiple range test tables for arcsine transformed proportion of minijacks among males for all treatments (Early/High, Early/Low, Late/High, Late/Low) in growth modulation experiment. N = 4 replicate tanks per treatment. Significance level = 0.05………………………………………..40

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Table 8 series. ANOVA, mean, and multiple range test tables for arcsine transformed proportion of precocious parr among males for all treatments (Early/High, Early/Low, Late/High, Late/Low) in growth modulation experiment. N = 4 replicate tanks per treatment. Significance level = 0.05………………..………………………41

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INTRODUCTION

Precocious male maturation has been a topic of numerous basic and applied studies in Atlantic salmon (Myers 1984, Myers et al. 1986; Thorpe 1986; 1987; 1991; Hutching and Jones 1998) where rates are reported to range from 0 to 100% depending on the stock (Hutching and Jones 1998; Fleming 1998). In Chinook salmon, this subject has received far less attention. Both sub-yearling and yearling precocious males exist in some spring Chinook salmon populations (Robertson 1957; Gebhards 1960; Taylor 1989; Foote et al. 1991; Mullan et al. 1992; Clarke and Blackburn; 1994; Pearsons et al. 2004). The incidence of precocious maturation in wild stocks of spring Chinook salmon is poorly characterized, but thought to be less than 5% (Gebhards 1960; Mullan et al. 1992). In contrast, the proportion of males maturing at two years of age in some hatchery and experimental populations of spring Chinook salmon ranges from 11 to 80% (Foote et al. 1991; Mullan et al. 1992; Clarke and Blackburn 1994).

Age of maturation in salmon is influenced by genetic, biotic, and abiotic factors (Power 1986) including energy stores (whole body lipid), size and/or growth rate at specific times of year (Rowe et al. 1991; Hopkins and Unwin 1997; Silverstein et al. 1998; Shearer and Swanson 2000; Shearer et al. in press; Campbell et al. 2003). Studies in spring Chinook salmon have shown that male maturation is physiologically initiated in the fall, approximately 10 months prior to autumn spawning (Silverstein et al. 1998; Shearer and Swanson, 2000; Shearer et al. in press). External characteristics of maturation (olive pigmentation, deep body shape, and dark fin margins) as well as very large white gonads typical of the later stages of maturation may not be evident until mid-summer prior to autumn spawning. These obvious external signs of early maturity are often not seen in the hatchery since most spring Chinook hatcheries release fish in March and April. This fact may explain, in part, why this issue has historically received only modest attention.

This project established that approximately 40-50% of the male spring Chinook salmon produced by the Cle Elum Supplementation and Research Facility mature precociously at two years of age. High rates of precocious maturation are undesirable in a supplementation program because they may have potentially adverse genetic, ecological and demographic effects and can result in a reduction in the number of returning anadromous adult fish for harvest and broodstock (Larsen et al. 2004a). This report addresses four central objectives: 1) estimate the incidence of precocious maturation in wild Yakima spring Chinook for comparison to the hatchery fish, 2) monitor the incidence of yearling precocious maturation in the hatchery population, 3) conduct a series of growth modulation experiments to control precocious maturation in the Cle Elum hatchery population, and 4) collaborate with tribal and state biologists in developing and implementing production scale growth modulation studies designed to reduce precocious male maturation while producing a successful smolt. Our ultimate goal is to develop rearing protocols to produce fish with morphological, physiological, and life-history attributes similar to naturally reared cohorts.

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OBJECTIVE 1: ESTIMATE INCIDENCE OF PRECOCIOUS MALE MATURATION IN WILD YAKIMA RIVER SPRING CHINOOK SALMON - 2005. The purpose of this objective was to assess the incidence of precocious maturation of naturally rearing Yakima River spring Chinook salmon in 2005 (brood year 2003). Wild fish were collected from the Roza Dam smolt trap during their mid-winter redistribution migration in January, and wild and hatchery fish were collected at the Chandler smolt bypass facility at Prosser Dam during smolt out migration in the spring (April and May). Validation of GSI in Mid-Winter to Screen for Precocious Male Maturation The most accurate and least expensive method for determining precocious male development in migrating fish is gonadosomatic index (GSI) [GSI; (gonad weight / body weight) x 100)] rather than measurement of the sex steroid 11-ketotestosterone (which is used to determine maturation in fish at the hatchery prior to release-see Objective 2 and see Larsen et al. 2004a). In Larsen et al. (2004a) log11-KT and log GSI were relatively interchangeable in mid-March for estimating which fish were immature male parr and which were precociously maturing minijacks. However, in all males, both log11-KT and log GSI values initially cluster together in a frequency distribution in the first year of life. In the winter-spring of their second year both 11-KT and GSI values of maturing males diverge from that of non-maturing males. It is somewhat unpredictable when clear separation between these two groups occurs and the threshold discriminating between non-maturing and maturing fish will change as maturing fish develop further. The purpose of this validation was to refine our understanding of how early in development GSI would provide a useful indicator of age-2 male maturation. Studies in winter 2005 were conducted to confirm that log GSI is an acceptable proxy for log 11-KT levels in migrating fish in January and February as has been the case for fish sampled later in the spring. Extra male hatchery fish were collected at the Cle Elum Supplementation and Research Facility (CESRF) on 10 January and 16 February 2005 and sampled for body weight, gonad weight for determination of GSI, and blood was collected to determine plasma 11-KT levels according to the method of Cuisset et al (1994). While it is known that GSI changes in fish as maturation progresses (Shearer et al. in press), samples collected in July from the Growth Modulation Experiment described in Objective 3 (see below) indicated that immature males consistently had a GSI 0.1% and precocious males > 0.1 % (Figure 21). Previously we used a GSI of 0.06% to designate the threshold between immature and maturing males (Larsen et al. 2004a), but subsequent evaluation of data from Objective 2 and previous collections from Roza and Prosser Dam in 2003 and 2004 have resulted in a modification to the more conservative 0.1% threshold. Thus, for this test and all past (2003-2004) and present (2005) collections at Roza and Prosser Dam this new more conservative threshold was used to screen migrating fish for precocious male maturation rates. Frequency distributions for GSI and 11-KT show some bi-modality in both January and February, being more distinctly bimodal for plasma 11-KT than GSI at these early sampling dates. Using the 0.8 ng/ml threshold for 11-KT (Larsen et al. 2004a) maturation rates were 38%

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and 26% precocious males in January and February, respectively. By contrast, using the 0.1% threshold for GSI (as described above) maturation rates were 6.5% and 10% in January and February, respectively. Furthermore, a scatter plot of the relationship between 11-KT and GSI in January and February demonstrates that 11-KT levels are increased beyond their threshold before that of GSI (Figure 2). Maturing males are not clearly distinguishable at this early date. These data indicate that if one uses the conservative threshold of 0.1%, GSI underestimates precocious male maturation rates in mid-Winter and bi-modality, like that seen for 11-KT, is less evident. 11-KT levels are suppressed in migrating fish (Larsen et al. 2004a), but unfortunately using GSI one does not know how many of the fish with GSI's below 0.1% are in fact destined for precocious male maturation. Thus, estimates of precocious male maturation from wild migrating fish in mid-winter are inaccurate and probably underestimate precocious male maturation rates.

Figure 1. Frequency distributions of Log10 plasma 11-Ketotestosternone (11-KT) and gonadosomatic index (GSI) from Cle Elum Hatchery , CESRF spring Chinook salmon sampled in January and February, 2005. Minijacks were designated by 11-KT using a threshold of 0.8 (ng/ml) according to the method of Larsen et al (2004a) and GSI using a threshold of 0.1%.

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Figure 2. Scatter plot of Log10 gonadosomatic index (GSI) and plasma 11-Ketotestosterone (11-KT) levels in male Cle Elum Hatchery (CESRF) spring Chinook salmon sampled in January (a) and February (b) 2005. Minijacks were designated by 11-KT using a threshold of 0.8 (ng/ml) according to the method of Larsen et al (2004a) and GSI using a threshold of 0.1%. However, screening of CESRF fish prior to release on 7 March 2005 showed a closer correlation between 11-KT and GSI for designating rates of precocious male maturation as previously seen in Larsen et al (2004a). Maturation rates were 37% based on plasma 11-KT and 31% based on GSI (Figure 3). So, GSI sill underestimates rates of maturation compared to 11-KT, but they differed by only 17% in March compared to 83% and 38%

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26% Precociously maturing males0.8 (ng/ml) threshold

5

in January and February, respectively. As the maturation process progresses in the spring, there is closer agreement between 11-KT and GSI, thus after mid-March GSI provides a relatively reliable tool for estimating precocious male maturation rates.

