Use of Ultrasound Imaging and Steroid Concentrations to Identify Maturational Status in Adult Steelhead

Transcription

1 North American Journal of Fisheries Management 24: , 2004 Copyright by the American Fisheries Society 2004 Use of Ultrasound Imaging and Steroid Concentrations to Identify Maturational Status in Adult Steelhead ALLEN F. EVANS* 1 Columbia River Inter-Tribal Fish Commission, 729 Northeast Oregon Street, Suite 200, Portland, Oregon 97232, USA MARTIN S. FITZPATRICK Oregon Department of Environmental Quality, 811 Southwest Sixth Avenue, Portland, Oregon 97204, USA LISBETH K. SIDDENS Department of Fisheries and Wildlife, Oregon State University, Corvallis, Oregon 97331, USA Abstract. We sought to develop a rapid, noninvasive, and accurate method for distinguishing prespawn (mature or maturing) adult steelhead (anadromous rainbow trout Oncorhynchus mykiss) from postspawn adults (kelts). Ultrasound images of gonads were collected, and levels of plasma testosterone (T), 11-ketotestosterone (11-KT), and 17 -hydroxy-20 -dihydroxyprogesterone (DHP) were determined for adult steelhead before and after spawning. Results demonstrated that ultrasound images provided quantifiable selection criteria (number of eggs or gonad size) for the identification of prespawn versus postspawn adults. Mature females were easily identifiable by the presence of numerous, well-developed eggs. Conversely, only a few mature eggs remained in the body cavities of female kelts. Ultrasound investigation of the maximum testis area demonstrated that testes undergo substantial size changes following spawning. Mean maximum testis area was 2.86 cm 2 in prespawn males and 0.62 cm 2 male kelts. Distributions of testis measurements between the two maturational types did not overlap, and results of a discriminant function analysis suggested that a classification criterion (threshold) of 1.25 cm 2 could accurately distinguish prespawn males from kelts. Median concentrations of T, 11-KT, and DHP were significantly higher in prespawn males (49.6, 78.4, and 13.0 ng/ml, respectively) than in kelts ( 1.2, 6.6, and 1.6 ng/ml), providing an independent verification of the ultrasound technique. Despite the high degree of corroboration between ultrasound measurements of gonad size and plasma steroid levels, application of ultrasound imaging may result in some classification error (a few percentage points) if substantial gonad mass is retained in the body cavity after spawning. Use of ultrasound to accurately identify postspawn steelhead is an important first step towards development of effective kelt management practices in the Columbia River basin. Steelhead (anadromous rainbow trout Oncorhynchus mykiss), unlike most other anadromous Pacific salmonid species, are iteroparous. The upstream migration of maturing or prespawn adult steelhead and the downstream migration of postspawn steelhead (kelts) can geographically and temporally overlap in the Snake River (Whitt 1954). The majority of adult summer-run steelhead (freshwater maturing; Busby et al. 1996) navigate Snake River dams in September and October but do not spawn until the subsequent spring (Whitt 1954; Robards and Quinn 2002). A sizable portion of the steelhead run overwinters in the Snake River * Corresponding author: aevans@realtimeresearch.org 1 Present address: Real Time Research, Inc., 201 Yellowtail Hawk Avenue, Bend, Oregon 97701, USA. Received June 24, 2003; accepted December 1, 2003 before traveling to natal streams for spawning (Whitt 1954). Following natural spawning, surviving individuals must emigrate to the Pacific Ocean to undergo the gonadal recrudescence necessary for repeat spawning. Thus, between March and May, some prespawn fish move to their spawning grounds while kelts are leaving their spawning grounds en route to the Pacific Ocean. Each spring, thousands of adult steelhead many of which are now federally listed as endangered or threatened (NMFS 1997) are observed in the juvenile bypass facilities at Snake River and Columbia River dams. Bypass facilities in the Columbia River basin are designed to divert migratory and resident fish around hydroelectric facilities via large screens that partially block turbine intakes (Muir et al. 2001). Although bypass facilities were specifically designed for the nonturbine 967