Figure 3. Scatter plot of Log10 gonadosomatic index (GSI) and plasma 11-Ketotestosterone (11-KT) levels in male Cle Elum Hatchery (CESRF) spring Chinook salmon sampled on 7 March 2005. Minijacks were designated by 11-KT using a threshold of 0.8 (ng/ml) according to the method of Larsen et al (2004a) and GSI using a threshold of 0.1%. Precocious male maturation rates were 37% of males using 11-KT and 31% using GSI. The solid line defines separation between phenotypes. 2005 Dam Collections (Brood Year 2003) On 26 January 2005, 300 wild Yakima River spring Chinook salmon were collected at Roza Dam to collect data on gender ratios, size and precocious male maturation rates of wild fish. As noted above precocious male maturation rates based on GSI in January and February in this and previous years are probably underestimates, but are relatively useful for comparisons of GSI with hatchery fish collected at the same time of year. In previous years approximately 600 fish were surveyed over two dates in January and February combined. However, due to low river flow conditions in winter 2005 the trap at Roza Dam was only operated on a very limited basis. In April and May approximately 300 wild and 300 Low Growth and 300 High Growth (see Objective 2) treatment fish were sampled at Prosser Dam. as with the Roza collections, low river flow limited the number of dates sampling was possible. Fish sampled at Roza and Prosser Dam were sacrificed for determination of length, weight, gonadal development and gonad weight to determine GSI.

-2.2

-2

-1.8

-1.6

-1.4

-1.2

-1

-.8

-.6

-.4

-.2

Log G

SI

-3 -2.5 -2 -1.5 -1 -.5 0 .5 1 1.5

Log 11-KT

31% Precociously maturing males 0.1% threshold

37% Precociously maturing males0.8 (ng/ml) threshold

Red line definesphenotypic modes

6

Figure. 4 Frequency distribution of log10transformed gonadasomatic index (GSI) of wild male fish collected at Roza Dam on the Yakima River on 26 January 2005. Male fish were classified as minijacks if their GSI exceeded 0.1% (log -1.0) according to a modification of the method of Larsen et al. (2004a). The total number of male and female fish sampled (N) and female:male ratios are presented. Table 1. Fork length, weight, total number of fish sampled (N), number of females and males, female:male ratio, X2 value for F:M ratio comparison to a 50:50 ratio, and percent of all males categorized by GSI as precociously maturing among wild Yakima Spring Chinook salmon captured at Roza Dam during the mid-winter re-distribution 2003-2005. NS = not significantly different from a 50:50 gender ratio.

YR Date Length (mm±SE)

Weight (g±SE)

N Female #

Male #

F:M ratio

X2

% prec. males

1/23 103.3±0.7 11.2±0.3 283 140 143 49:51 NS 0% 2003

2/13 95.4±0.5 8.6±0.1 317 163 154 51:49 NS 8.4%

1/28 103.6±0.5 11.1±0.2 310 174 136 56:44 4.7* 11% 2004

2/27 105.5±0.6 11.8±0.2 308 176 132 57:43 6.3* 5.3%

2005 1/26 100.2±0.6 10.±0.2 300 167 133 56:44 3.9* 7.5%

0

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-2.2 -2 -1.8 -1.6 -1.4 -1.2 -1 -.8 -.6

Log GSI

6% PutativeMinijacks

94% ImmatureMales

Roza Dam 26 January 2005 N = 300

Female:Male56:44

7

On 23 January 2005, 300 fish were sampled at Roza Dam (see Table 1); 167 were female and 133 were male for a 56:44 female to male ratio that is significantly different from 56:44 (X2 = 3.9, = 0.05). Among the 167 males 7.5% initiated precocious male maturation (Figure 1). Gender ratios may provide an indication of the number of 0-age precocious males in the population (theoretically, 0-age fish would have matured the previous fall and dropped out of the population that migrates to the lower Yakima River mid-winter and then skewed the gender ratio in favor of females). To the extent that these samples are representative of the population, the 56:44 gender ratios observed in these collections suggest that 0-age precocious males made up approximately 5% of the male population. Table 1 presents the size of wild spring Chinook salmon captured at Roza Dam in the winter 2005 as well as collections made in 2003 and 2004 for comparison. It should be stressed that point estimates of sex ratio in wild fish on a single date may not be indicative of sex ratios in the whole population, but rather give some indication as to how close the population is to a 50:50 ratio. Sex ratios differing from 50:50 could be due to precious male maturation, differential mortality, or gender specific differences in migration timing. On 20 April 2005 148 wild fish were sampled at Prosser Dam in the lower Yakima River (see Table 2); 79 were female and 69 were male for a 53:47 female to male ratio that was not significantly different from 50:50 (see summary Table 2). Among the 69 males 1.4% were minijacks (Figure 2a). On 28 April 2005 150 wild fish were sampled at Prosser Dam, 94 were female and 56 were male for a 63:37 female to male ratio that was skewed in favor of females (X2 = 9.6, = 0.05). Among the 37 males 1.8% were minijacks (Figure 3a). Gender ratios and minijack rates of wild fish collected at Prosser Dam in 2003 and 2004 are presented for comparison (Table 2). On 20 April 150 Modified (Low) Growth hatchery fish were sampled at Prosser Dam; 64 were female and 86 were male for a 43:57 female to male ratio that was not significantly different from 50:50. Among the 86 males 10.4% were minijacks (Figure 2b). On 28 April 2005 150 Modified (Low) Growth hatchery fish were sampled at Prosser Dam; 79 were female, 71 were male for a 53:47 female to male ratio that was not significantly different from 50:50. Among the 71 males 11% were minijacks (Figure 3b). On 20 April, 2005 150 Conventional (High) Growth hatchery fish were sampled at Prosser Dam; 75 were female and 75 were male for a 50:50 female to male ratio that was not significantly different from 50:50 (Table 2). Among the 75 males 25% were minijacks (Figure 2c). On 28 April 2005 150 Conventional (High) Growth hatchery fish were sampled at Prosser Dam; 71 were female and 79 were male for a 47:53 female to male ratio that was not significantly different from 50:50. Among 79 males 14% were minijacks (Figure 3c.). Gender ratios and minijack rates for hatchery fish collected at Prosser Dam in 2003 and 2004 (Larsen et al. 2005) are presented for comparison (Table 2).

8

Table 2. Fork length, weight, total number of fish sampled (N), number of females and males, female:male ratio, X2 value for F:M ratio comparison to a 50:50 ratio, and percent of all males categorized by GSI as precociously maturing among hatchery (Conventional and Modified) and Wild Yakima Spring Chinook salmon captured at Prosser Dam during the spring smolt migration 2003-2005. NS = not significantly different from a 50:50 gender ratio. Data from 2003 and 2004 (BY 2001 and 2002) fish modified from Larsen et al. (2005).

Treatment YR Date Length (mm±SE)

Weight (g±SE)

N F# M# F:M X2 % prec. males

Mean Prec. males

4/22 124.0±0.5 19.2±0.2 202 98 104 49:51 NS 0 2003

5/5 125.5±0.6 20.3±0.3 147 81 66 55:45 NS 0 0%

4/21 124.5±0.8 19.6±0.4 149 100 49 67:33 17.5* 0%

4/28 124.0±0.6 18.8±0.4 144 86 58 60:40 5.4* 3.4%

5/4 122.6±0.6 18.2±0.3 138 74 64 54:46 NS 9.5% 2004

5/13 121.1±0.6 18.4±0.3 150 96 54 64:36 11.8* 1.9%

3.7%

4/20 134.5±0.5 25±0.3 148 79 69 53:47 NS 1.4%

Wild

2005 4/28 120.5±0.7 17.7±0.3 150 94 56 63:37 9.6* 1.8%

1.6%

4/21 122.4±0.5 17.4±0.3 150 77 73 51:49 NS 13.7%

4/28 126.8±0.5 18.5±0.3 150 84 66 56:44 NS 13.6%

5/4 125.7±0.5 18.8±0.7 150 72 78 48:52 NS 13% 2004

5/13 129.4±0.6 20.6±0.7 147 76 71 52:48 NS 5.7%

12%

4/20 128.0±0.4 20.4±0.2 150 64 86 43:57 NS 10.4%

Modified (Low) Growth

2005 4/28 119.0±0.8 15.6±0.2 150 79 71 53:47 NS 11%

10.7%

4/22 131.4±0.5 22.0±0.3 203 124 79 61:39 9.98* 34% 2003

5/5 131.4±0.6 22.2±0.3 150 97 53 65:35 12.9* 3.7% 19%

4/21 128.4±0.6 20.1±0.3 150 99 51 66:34 15.4* 25%

4/28 131.3±0.5 20.3±0.3 150 84 66 56:44 NS 18.2%

5/4 131.7±0.6 20.9±0.3 150 87 63 58:42 3.84* 20.6% 2004

5/13 132.8±0.6 20.9±0.3 152 97 55 64:36 11.6* 3.7%

17%

4/20 133.6±0.5 23.1±0.3 150 75 75 50:50 NS 25%

Con-ventional (High) Growth

2005 4/28 125.0±0.5 18.1±0.3 150 71 79 47:53 NS 14%

20%

9

Figure. 5 Frequency distribution of log10transformed gonadasomatic index (GSI) of male wild fish collected at Prosser Dam on the Yakima River on 20 April 2005. Male fish were classified as minijacks if their GSI exceeded 0.1 (log -1.) according to a modification of the method of Larsen et al. (2004a).

Prosser Dam

4/20/05

02468

10

121416182022

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- Hatchery

02468

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121416182022

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-1.8 -1.6 -1.4 -1.2 -1 -.8 -.6 -.4 -.2 0

02468

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Log GSI

Modified (Low) Growth Treatment

- Hatchery

Wild Yakima spring Chinook

25% Minijacks

75% Immature Males

10.4% Minijacks

89.6% Immature Males

1.4% Minijacks

98.6% Immature Males

a)

b)

c)

10

Figure. 6 Frequency distribution of log10transformed gonadasomatic index (GSI) of male hatchery fish collected at Prosser Dam on the Yakima River on 28 April 2005. Male fish were classified as minijacks if their GSI exceeded 0.1 (log -1.) according to a modification of the method of Larsen et al. (2004a).

Prosser Dam28 April 2005

Log GSI

14% Minijacks

86% Immature Males

11% Minijacks

89% Immature Males

1.8% Minijacks

98.2% Immature Males

0

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Conventional (High)Growth Treatment

- Hatchery

Modified (Low) Growth Treatment

- Hatchery

Wild Yakima spring Chinook

a)

b)

c)

11

Assessment of average gender ratios and proportions of precociously maturing males in collections at Prosser Dam in 2005 corroborated findings from previous years (Table 2). First, minijacks made up a smaller percentage of all wild males captured (1.6%) compared with either High (20%) or Low (10.7%) growth hatchery treatments. Gender ratios were not significantly different from 50:50 in any group except on 28 April 2005 they were skewed in favor of females in the wild population.

12

OBJECTIVE 2: ESTIMATE THE INCIDENCE AGE-2 PRECOCIOUS MALE MATURATION IN THE CLE ELUM HATCHERY POPULATION The purpose of this objective was to accurately assess the incidence of age-2 precocious male maturation for brood years 2003 Cle Elum hatchery spring Chinook released from the Clark Flat, Easton and Jack Creek acclimation sites distributed throughout the Yakima River basin. In cooperation with Ray Brunson (USFWS pathologist) we sacrificed fish from each raceway for length, weight, visual assessment of gonadal development, and plasma 11-ketotestosterone (11-KT) levels (a reproductive steroid that indicates initiation of the maturation process) (Larsen et al. 2004a). Samples were collected in March of each year during the routine pathogen screening prior to the opening of the raceways for volitional release of all fish. 2005 Acclimation Site Collections (Brood Year 2003) In 2005, 1080 fish were sampled from the Yakima CESRF acclimation sites prior to the start of volitional release: 360 fish from each of Clark Flat, Easton, and Jack Creek acclimation sites. In an effort to reduce the number of precociously maturing male fish in the hatchery population the Yakima Fisheries Program initiated a production scale growth modulation experiment with brood year 2002 fish using the best available data to date from laboratory growth modulation experiments conducted in 2002 (see Objective 3). Thus, 50% of the fish in the program were reared according to conventional rearing methods that have been used since the inception of the project with regard to ration and growth rate. The other half of the population was reared on a modified reduced ration during the autumn period, one year prior to release (see Larsen et al. in press). In brief, fish were grown to a target size of either 15 grams (30 fish per pound) for the High Growth treatment or 10 grams (45 fish per pound) for the Low Growth treatment by the start of tagging on October 15 of each year. The minimum size of the Low Growth treatment was chosen in part because this was believed to be near the minimum size that could still be tagged with coded wire tags in various body locations and elastomer eye tags. Data collected for the High and Low Growth treatment groups at each acclimation site are presented in Figures 7-9 and Table 3. At Clark Flat acclimation site the female:male ratio was 42:58 for the High Growth treatment significantly skewed in favor of males (X2, p = 4.35, =0.05) and 52:48 for the Low Growth treatment. Precocious male maturation rates at Clark Flat were 25% and 12.7% for the High and Low Growth treatments, respectively (Figure 7). At the Easton acclimation site the female:male ratio was 54:46 for the High Growth treatment and 50:50 for the Low Growth treatment. The precocious male maturation rates were 30.1% and 17.8% for the High and Low Growth treatments, respectively (Figure 8). Finally, at the Jack Creek acclimation site the female:male ratio was 62:38 for the High Growth treatment and significantly skewed in favor of females (X2, p=17.4, =0.05) and 43:57 for the Low Growth treatment. The precocious male maturation rates were 26.9% and 8.9% for the High and Low Growth treatments, respectively (Figure 9). When data from each treatment was pooled from all acclimation sites gender ratios were not significantly different from a 50:50 ratio (Table 3). Overall, the precocious male maturation rate of the brood year 2003 Cle Elum hatchery fish under the Conventional ( High) Growth treatment was 27% while that of the

13

Modified (Low) Growth treatment was 13% representing a 52% reduction in precocious male maturation. For comparison, the precocious male maturation rate of the brood year 2002 Cle Elum hatchery fish under the High Growth treatment was 43% while that of the Low Growth treatment was 29% representing a 33% reduction in precocious male maturation. Size between the experimental treatments differed at all acclimation sites at the initiation of volitional release in mid-March 2004 (Table 3). All sites combined in 2005, the Low Growth fish averaged 11.6±0.24 grams and the High Growth fish averaged 15.5±0.15 grams. Data from brood years 2001 and 2002 sampled in 2003 and 2004, respectively, are presented for comparison as well (Table 3).

14

Figure. 7 Frequency distribution of log10transformed plasma 11-ketotestosterone (11-KT) levels of male High Growth and Low growth treated hatchery spring Chinook salmon collected at the Clark Flat Acclimation site on the Yakima River in March 2005. Male fish were classified as minijacks if their plasma 11-KT level exceeded 0.8 ng/ml (Log -0.09) according to the method of Larsen et al. (2004a). The total number of male and female fish sampled on each date (N) and female:male ratios are presented for each acclimation site.

0

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Clark FlatHigh Growth

Immature Male75% Age-2 Precocious

male25%

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Clark Flat:Low Growth Immature Male

87.3% Age-2 Precocious Male12.7%

N = 180F:M

42:58

N = 180F:M

52:48

15

Figure. 8 Frequency distribution of log10transformed plasma 11-ketotestosterone (11-KT) levels of male High Growth and Low growth treated hatchery spring Chinook salmon collected at the Easton Acclimation site on the Yakima River in March 2005. Male fish were classified as minijacks if their plasma 11-KT level exceeded 0.8 ng/ml (Log -0.09) according to the method of Larsen et al. (2004a). The total number of male and female fish sampled on each date (N) and female:male ratios are presented.

0

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Log 11-KT

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Log 11-KT

EastonHigh Growth

EastonLow Growth

Immature Male69.9%

Age-2 Precocious Male30.1%

Immature Male82.2% Age-2 Precocious

Male17.8%

N = 180F:M

50:50

N = 180F:M

54:46

16

Figure. 9 Frequency distribution of log10transformed plasma 11-ketotestosterone (11-KT) levels of male High Growth and Low Growth treated hatchery spring Chinook salmon collected at the Jack Creek Acclimation site on the Yakima River in March 2005. Male fish were classified as minijacks if their plasma 11-KT level exceeded 0.8 ng/ml (Log -0.09) according to the method of Larsen et al. (2004a). The total number of male and female fish sampled on each date (N) and female:male ratios are presented.