2 968 EVANS ET AL. passage of juvenile salmonids, adult salmonid fallbacks (fish that initially pass the dam via fishways but subsequently fall back downstream) and downstream-migrating adults (e.g., kelts) are also encountered in the collection areas of juvenile bypass facilities each spring. From 1996 to 2000, an average of 5,050 adult steelhead (ranging from 4,182 to 6,504) were counted in the juvenile bypass facility at Lower Granite Dam on the Snake River (694 river kilometers [rkm] from the Pacific Ocean) between late March and June of each year (USACE ). Currently, there is no reliable method for distinguishing a prespawn adult steelhead fallback from a downstream-migrating kelt. Because steelhead kelts might be a valuable resource for rebuilding depleted runs on the Snake River and elsewhere, fisheries researchers need access to a rapid, noninvasive, accurate way to distinguish prespawn steelhead from kelts. One potential method of identifying maturational status in adult steelhead is ultrasound. Ultrasonic waves (acoustic energy measured in megahertz) produce images of the size, shape, and location of soft tissues (Martin et al. 1983) within biological organisms. Ultrasound technology is an identification method that has been used, albeit not widely, since the early 1980s to determine the sex and maturational status of various freshwater and marine fishes (Martin et al. 1983; Reimers et al. 1987; Shields et al. 1993; Blythe et al. 1994; Arkush and Petervary 1998). When operators are properly trained in use of the equipment and are knowledgeable about specimen anatomy, ultrasound has the potential to be a highly accurate, noninvasive diagnostic tool in fisheries science. For example, in ultrasound studies conducted on adult rainbow trout, Reimers et al. (1987) were able to distinguish with 100% accuracy the sex of specimens 5 months before spawning. Similarly, an ultrasound examination of striped bass Morone saxatilis allowed identification of the sex of fish with 100% accuracy throughout the entire reproductive cycle (Blythe et al. 1994). In addition to sex identification, ultrasound can also be used to distinguish immature from mature specimens based on the maximum diameter of the gonad (Blythe et al. 1994; Arkush and Petervary 1998). Fish with large, well-developed gonads were readily identifiable as mature relative to immature specimens, which had small gonads. Even fish as small as the Pacific herring Clupea pallasi can be segregated by sex and maturational status with ultrasound (Bonar et al. 1989). Despite ultrasound use in identifying both sex and maturation in other fish species, the determination of maturational status in adult steelhead via ultrasound has yet to be reported in published literature. A potential obstacle to the use of ultrasound for identifying kelts relates to the varying size of gonads following spawning. For example, if a male fish retains substantial gonad mass after spawning, then it may be difficult to distinguish a prespawn fish from a kelt. However, studies of gonad development in salmonids provide information to suggest that the physical size of gonads differs among maturational stages. Although the duration of spermatogenesis and spermiation in salmonids is long (Gjerde 1984; Munkittrick and Moccia 1987), the physical size of testes undergoes wide seasonal variation (Billard 1983). Large variation in testis size has been attributed to the cyclical expansion and contraction of interstitial cells (Billard 1983), the loss of mass via ejaculation (Munkttrick and Moccia 1987), and the subsequent degeneration and loss of dead Leydig cells (Cauty and Loir 1995). Therefore, despite the long duration of testicular growth in male steelhead, changes in gonad size (i.e., a decrease) should be detectable with ultrasound imaging. Along with changes in the gonad size, plasma hormone levels also vary throughout gonad development and may be used as indicators of maturational status (Schreck 1972; Scott et al. 1980; Baynes and Scott 1985; Fitzpatrick et al. 1986). For example, the steroid hormones testosterone (T), 11- ketotestosterone (11-KT), and 17 -hydroxy-20 dihydroxyprogesterone (DHP), which are produced in the gonads and circulate within the blood system (Bone et al. 1995), are correlated with sperm formation and production in male rainbow trout (Campbell et al. 1980; Scott et al. 1980; Baynes and Scott 1985; Vizziano et al. 1996). Testosterone, 11-KT, and DHP levels increased steadily with the amount of spermatozoa being produced in three different strains of rainbow trout studied by Baynes and Scott (1985). Following spermiation, the steroid levels dramatically declined in all three strains (Baynes and Scott 1985). Vizziano et al. (1996) demonstrated that plasma concentrations of DHP were positively correlated to the volume of milt produced in male testes. In a similar study of steroid concentrations, Fitzpatrick et al. (1986) recorded higher levels of DHP in milt-producing coho salmon O. kisutch relative to non-milt-producing males. Although the complete effects of the three hormones during reproductive development are complex and only partially understood (Vizziano et al. 1996), the hormones are clearly associated with