0

5

10

15

20

25

Cou

nt

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0

5

10

15

20

25

Cou

nt

-3 -2.5 -2 -1.5 -1 -.5 0 .5 1 1.5Log 11-KT

Jack CreekHigh Growth

Jack CreekLow Growth

Immature Male73.1%

Age-2 Precocious Male26.9%

Immature Male91.1%

Age-2 Precocious Male8.9%

N = 180F:M

43:57

N = 180F:M

62:38

17

Table 3. Fork length, weight, total number of fish sampled (N), number of females (F) and males (M), female:male ratio, X2 value for F:M ratio comparison to a 50:50 ratio, and percent of all males categorized by plasma 11-KT levels as precociously maturing Cle Elum Hatchery spring Chinook salmon sampled prior to volitional release form Clark Flat, Easton, and Jack Creek acclimation sites in 2003- 2005 (brood years 2001 - 2003). Brood year 2002 and 2003 were reared under either Low or High Growth treatments to control precocious male maturation rates. NS = not significantly different from a 50:50 gender ratio. Data from BY 2001 and 2002 fish modified from Larsen et al. (2005).

Brood Year

Acc. Site TRT.

Length (mm±SE)

Weight (g±SE) N F# M# F:M X2

% prec. male

Clark Flat

High 118.8±0.79 19.0±0.42 120 64 56 53:47 NS 61%

Easton High 119.2±0.58 19.1±0.30 180 95 85 53:47 NS 67% 2001

Jack Creek

High 118.7±0.43 18.8±0.22 360 188 172 52:48 NS 45%

Low 103.2±0.54 11.1±0.18 180 83 97 46:54 NS 30% Clark Flat

High 114.7±0.57 15.8±0.25 180 98 82 54:46 NS 40%

Low 103.6±0.51 11.6±0.18 180 91 89 51:49 NS 27% Easton

High 113.3±0.57 15.6±0.25 180 99 81 55:45 NS 49%

Low 106.6±0.57 12.4±0.22 180 88 92 49:51 NS 30%

2002

Jack Creek High 113.4±0.65 15.4±0.29 180 90 90 50:50 NS 41%

Low 102.1±0.47 11.1±0.16 180 94 86 52:48 NS 13% Clark Flat

High 112.6±0.59 15.3±0.24 180 76 104 42:58 4.35 25%

Low 104.1±0.52 12.9±0.67 180 90 90 50:50 NS 18% Easton

High 113.2±0.78 15.8±0.26 180 98 82 54:46 NS 30%

Low 100.0±0.49 10.8±0.17 180 78 102 43:57 NS 9%

2003

Jack Creek High 112.0±0.60 15.4±0.26 180 112 68 62:38 17.4 27%

18

OBJECTIVE 3: EXPERIMENTAL CONTROL OF PRECOCIOUS MALE MATURATION THROUGH GROWTH RATE MODULATION Background The purpose of Objective 3 is to conduct a series of experiments (in 1.4m diameter circular tanks) to examine the effect of growth rate modulation on the incidence of age-2 precocious male maturation in Cle Elum hatchery spring Chinook salmon. The first iteration of these experiments tested the effect of altering growth rate in the summer and fall in a 2X2 factorial design. Results indicated that manipulating growth rate could alter the rate of precocious maturation. The highest growth group (High summer/High autumn growth) had 69% male maturity while the lowest growth group (Low summer/Low autumn growth) had 42% maturity. The maturity level of BY 2001 production fish sampled during pathology screening was approximately 60%. In other years the production fish have been as low as 40%. The Cle Elum facility is currently rearing 50% of its production fish under the normal rearing regime with a target weight of approximately 15 grams at tagging in mid-to late October and 50% of its production fish similar to the Low/Low treatment with a size of approximately 10 grams by mid-October to try and reduce precocious maturity while maintaining a large enough fish to tag in October (see Objective 4). Results from that study are in press in Transactions of the American Fisheries Society under the following citation: Larsen, D.A., Beckman, B.R., Strom, C.R., Parkins, P.J., Cooper, K.A., Fast, D.E., and

Dickhoff, W.W. (In press). Growth modulation alters the incidence of early male maturation and physiological development of hatchery reared spring Chinook salmon: a comparison with wild fish. Transactions of the American Fisheries Society.

Obviously, reducing the maturation rate by 40% in the production fish by rearing them smaller represented progress, but our goal is to determine if it is possible to rear Yakima spring Chinook to have a male precocious male maturation rate more in line with that of the wild fish (approximately 5-10%). The second iteration of this objective built on the results of the previous study and explored the effect of altering emergence timing and whole body lipid levels during early development to further reduce the level of age-2 male maturation in the Cle Elum Supplementation and Research Facility (CESRF) spring Chinook stock. This experiment tested the following null hypotheses: H01: Early fry emergence has no effect on physiological development or incidence of precocious male maturation of supplementation hatchery spring Chinook. H02: Low lipid diet during early development has no effect on physiological development or incidence of precocious male maturation of supplementation hatchery spring Chinook.

19

BY 2003 Growth Modulation Experiment Analysis of data from our BY 2001 experiment suggested that adjustment of pond timing (to an earlier date) and reduction in dietary lipid content may further reduce the level of precocious male maturation in the CESRF population. Treatments included fish ponded in early March on high (current commercial formulation diet) and low fat diets and fish ponded in mid- April (current production time) on high and low fat diets in a 2x2 factorial design with 4 replicates per treatment. The treatments were all grown to a target size of 10 grams (the programs minimal size for tagging) by October 15. This involved production of low fat and high fat (conventional) experimental diet for these fish at our research facility with the following formulations: High fat diet Ingredient % Amount for 5kg Dry fish meal mix 84% 4200g Fish Meal 80% Choline Chloride 1% Vitamin mix 2% Mineral mix 1% Guar Gum 0.5% 25g Carboxymethylcellulose 0.5% 25g Vitamin C 1% 50g Wheat Middlings 4% 200g Fish Oil 10% 500g (or mls.) Water 500ml Low fat diet Ingredient % Amount for 5kg Dry fish meal mix 84% 4200g Fish Meal 80% Choline Chloride 1% Vitamin mix 2% Mineral mix 1% Guar Gum 0.5% 25g Carboxymethylcellulose0.5% 25g Vitamin C 1% 50g Wheat Middlings 14% 700g Fish Oil 0% 0g (or mls.) Water 1250ml 5000 BY 2003 eggs were collected from broodstock throughout the adult run (50 eggs from multiple pairs). Half of the eggs were incubated at elevated temperature (8 C) in a separate egg stack to induce ponding in early March. The remaining eggs were incubated with the production eggs at 5C. Approximately 300 randomly selected fish

20

were distributed among 16 1.4 m diameter tanks. After ponding all fish were fed commercial starter feed. After a few weeks experimental feeds were used (4 tanks low fat, 4 tanks high fat). In April the same process was repeated with the late emerging groups (4 tanks low fat, 4 tanks high fat). The four treatments were designated Early /High, Early /Low, Late /High, Late /Low. Throughout the experimental period representative production fish were also sacrificed to monitor physiology. The following parameters were measured approximately monthly in both Experimental and Production fish: weight, length, visual gonadal development, gill Na+/K+-ATPase activity (McCormick, 1993), plasma insulin-like growth factor-I (Shimizu et al. 2000) and whole body lipid (AOAC, 1975) (indicators of energetic and growth status). In June of 2005 the remaining experimental fish (approximately 250/tank) were sacrificed, weighed, measured for fork length, assessed for gender and gonads were weighed for determination of GSI to determine precocious male maturation rate.

Expectations: The expectations from this experiment were that fish ponded at the normal April ponding date and fed a high fat commercial diet will be most similar to the current small production fish and have precocious male maturation rates in excess of 40%. These same fish on a low fat diet may have lower rates of precocity. Previous work by Silverstein et al. (1998) has suggested that precocity can be modulated in spring Chinook by lowering dietary lipid levels. The fish ponded earlier may be able to obtain sufficient body size for tagging via a longer growth period but able to have slow autumn growth rate to prevent excessive precocious male development. Finally, the fourth treatment group includes both early pond timing and a low fat diet because this group most closely matches the growth and energetic stores of wild fish that have lower precocious male maturation rates. Since this fourth group involves the most significant and expensive alterations in hatchery practice (altered incubation and feed quality) we believe it is important to explore the other less invasive hatchery alterations outlined above before changing protocols at the production scale. Results Laboratory scale growth modulation experiment: comparison with hatchery production fish: The growth modulation experiment explored the effect of altering pond timing and dietary lipid level on physiology and precocious male maturation levels in an experimental subset of CESRF spring Chinook salmon. Data were analyzed by ANOVA for each parameter measured with P values presented on each figure (�=0.05). Furthermore, accompanying each figure, data is replotted with data from hatchery production fish from the production growth modulation experiment (Objective 4) for comparison to experimental fish. As of the time of this report, whole body lipid analysis was not completed. Note: No statistical analysis was conducted (or presented) for the figures showing the data from both the growth modulation experiment and the hatchery production fish. See Objective 4 for statistical comparisons between High and Low production hatchery fish.

21

Overall, there were no significant differences in weight among treatments (p=0.841) (Figure 10). Weight of production fish on either the Low or High Growth treatments were generally higher than that of all experimental fish throughout the Autumn (Figure 11). In winter weight of the High Growth treatment continued to be higher than that of the Low Growth and Experimental fish. The data for fork length mirrored that of weight for both Experimental and Production fish (Figures 12 and 13). There was no significant difference in fork length among treatments (p=0.4383) (Figure 12). Condition factor provides information on change in body shape throughout development. Condition factor increased through the summer, peaked in autumn, declined in winter and increased again in spring just prior to smoltification among all treatments (Figure 14). There was no significant difference in condition factor among all treatments (p=0.5555). Condition factor peaked earlier in the summer and declined lower in the winter in Production High and Low growth treatments compared with the four Experimental groups (Figure 15). Finally, plasma insulin-like growth factor-I levels provide a reliable metric of growth related physiology in salmonids. As with all other growth indicators above, there was no significant difference in plasma IGF-I levels among all treatments (p=0.6337) (Figure 16). Plasma IGF-I levels of the Production High Growth treatment were higher than that of the Experimental treatments or the Production Low Growth fish in September (Figure (17) and peaked in both Production treatments above that of the Experimental fish in early October before declining throughout the winter. Winter plasma IGF-I levels were lower in both Production treatments relative to that of the Experimental treatments throughout the winter. Finally, plasma IGF-I levels increased throughout the spring in all groups in association with high spring growth and smoltification.

22

Figure 10. Weight (g) of treatment groups in growth modulation experiment examining early and late ponding date and high and low fat diet: (Early/High, Early/Low, Late/High, and Late/Low). Magnification of data February to July is included to aid in viewing early treatment differences. Dashed lines fit by eye.

Wei

ght

(g)

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F M A M J J A S O N D J F M A M J J2004 2005

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F M A M J J

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Early pond3/1/04

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Late / Low

Late / High

Early / Low

Early / High

P =0.841

23

Figure 11. Weight (g) of treatment groups in growth modulation experiment examining early and late ponding date and high and low fat diet: (Early/High, Early/Low, Late/High, and Late/Low) and Cle Elum hatchery Production fish reared under High and Low Growth treatments (Prod./ High and Prod./Low). Magnification of data from February to July is included to aid in viewing early treatment differences. Dashed lines fit by eye.

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Late / High

Early / Low

Early / High

24

Figure 12. Fork length (mm) of treatment groups in growth modulation experiment examining early and late ponding date and high and low fat diet: (Early/High, Early/Low, Late/High, and Late/Low). Dashed lines fit by eye.

For

k L

engt

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m)

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F M A M J J A S O N D J F M A M J J2004 2005

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120

140

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Early pond3/1/04

Late pond4/14/05

P =0.4383

25

Figure 13. Fork length (mm) of treatment groups in growth modulation experiment examining early and late ponding date and high and low fat diet: (Early/High, Early/Low, Late/High, and Late/Low) and Cle Elum hatchery Production fish reared under High and Low Growth treatments (Prod./ High and Prod./Low). Dashed lines fit by eye.

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m)

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F M A M J J A S O N D J F M A M J J2004 2005

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Early pond3/1/04

Late pond4/14/05

26

Figure 14. Condition Factor (%) of treatment groups in growth modulation experiment examining early and late ponding date and high and low fat diet: (Early/High, Early/Low, Late/High, and Late/Low).

Con

diti

on F

acto

r (%

)

Late / Low

Late / High

Early / Low

Early / High

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F M A M J J A S O N D J F M A M J J2004 2005

0.9

1

1.1

1.2

1.3

Early pond3/1/04

Late pond4/14/05

P =0.5555

27

Figure 15. Condition factor (%) of treatment groups in growth modulation experiment examining early and late ponding date and high and low fat diet: (Early/High, Early/Low, Late/High, and Late/Low) and Cle Elum hatchery Production fish reared under High and Low Growth treatments (Prod./ High and Prod./Low).

Con

diti

on F

acto

r (%

)

Prod. / Low

Prod. / High

Late / Low

Late / High

Early / Low

Early / High

Month

F M A M J J A S O N D J F M A M J J2004 2005

0.9

1

1.1

1.2

1.3

Early pond3/1/04

Late pond4/14/05

28

Figure 16. Plasma IGF-I (ng/ml) of treatment groups in growth modulation experiment examining early and late ponding date and high and low fat diet: (Early/High, Early/Low, Late/High, and Late/Low).

4

8

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20

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ng/m

l)

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Early / Low

Early / High

Month

F M A M J J A S O N D J F M A M J J

2004 2005

Early pond3/1/04

Late pond4/14/05

P = 0.6337

29

Figure 17. Plasma IGF-I (ng/ml) of treatment groups in growth modulation experiment examining early and late ponding date and high and low fat diet: (Early/High, Early/Low, Late/High, and Late/Low) and Cle Elum hatchery Production fish reared under High and Low Growth treatments (Prod./ High and Prod./Low).

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F M A M J J A S O N D J F M A M J J

2004 2005

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30

Gill Na+/K+-ATPase activity was measured throughout the investigation to

monitor smolt related physiology in the Experimental groups (Figure 18). There were no significant differences in gill ATPase activity among treatments (p=0.4837). Furthermore, gill ATPase activity levels were similar among all experimental treatments and Production High and Low Growth treatments as well (Figure 19). Taken together these data indicate that none of the treatments (4 experimental and 2 production) were detrimental to the smoltification process, most notably in the Production groups that were grown at relatively different rates and final release sizes throughout development.

Figure 18. Gill Na+/K+-ATPase activity (μmoles PO4 / mg protein x hr) of treatment groups in growth modulation experiment examining early and late ponding date and high and low fat diet: (Early/High, Early/Low, Late/High, and Late/Low).

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Early / High

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P =0.4837

31

Figure 19. Gill Na+/K+-ATPase activity (μmoles PO4 / mg protein x hr) of treatment groups in growth modulation experiment examining early and late ponding date and high and low fat diet: (Early/High, Early/Low, Late/High, and Late/Low) and Cle Elum hatchery Production fish reared under High and Low Growth treatments (Prod./ High and Prod./Low).

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ase

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32

In July 2005 all remaining fish from each of the four experimental treatments were sacrificed, sampled for gender, length, weight, and the gonads were weighed in all males to determine gonadosomatic index (GSI) (Table 4). ANOVA analysis comparing final fork length and weight data from the experiment revealed significant differences among some treatments (see Table 5 series and Table 6 series). However, they were relatively small differences and not believed to be biologically relevant. Table 4. Final demographic data (fork length, weight, # males and females, female:male ratio and precocious male maturation rates) from fish sampled on the final sampling date of 6 July, 2005 from the growth modulation experiment examining the effect of early and late fry ponding date and high and low lipid diet on age-1 and age-2 male maturation in Cle Elum Hatchery spring Chinook salmon. Treatment Fork

Length (mm)

Weight (g)

# females

# males

F:M ratio

% age-1 precocious males

% age-2 precocious males

Early / High 132.9±0.2 26.4±0.3 391 405 49:51 1.0% 14.4%

Early / Low 134.3±0.4 27.2±0.3 423 403 51:49 3.9% 12.0%

Late / High 132.1±0.4 25.9±0.3 448 481 48:52 0% 16.1%

Late / Low 133.9±0.4 27.3±0.3 434 446 49:51 0% 18.6%

33

Table 5 series. ANOVA, mean, and multiple range test tables for mean weights of fish from treatments (Early/High, Early/Low, Late/High, Late/Low) in the growth modulation experiment sampled at the end of the experiment on 6 June 2005. Significance level = 0.05.