3 MATURATIONAL STATUS IN STEELHEAD 969 the maturation process. If blood steroid levels of T, 11-KT, and DHP differ significantly among fish of varying maturational states, and if gonad diameter is a good indication of maturational status, then plasma steroid levels may strongly corroborate the ultrasound data. Thus, reproductive hormones could provide an independent assessment of ultrasound imaging of gonads. For example, male steelhead with large testes may also have elevated concentrations of steroids and vice versa. If steroid concentrations measured in fish of known maturational status were found to be positively correlated with gonad size as determined by ultrasound imaging, then they would help to confirm the accuracy of ultrasound classification. The primary goal of this study was to develop criteria for identifying maturational status of adult steelhead based on gonad size and to determine if plasma steroid levels corroborate the ultrasound data. If quantifiable differences exist among maturational types, then ultrasound techniques will give fisheries researchers access to a noninvasive, rapid, accurate tool for use both in the field and in the laboratory. Lastly, numerous authors have reported plasma hormone concentrations in nonmigratory, captive stocks of rainbow trout (Campbell et al. 1980; Scott et al. 1980; Baynes and Scott 1985; Scott and Sumpter 1989; Holloway et al. 1999). However, hormone levels in anadromous steelhead have not been reported in the literature, and are described in the present study. Methods A portable Aloka SSD-500v ultrasound machine equipped with a 7.5-MHz linear probe was used to examine the gonads of prespawn steelhead and kelts. The probe was capable of imaging up to 9 cm of tissue depth with a horizontal plane of 4 cm. The examination consisted of gently placing the ultrasound probe along each fish s abdomen and then moving the probe along the abdominal surface until the gonads were located. For females, the presence or absence of eggs within the body cavity was noted. For males, the maximum crosssectional area (cm 2 ) of the single largest testis within the ultrasound s range was measured within the body cavity. Precise measurements of the testis were taken by use of an elliptical trackball function that is commonly found on ultrasound units. Once the gonads were located and a clear ultrasound image was obtained, pictures from each fish were recorded on a standard diskette in a Mavicap digital adaptor and stored for later viewing. The first test with ultrasound on adult steelhead was conducted in March 1999 at the Minthorn Hatchery, located on the Umatilla River (UR; 465 rkm from the mouth of the Columbia River), Oregon. In all, 12 steelhead (6 females and 6 males) were examined with ultrasound before and after artificial stripping of the eggs and milt. Following artificial spawning, autopsies were conducted to correlate anatomical structures with ultrasound images. In 2000, we focused on males to both confirm the limited number of observations made in 1999 and to develop a more comprehensive database. Adult steelhead were again sampled from the UR, and additional samples were collected from the Wallowa Hatchery on the Wallowa River (WR; 293 rkm from the mouth of the Snake River), Oregon, and from the Prosser Hatchery on the Yakima River (YR; 539 rkm from the mouth of the Columbia River), Washington. Steelhead returning to the WR and UR were examined with ultrasound just before artificial stripping on 29 March and 4 April 2000, respectively. All steelhead examined at WR and UR facilities produced milt via a gentle stripping of the abdominal cavity and were thus considered to be spermiating. Yakima River steelhead examined via ultrasound were from a subsample of individuals that had spawned naturally and that were held captive for kelt reconditioning experiments. Unlike the mature specimens examined at the WR and UR, postspawn YR fish did not produce milt upon gentle stripping of the abdomen. The YR fish were examined between 27 March and 18 May In addition to the naturally spawned kelts examined from the YR, autopsies were conducted on seven kelts (three males and four females) that died during the reconditioning experiment on 15 June Adult steelhead awaiting artificial spawning at the UR (n 13) and WR (n 42) hatcheries were killed before sampling. Kelts removed from the YR (n 34) were transferred via dip net to a nearby 190-L sampling tank containing fresh river water, where they were anesthetized in a buffered solution of tricaine methanesulfonate (MS-222) at 60 mg/l and released into the reconditioning tanks following ultrasound examination and measurement of fork-length. Ultrasound examination time, including the time needed to anesthetize specimens from the YR, was approximately 4 min/fish. For plasma hormone levels, a blood sample was removed from the caudal vein of each male (UR: n 12; WR: n 15; YR: n 13) by use of a Vacutainer containing heparin to prevent coagulation. Blood samples were kept on ice until the plasma could be separated by centrifugation and

4 970 EVANS ET AL. stored at 70 C. All plasma samples were analyzed for T, 11-KT, and DHP by radioimmunoassay based on the procedures of Fitzpatrick et al. (1986). Each sample was assayed in duplicate, and the intra-assay coefficient of variation (CV 100 SD/mean) was calculated for the duplicates. Samples with intra-assay CVs in excess of 10% (n 3 or 2.5% of all samples collected) were considered contaminated and were removed from the data set. Average extraction efficiency for all three steroids was 88%, with a minimum assay detection threshold of 1.2 ng/ml for T, 1.1 ng/ml for 11- KT, and 0.7 ng/ml for DHP. The intra-assay CV was below 3% for all three steroids assayed. Reported steroid levels were corrected for extraction efficiencies. Ultrasound data on maximum testis cross-sectional area (cm 2 ) from males were compared between sample populations and maturational types by use of one-way analysis of variance. Discriminant function analysis (DFA) was used to develop a testis size classification rule based on the variation in testis area measurements observed between maturational types. The variance covariance matrices between mature and kelt groups were not considered equal (P 0.01, chi-square); therefore, a quadratic DFA was used to compare testis measurements (Khattree and Naik 2000). To evaluate the classification rule, a cross-validation technique was used to estimate classification error based on the number of misclassified individuals for each maturational type and for the sample as a whole (overall error). Distributions of the steroid concentrations were nonnormal in mature steelhead due to the presence of outliers, and steroid concentrations in kelts often were below the assay detection level. Thus, steroid levels were compared by use of the Wilcoxon rank-sum procedure, a distribution-free and outlier-sensitive test (Ramsey and Schafer 1997). Classification criteria for plasma hormone concentrations were again calculated via DFA, based on a nonparametric approach (k-nearest-neighbor method; Khattree and Naik 2000) to account for data nonnormality. Error rates were again estimated by use of the cross-validation approach (Khattree and Naik 2000). Simple linear regression was used to correlate testis area with logtransformed steroid concentrations. All statistical tests were run in the Statistical Analysis System for Windows release 8.0 (SAS Institute 1999). We report means with SD values. Statistical significance was set at 0.05, although exact P-values are reported where available. Results Ultrasound Examination The region just posterior to the pectoral fins provided the most-diagnostic images of testes or ovaries. However, other locations along the abdomen (e.g., just posterior to the pelvic fins) also provided useful diagnostic images. In some circumstances, more than one area within the fish s body cavity had to be examined before diagnostic images could be obtained. The imaging of visceral organs (e.g., liver, spleen, pyloric caecae, and gallbladder) just posterior to the transverse septum served as landmarks for locating the gonads. Identification of visceral organs was an important first step in making gonad measurements because these structures were present in every fish regardless of sex or reproductive developmental stage. Each fish s swim bladder consistently blocked ultrasound images near the posterior end of the gonad because acoustic waves reverberated off the swim bladder, thus distorting ultrasound imaging. Fortunately, gonad measurement was still possible because the majority of the gonad resides anterior to the swim bladder. Results of autopsies from the seven YR kelts that died during the reconditioning experiment at the Prosser Hatchery demonstrated that kelt testes smaller than 0.20 cm 2 were indiscernible from the midgut and surrounding pyloric caecae due to gonad atrophy. Autopsies of female kelts revealed the presence of new oocytes, often located with a few remnant overripe eggs. Similar to kelt testes smaller than 0.20 cm 2, the new pair of female kelt ovaries could not be distinguished via ultrasound imaging because of their small size (oocyte diameter 1 mm). Prespawn females were easily distinguished from freshly postspawned females by the presence of a mature egg mass. The ultrasound was sensitive enough and provided such high-resolution images that individual eggs within the female body cavity could be easily discerned (Figure 1). The egg mass in prespawn steelhead was so dominant that visceral organs were often obstructed from view by the numerous, highly reflective, well-developed eggs. Conversely, female kelts were characterized by the presence of few remnant eggs within the body cavity (Figure 2), often lodged just underneath the abdominal muscle tissue or anterior to the urogenital papilla (an appendage used to extrude eggs during spawning). Eggs were often uniform in size (approximately 0.30 cm in diameter). Unlike female steelhead, some males retained