3 1196.895 398.965 6.486 .0002 19.457 .980

3427 210813.546 61.515

DF Sum of Squares Mean Square F-Value P-Value Lambda Power

Treatment

Residual

ANOVA Table for Weight

796 26.402 7.435 .264

826 27.182 8.453 .294

929 25.860 7.915 .260

880 27.267 7.526 .254

Count Mean Std. Dev. Std. Err.

Early/High

Early/Low

Late/High

Late/Low

Means Table for Weight

Effect: Treatment

-.781 .764 .0451 S

.542 .743 .1529

-.865 .752 .0242 S

1.322 .735 .0004 S

-.084 .745 .8245

-1.407 .723 .0001 S

Mean Diff. Crit. Diff. P-Value

Early/High, Early/Low

Early/High, Late/High

Early/High, Late/Low

Early/Low, Late/High

Early/Low, Late/Low

Late/High, Late/Low

Fisher's PLSD for Weight

Effect: Treatment

Significance Level: 5 %

Frequency distributions for Log-GSI of male fish showed distinct bimodality in each treatment that allowed for clear designation of fish as either immature or precociously maturing males (Figure 20). A small number (8 total) of age-1 precociously maturing males (precocious parr) were easily distinguished from visual examination from the early ponded treatments. A comparison of the frequency distributions between immature and age-2 and immature and age-1 precociously maturing males in June showed similar log-GSI values (Figure 21). However, the reproductive state of the testes was clearly different between the two precocious male age classes. The age-1 precociously maturing males would have matured initially in the previous autumn, survived through the winter and at the time of our sampling the gonads appeared to be in a state of regression. The testes of the age-2 precocious males were white, filled with milt, but not yet running. However, no histological analysis of the gonads was conducted to confirm this supposition. Taken together, these data were combined to estimate age-1 and age-2 precocious male maturation rates between the four treatments among all males (Figure 22) and among all fish (male and female) (Figure 23). All percentage data were

34

arcsine transformed and analyzed by ANOVA according to Zar (1984). There were no significant differences in age-2 male maturation rates as a percentage of all males among treatments (Table 7 series). However, there were significant differences among treatments in age-1 male maturation rates among males (Tables 8 series). Rates of age-1 precocious parr production were significantly different between all treatments with the exception of the Late/Low and Late/High treatments that both had no precocious parr. Table 6 series. ANOVA, mean, and multiple range test tables for mean fork lengths of fish from treatments (Early/High, Early/Low, Late/High, Late/Low) in growth modulation experiment sampled at the end of the experiment on 6 June 2005. Significance level = 0.05 indicated by S on third table.

3 2567.469 855.823 6.198 .0003 18.594 .974

3427 473209.924 138.083

DF Sum of Squares Mean Square F-Value P-Value Lambda Power

Treatment

Residual

ANOVA Table for Length

796 132.859 11.095 .393

826 134.272 12.402 .432

929 132.096 11.690 .384

880 133.876 11.763 .397

Count Mean Std. Dev. Std. Err.

Early/High

Early/Low

Late/High

Late/Low

Means Table for Length

Effect: Treatment

-1.413 1.144 .0155 S

.763 1.113 .1786

-1.017 1.127 .0770

2.177 1.102 .0001 S

.396 1.116 .4864

-1.780 1.084 .0013 S

Mean Diff. Crit. Diff. P-Value

Early/High, Early/Low

Early/High, Late/High

Early/High, Late/Low

Early/Low, Late/High

Early/Low, Late/Low

Late/High, Late/Low

Fisher's PLSD for Length

Effect: Treatment

Significance Level: 5 %

35

Figure 20. Frequency distribution of log10-GSI of male Yakima spring Chinook salmon reared under (Early/High, Early/Low, Late/High, Late/Low) experimental growth treatments. Fish were sampled on 6 June, 2005. Percentages of age-2 precociously mature males are designated for each treatment.

0

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Cou

nt

-2.5 -2 -1.5 -1 -.5 0 .5 1Log GSI

Early/High

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nt

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Late/Low

18.6±3.9%16.1±2.0%

12.0±2.2%14.4±0.3%

36

Figure 21. Comparison of frequency distribution of log10GSI for immature and age-2 (left graph) and immature and age-1 (right graph) precociously mature males from Yakima spring Chinook salmon reared in the growth modulation experiment. Fish were sampled on 6 June 2005. Arrow designates the 0.1% GSI threshold below and above which fish were designated as immature or precociously mature, respectively. Note 1, Immature males are the same fish plotted on each graph. Note 2, to aid in viewing, the y-axis for the right plot is on a log10 scale.

Age-1“Precocious Parr”

Male

0

50

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150

200

250

300

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Cou

nt

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-2.5 -2 -1.5 -1 -.5 0 .5 1

Log GSI

ImmatureMale

Age-2 “Minijack”

Male

ImmatureMale

GSI threshold 0.1% GSI threshold 0.1%

37

Figure 22. Percentages of immature, and age-1 and age-2 precociously mature males among all males in the Early/High, Early/Low, Late/High, Late/Low treatment groups from Cle Elum Hatchery spring Chinook salmon growth modulation experiment (averaged from 4 replicate tanks per treatment). Percentages ± SE are presented above each category.

0

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Ear

ly/H

igh

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ow

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e/H

igh

Lat

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ow

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Immature Male

Age-2 (“Minijack”)

Age-1 (“Prec. Parr”)

84.8±0.4

03.9±1.11.0±0.4

18.6±3.916.1±2.012.0±2.214.4±0.3

84.1±2.4 83.9±2.0 81.4±3.9

0

38

Figure 23. Percentages of female, immature, and age-1 and age-2 precociously mature males among all fish in the Early/High, Early/Low, Late/High, Late/Low treatment groups from Cle Elum Hatchery spring Chinook growth modulation experiment (averaged from 4 replicate tanks per treatment). Percentages ± SE are presented above each category.

Preliminary analysis of results from this experiment provided four significant

results: 1) With the exception of differences in growth during the very early stages of this experiment (where pond time and dietary lipid levels varied the most), there were no overall physiological differences between treatments throughout most of this study. 2) Lower growth in their first summer reduced rates of age-2 precocious male maturation in all treatment groups (range 12-19% of males) compared with our previous study (42-69%-Larsen et al. in press). 3) In contrast with previous studies (Rowe et al. 1991; Silverstein et al. 1997, 1998; Shearer and Swanson 2000; Shearer et al. in press) the low lipid diet had no significant effect on precocious male maturation rate. However, it should be noted that whole body lipid analysis is not yet complete to determine if different diets had an effect on lipid levels. 4) Altering fry emergence timing by manipulating egg incubation temperature produced age-1 precocious parr (1-4% of males) for the first time in our experiments. It is worth noting that while these rates of age-1 maturation were minimal, they were observed in fish grown to a relatively small size in their first autumn (8-9gms). Any increase in autumn growth rate beyond the level

0

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100

Ear

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Ear

ly/ L

ow

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Lat

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Female

Immature Male

Age-2 (“Minijack”)

Age-1 (“Prec. Parr”)

49.3±1.1

7.3±0.2

41.1±2.143.4±0.941.0±1.9

42.9±1.0

49.4±1.548.2±1.051.2±1.6

8.4±1.15.9±1.2

1.9±0.50.5±0.2 00

9.5±1.9

39

in this experiment, coupled with the earlier emergence timing, may significantly increase the prevalence of age-1 “precocious parr”.

In this study we did not originally plan to produce fish of such a small size since program mangers view a parr less than 10 grams by mid-October as too small to tag. However, this work once again emphasized that growth pattern has a more significant effect on determining early male maturation than whole body lipid levels (at least in small fish) and demonstrates that rearing fish at a small size could result in smolts with a more “wild-like” life history pattern. However, from a fisheries management perspective, a significant concern is that by reducing the size of the fish during juvenile development precocious male maturation rates will be reduced, but there may be a survival disadvantage for smolts caused by small size. Numerous studies in hatchery and experimental fish (Martin and Wertheimer 1989; Virtanen et al. 1991; Farmer 1994; Lundqvist et al. 1994) and wild fish (Ward and Slaney 1988; Ward et al. 1989; Henderson and Cass 1991) have suggested that large smolt size is advantageous for survival. Thus, the next iteration of these growth modulation experiments will focus on the effect of earlier pond timing to increase early growth in fish prior to the autumn "decision period" in an effort to increase size while continuing to reduce precocious male maturation rates. But, these studies must be aware of the potential for increasing rates of age-1 maturation.

40

Table 7 series. ANOVA, mean, and multiple range test tables for arcsine transformed proportion of age-2 precocious males (minijacks) among males for all treatments (Early/High, Early/Low, Late/High, Late/Low) in growth modulation experiment. N = 4 replicate tanks per treatment. Significance level = 0.05.

3 57.094 19.031 1.329 .3109 3.986 .263

12 171.891 14.324

DF Sum of Squares Mean Square F-Value P-Value Lambda Power

Treatment

Residual

ANOVA Table for MJ Arcsine

4 22.275 .553 .277

4 20.025 3.816 1.908

4 23.537 3.099 1.550

4 25.200 5.729 2.865

Count Mean Std. Dev. Std. Err.

Early/High

Early/Low

Late/High

Late/Low

Means Table for MJ Arcsine

Effect: Treatment

2.250 5.831 .4169

-1.262 5.831 .6456

-2.925 5.831 .2959

-3.512 5.831 .2139

-5.175 5.831 .0771

-1.663 5.831 .5461

Mean Diff. Crit. Diff. P-Value

Early/High, Early/Low

Early/High, Late/High

Early/High, Late/Low

Early/Low, Late/High

Early/Low, Late/Low

Late/High, Late/Low

Fisher's PLSD for MJ Arcsine

Effect: Treatment

Significance Level: 5 %

41

Table 8 series. ANOVA, mean, and multiple range test tables for arcsine transformed proportion of age-1 (precocious parr) among males for all treatments (Early/High, Early/Low, Late/High, Late/Low) in growth modulation experiment. N = 4 replicate tanks per treatment. Significance level = 0.05.

3 337.226 112.409 22.187 <.0001 66.562 1.000

12 60.796 5.066

DF Sum of Squares Mean Square F-Value P-Value Lambda Power

Treatment

Residual

ANOVA Table for Parr Arcsine.2

4 4.987 3.453 1.727

4 11.165 2.888 1.444

4 0.000 0.000 0.000

4 0.000 0.000 0.000

Count Mean Std. Dev. Std. Err.

Early/High

Early/Low

Late/High

Late/Low

Means Table for Parr Arcsine.2

Effect: Treatment

-6.177 3.468 .0022 S

4.987 3.468 .0086 S

4.987 3.468 .0086 S

11.165 3.468 <.0001 S

11.165 3.468 <.0001 S

0.000 3.468 •

Mean Diff. Crit. Diff. P-Value

Early/High, Early/Low

Early/High, Late/High

Early/High, Late/Low

Early/Low, Late/High

Early/Low, Late/Low

Late/High, Late/Low

Fisher's PLSD for Parr Arcsine.2

Effect: Treatment

Significance Level: 5 %

Physiological comparison between immature and precociously maturing male spring Chinook salmon

In the final months of this experiment, measurement of plasma 11-KT levels allowed for the separation of immature and precociously maturing males among all fish sampled. Plasma 11-KT levels from all males sampled varied widely in January and February, but no clear separation above and below the previously established (Larsen et al. 2004a) 0.8 ng/ml threshold for designating precocious male maturation was evident (Figure 24). However samples collected in early-April clearly clustered above and below this threshold, corroborating previous findings that clear separation is not seen until approximately mid-March (see Objective 1 above). Average 11-KT levels for immature, age-1 maturing, and age-2 maturing fish showed that 11-KT levels of age-1 maturing males were intermediate between that of immature and age-2 maturing males (Figure 25). These reduced plasma 11-KT levels in the age-1 precocious males provide further evidence that these fish were in a state of reproductive regression. What is unclear is

42

whether these fish would have fully regressed and undergone smoltification or re-matured at age-2 as minijacks. Future studies will attempt to shed light on the ultimate fate of this life-history variant in spring Chinook salmon.

Figure 24. Scatter-plot of plasma 11-ketotestosterone (11-KT) levels in all male spring Chinook salmon sampled in the growth modulation experiment from January to June 2005 (open circles). 11-KT levels (mean±se) for maturing and immature males were determined by designating fish with levels above and below 0.8 ng/ml as maturing or immature, respectively according to the method of Larsen et al. (2004a). The dashed line indicates the 0.8ng/ml threshold.

-0.50

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etot

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Immature Males

Month

F M A M J J A S O N D J F M A M J2004 2005

Early pond3/1/04

Late pond4/14/05

0.8 ng/mlthreshold

43

Figure 25. Plasma 11-ketotestosterone levels from all immature, age-1 and age-2 precociously maturing spring Chinook salmon from the growth modulation experiment sampled from January to May, 2005. Different letters denote significant differences between male maturation types. Significance level = 0.05.

Size differences between immature and age-2 precociously maturing males were evident in early spring with precociously maturing males being both heavier (p<0.0001) (Figure 26) and longer (p<0.0001) (Figure 27) than immature males. The growth related changes are reflected in the plasma IGF-I levels, being higher in the larger maturing males than the smaller immature males throughout the spring (p=0.0046) (Figure 28). Previous studies have found elevated plasma IGF-I levels in maturing spring Chinook salmon (Shearer and Swanson 2000; Campbell et al. 2003) and these high IGF-I levels may, in part, result from the elevated 11-KT levels in these fish as observed in (Larsen et al. 2004b).

Throughout the spring, condition factor (CF) differed between immature and maturing males (p<0.0001), increasing in the precociously maturing males, but decreasing or remaining unchanged in the immature males. This result reflects the tendency for CF to decrease in immature fish during smoltification, as fish become more streamlined in preparation for downstream migration and ocean entry (Figure 29). In concert with these smoltification related morphological changes, gill Na+/K+-ATPase activity increased significantly throughout the spring in the immature males, but remained relatively unchanged in the precociously maturing males (p<0.0001) (Figure 30).

0

1

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3

4

ImmatureMale

Age-2Precocious

Male

Age-1 Precocious

Male

N=5

N=95

N=241Pla

sma

11-K

T (

ng/m

l)

a

b

c

44

Figure 26. Weight of immature and precociously maturing male spring Chinook salmon from the growth modulation experiment in spring. Precociously maturing males were designated based on having plasma 11-KT levels above 0.8 ng/ml according to the method of Larsen et al. (2004a). Asterisks indicate significant differences between immature and maturing fish. Significance level = 0.05.

5

10

15

20

25

30

35

Precociously Maturing Males

Immature Males

Wei

ght

(g)

Month

F M A M J J A S O N D J F M A M J J2004 2005

Early pond3/1/04

Late pond4/14/05

P < 0.0001

*

*

**

*

45

Figure 27. Fork length of immature and precociously maturing male spring Chinook salmon from the growth modulation experiment in spring. Precociously maturing males were designated based on having plasma 11-KT levels above 0.8 ng/ml according to the method of Larsen et al. (2004a). Asterisks indicate significant differences between immature and maturing fish. Significance level = 0.05.

90

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130

140

For

k L

engt

h (m

m)

Month

F M A M J J A S O N D J F M A M J J2004 2005

Early pond3/1/04

Late pond4/14/05

P < 0.0001

*

*

***

Precociously Maturing Males

Immature Males

46

Figure 28. Plasma IGF-I of immature and precociously maturing male spring Chinook salmon from the growth modulation experiment in spring. Precociously maturing males were designated based on having plasma 11-KT levels above 0.8 ng/ml according to the method of Larsen et al. (2004a). Asterisks indicate significant differences between immature and maturing fish. Significance level = 0.05.

5

10

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25

30

Month

F M A M J J A S O N D J F M A M J J2004 2005

Early pond3/1/04

Late pond4/14/05

Precociously Maturing Males

Immature Males

Pla

sma

IGF

-I (

ng/m

l)P = 0.0046

**

*

*

47

Figure 29. Condition factor of immature male and precociously maturing male spring Chinook salmon from the growth modulation experiment in spring. Precociously maturing males were designated based on having plasma 11-KT levels above 0.8 ng/ml according to the method of Larsen et al. (2004a). Asterisks indicate significant differences between immature and maturing fish. Significance level = 0.05.