5 MATURATIONAL STATUS IN STEELHEAD 971 FIGURE 1. Photograph of a prespawn female steelhead (left) and a cross-sectional ultrasound image (right) showing the tightly packed egg mass of a prespawn female. Egg membranes are hyperechoic and produce white rings around the darker, hypoechoic egg contents. The ultrasound image was made with the specimen inverted. substantial gonad tissue following spawning. Comparison of the images from male gonads between the two maturational types revealed two trends: differences in maximum testis area and differences in gonad echogeneity. Echogeneity refers to the acoustic properties of tissues and is depicted on the ultrasound monitor by different shades of black and white. The gonads of prespawn steelhead appeared more hypoechoic (dark) relative to the hyperechoic (white) gonads of kelts. Ultrasound images from a mature, prespawn male (Figure 3) and a male kelt (Figure 4) illustrate the typical differences in testis size and echogeneity between maturational types. Investigations of the maximum testis area in 55 prespawn male steelhead and 34 kelts demonstrated that testes underwent substantial size changes following spawning (Figure 5). Testis area measurements were normally distributed and bimodal, and did not overlap between the two groups (Figure 5). Testis size did not differ between prespawn samples at WR versus UR(P 0.25), but highly significant differences in mean testis area were detected between prespawn males and YR kelts (P 0.001). Mean testis area was cm 2 in kelts compared to cm 2 in prespawn males, a nearly threefold difference. Based on results from the DFA, the generalized square distances between maturational types were calculated as for male kelts and for mature, prespawn males, demonstrating a high degree of separation. Based on results of the quadratic DFA, the classification rule (threshold) for assigning individual observations was 1.25 cm 2. In other words, the prediction was that all males with a testis area of 1.25 cm 2 or greater were prespawners, whereas males with a testis area less than 1.25 cm 2 were kelts. Posterior probability of mem- FIGURE 2. Photograph of a postspawn female steelhead (left) and a cross-sectional ultrasound image (right) showing three remnant eggs just underneath the specimen s abdominal muscle tissue. Egg membranes are hyperechoic and produce white rings around the darker, hypoechoic egg contents. The ultrasound image was made with the specimen inverted.

6 972 EVANS ET AL. FIGURE 3. Photograph of a prespawn male steelhead (left) and a cross-sectional ultrasound image (right) showing the testes (two dominant lobes) of a prespawn male. The testis on the ultrasound image appears as a grayish (hypoechoic), elliptical mass in the center of the image and is highlighted by an arrow. The fish s liver and fluidfilled gallbladder (GB) are also depicted on the ultrasound image. The testis measurement (2.89 cm 2 ) is shown along the right margin of the ultrasound image. bership, a measure of the degree of classification confidence, ranged from 62.36% to 99.98% among kelt samples; the probability of correct classification was greater than 95% in individuals with a testis area of 1.02 cm 2 or less. Of the 34 individual kelt testis measurements obtained, only two measurements (6%) were greater than 1.02 cm 2. Posterior probability of membership ranged from 96% to 100% among prespawn fish, and individuals with a testis area greater than 1.41 cm 2 had a higher than 95% probability of correct classification. Of the 55 individual prespawn testis measurements obtained, 100% were greater than 1.41 cm 2. Crossvalidation techniques in the DFA estimated an overall ultrasound classification error rate of for male specimens. Plasma Hormone Profiles Measurements of T, 11-KT, and DHP demonstrated that prespawn male steelhead and kelts possessed significant differences in plasma hormone levels (Figure 6). All three steroid hormones measured in kelt samples were significantly lower than those from prespawn male samples (P 0.001). Plasma concentrations among prespawn males were highly variable. Testosterone levels in prespawn males ranged from 11.6 to 78.7 ng/ml, 11- KT levels ranged from 25.7 to ng/ml, and DHP levels ranged from 2.8 to 54.2 ng/ml (Figure 6). Despite the variation in plasma hormones found among prespawn males, values were substantially higher than those found in male kelts. In fact, plas- FIGURE 4. Photograph of a postspawn male steelhead (left) and a cross-sectional ultrasound image (right) showing the testis (located at tip of probe) of a male postspawner. The testis on the ultrasound image appears as a white (hyperechoic), elliptical mass in the center of the image and is highlighted by an arrow. The testis measurement (0.76 cm 2 ) is shown along the right margin of the ultrasound image.