1

1.1

1.2

1.3

1.4

Con

diti

on F

acto

r

Month

F M A M J J A S O N D J F M A M J J2004 2005

Early pond3/1/04

Late pond4/14/05

P < 0.0001

*

**

*

Precociously Maturing Males

Immature Males

48

Figure 30. Gill Na+/K+-ATPase activity of immature male and precociously maturing male spring Chinook salmon from the growth modulation experiment in spring. Precociously maturing males were designated based on having plasma 11-KT levels above 0.8 ng/ml according to the method of Larsen et al. (2004a). Asterisks indicate significant differences between immature and maturing fish. Significance level = 0.05. Taken together, results from this physiological comparison between immature and precociously maturing males clearly demonstrated that minijacks are larger, less stream lined and less well adapted as smolts. These findings corroborate those found in earlier studies in Atlantic salmon (Shrimpton and McCormick, 2002) and spring Chinook salmon (Larsen et al. in press). Despite this, many of these fish do in fact migrate hundreds of kilometers downstream in the spring (Larsen et al. 2004a, Objective 1 this report) and back up-stream later in the summer in an attempt to reach their natal spawning grounds (Beckman and Larsen, 2005). However, many of the lower reaches of Columbia River tributaries including the Yakima River have thermal blocks in late summer that may prevent minijacks from making it back to headwater areas. Thus, these fish may compete with other native stocks for habitat and space and stray in to more accessible non-natal streams to spawn.

1

2

3

4

5

Precociously Maturing Males

Immature Males

Gill

Na+ /

K+ -

AT

Pas

e

Month

F M A M J J A S O N D J F M A M J J2004 2005

Early pond3/1/04

Late pond4/14/05

P < 0.0001 *

*

49

OBJECTIVE 4: PRODUCTION SCALE GROWTH MODULATION STUDIES

Along with biologists from the Yakama Nation and the WDFW we are currently implementing a production scale experiment aimed at reducing precocious maturation in Yakima spring Chinook (see Objective 2). We designed a rearing regime using the best available information from our first growth modulation experiment described in Larsen et al. (2004a). As described in Objective 2 half of the production fish are being grown at the normal production size of 15 grams and half at 10 grams by Oct. 15 for tagging. These treatment groups are differentially tagged in order to monitor juvenile survival during spring smolt migration at various Yakima and Columbia River dams and smolt-to-adult survival. The concern is that by reducing the size of the fish during juvenile development precocious maturation will be reduced, but there may be a predation survival disadvantage caused by small size. Numerous studies in hatchery and experimental fish (Martin and Wertheimer 1989; Virtanen et al. 1991; Farmer 1994; Lundqvist et al. 1994) and wild fish (Ward and Slaney 1988; Ward et al. 1989; Henderson and Cass 1991) have suggested that large smolt size is advantageous for survival. We monitored the growth rate of the production fish by, sampling groups periodically, and just prior to release examining fish for indications of precocious maturation at the acclimation sites. Data regarding size, gender, and percent precocious male maturation collected at both the Yakima acclimation sites prior to volitional release and at Prosser Dam during the spring smolt migration for this objective are presented in both Objective 1 and Objective 2 above.

Several parameters were monitored throughout this investigation to compare physiological responses to the two treatments. As expected, compared to the Modified (Low) growth treatment, the Conventional (High) growth fish were significantly heavier (p<0.0001) (Figure 31), longer (p<0.0001)(Figure 32) and had higher IGF-I levels (p<0.0025) (Figure 33) and CF's (p<0.0143) (Figure 34). Similar to the results presented in Objective 3 (above) regarding comparisons between immature and precociously maturing males, the higher IGF-I and CF of the High growth fish may, in part, be a result of the higher number of precociously mature males found in that treatment (Objective 2, Figures 7-9 and Table 2). However, there was no significant effect of these treatments on the smolt indicators of gill Na+/K+-ATPase (p=0.4223) (Figure 35) and plasma thyroxine (a developmental hormone commonly associated with the smoltification process) (p=0.3212)(Figure 36) suggesting that the Low growth treatment may not be detrimental to the smoltification process. Taken together, in two years of testing, the Modified (Low) growth treatment reduced the level of precociously maturing males compared with the Conventional (High) growth treatment by 33% (43 vs. 29%) and 52% (27 vs. 13%) in 2004 and 2005, respectively (Objective 2, Table 3). Furthermore, physiological monitoring of these two treatments indicates that the Modified treatment does not compromise the development or smoltification process despite the smaller size of these fish. The ultimate success of this treatment will be revealed when adult return rates are compiled over the four years of this experiment (brood years 2002-2005).

50

Figure 31. Weight of Conventional (High) growth and Modified (Low) growth CESRF spring Chinook salmon from production scale growth modulation experiment. To aid in viewing insert shows magnification of early growth period. Asterisk indicates significant differences between treatments at a given date. Dashed line fit by eye. Significance level =0.05.

Wei

ght

(g)

Production - Low

Production - High

Month

F M A M J J A S O N D J F M A M J J2004 2005

Wei

ght

(g)

F M A M J J

0

10

20

30

40

0

1

2

3

4

5

Pond4/14/05

P<0.0001

*

*

**

*

*

*

**

51

Figure 32. Fork length of Conventional (High) growth and Modified (Low) growth CESRF spring Chinook salmon from production scale growth modulation experiment. Dashed line fit by eye. Significance level =0.05.

For

k L

engt

h (m

m)

Month

F M A M J J A S O N D J F M A M J J2004 2005

20

40

60

80

100

120

140

160

Pond4/14/05

***

**

*

*

*

P < 0.0001

Production - Low

Production - High

52

Figure 33. Plasma IGF-I levels of Conventional (High) growth and Modified (Low) growth CESRF spring Chinook salmon from production scale growth modulation experiment. Asterisk indicates significant differences between treatments at a given date. Significance level =0.05.

4

8

12

16

20

24

28

Pla

sma

IGF

-I (n

g/m

l)

Month

F M A M J J A S O N D J F M A M J J

2004 2005

Early pond3/1/04

Late pond4/14/05

P = 0.0025

*

** *

Production - Low

Production - High

53

Figure 34. Condition factor of Conventional (High) growth and Modified (Low) growth CESRF spring Chinook salmon from production scale growth modulation experiment. Asterisk indicates significant differences between treatments at a given date. Significance level =0.05.

Con

diti

on F

acto

r (%

)

Month

F M A M J J A S O N D J F M A M J J2004 2005

0.9

1

1.1

1.2

1.3

Early pond3/1/04

Late pond4/14/05

P=0.0143

*

* *

*

*

*

Production - Low

Production - High

54

Figure 35. Gill Na+/K+-ATPase activity of Conventional (High) growth and Modified (Low) growth CESRF spring Chinook salmon from production scale growth modulation experiment. Significance level =0.05.

0

1

2

3

4

5

Gill

Na+

/K+ -

AT

Pas

e

Month

F M A M J J A S O N D J F M A M J J2004 2005

Pond4/14/05

P = 0.4223

Production - Low

Production - High

55

Figure 36. Plasma thyroxine (T4) levels of Conventional (High) growth and Modified (Low) growth CESRF spring Chinook salmon from production scale growth modulation experiment. Significance level =0.05.

5

10

15

20

25

Thy

roxi

ne (

ng/m

l)

Month

F M A M J J A S O N D J F M A M J J2004 2005

Pond4/14/05

P=0.3212

Production - Low

Production - High

56

ACKNOWLEDGMENTS This work was conducted in cooperation with Bill Bosch (Yakama Nation Fisheries, Toppenish, WA), Todd Pearsons (Washington Department of Fish and Wildlife, Ellensburg, WA), Steve Schroder and Craig Busack (Washington Department of Fish and Wildlife, Olympia, WA) and Curt Knudsen (Oncorh Consulting, Olympia, WA). We thank Joe Hoptowit, Gerald Lewis, and Leroy Senator (Yakama Nation Fisheries, Toppenish, WA) for assistance with fish collections at Roza and Prosser Dams and staff of the Cle Elum Supplementation and Research Hatchery (Cle Elum, WA) including D.J. Brownlee, Vernon Bogar, Simon Goudy, Annie Joe Parrish, and Greg Strom for rearing of Experimental and Production fish. Assistance in sampling was provided by Jon Dickey and Larissa Felli, (School of Aquatic and Fisheries Science, University of Washington, Seattle, WA), and Andrew Dittman (National Marine Fisheries Service, Seattle, WA), Samples were collected during pathology screening with extensive assistance from Ray Brunson, Joy Evered, Chris Paterson, and Sonia Mumford (U.S. Fish and Wildlife Service, Olympia, WA).

57

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