7 MATURATIONAL STATUS IN STEELHEAD 973 FIGURE 5. Maximum testis area distributions for postspawn male steelhead from the Yakima River, Washington, and prespawn male steelhead from the Wallowa and Umatilla rivers, Oregon. Measurements were based on ultrasound examination and represent the largest testis area found along the length of the gonad. ma levels in kelts were often below the minimum assay detection threshold (e.g., T), and only trace amounts of 11-KT and DHP could be detected in fish following spawning (Figure 6). Of the three steroids measured, only concentrations of DHP overlapped between the two maturational categories: one prespawn individual had a value typical of DHP within the kelt group (Figure 6). Median T levels for prespawn males from the WR and UR were 46.2 and 50.7 ng/ml, respectively, and were not significantly different (P 0.68). Testosterone levels from YR male kelts were below the minimum assay detection level and were always less than 1.2 ng/ml. Median levels of DHP for prespawn fish were 16.0 ng/ml for WR samples and 10.0 ng/ml for UR samples; the two means differed significantly (P 0.02). Similar to the results for T, DHP levels from the 13 kelts were often below the assay detection level. The median DHP level for kelts was 1.6 ng/ml. Median 11-KT levels for prespawn fish from the WR (116.0 ng/ml) and UR (65.5 ng/ml) were significantly different (P 0.01). The median 11-KT concentration from kelt samples was 6.6 ng/ml, and all 13 measurements were within the limits of the assay s standard curve (Figure 6). Nonparametric DFA of the steroid data showed a high degree of separation between the maturational types. The classification rules for assigning individual observations were 15.3 ng/ml for 11- KT and 5.3 ng/ml for DHP. The DFA was not conducted on T concentrations because all kelt samples were below the assay detection threshold ( 1.2 ng/ml); however, a minimum gap of 10.3 ng/ml existed between kelt and prespawn T concentrations. Cross-validation techniques in the DFA resulted in a 100% (n 35) discrimination of 11-KT concentrations between the two maturational types, and the minimum gap between types was 20.7 ng/ml. Cross-validation assignment resulted in an estimated error of 0.02 for DHP samples; one prespawn individual was erroneously classified as a kelt. There was no evidence to suggest that maximum prespawn testis area was associated with prespawn concentrations of plasma hormones. Regression slopes of testis area versus log-transformed values of T, 11-KT, and DHP were not significantly different from zero (WR: P 0.71 [T], 0.73 [11-KT], 0.79 [DHP]; UR: P 0.38 [T], 0.25 [11-KT], 0.53 [DHP]). Similarly, kelts with larger maximum testis area did not have elevated concentrations of 11-KT or DHP (YR: P 0.46 [11-KT], 0.96 [DHP]). Comparison between kelt testis area and

8 974 EVANS ET AL. FIGURE 6. Distributions of plasma concentrations of testosterone, 17 -hydroxy-20 -dihydroxyprogesterone (DHP), and 11-ketotestosterone (11-KT) in postspawn male steelhead from the Yakima River (squares), Washington, and in prespawn male steelhead from the Wallowa River (WR diamonds) and Umatilla River (UR triangles), Oregon. Median values are indicated by arrows, and the minimum assay detection threshold for each steroid is denoted by a dotted line. T concentration was not possible because all T concentrations were below the assay s standard curve. Discussion Ultrasound proved to be a rapid, noninvasive way to distinguish between prespawn steelhead and kelts. Ultrasound images provided quantifiable selection criteria (number of eggs and/or size of the gonads) for the classification of spawning types among the samples examined. An obvious difference in gonad mass and egg number between prespawn females and female kelts was apparent, although kelts frequently retained a few eggs. Ultrasound readily detected the chorionic membrane surrounding each egg yolk as a distinctive ring. Male steelhead were readily identifiable by the presence of testicular tissue, but determination of their maturational status was more difficult relative to females because some males retained considerable gonad mass after spawning. However, ultrasound measurements of testis size provided overwhelming evidence of differences between prespawn males and kelts that had spawned naturally. The distributions of testis area measurements for the two maturational types did not overlap. In addition, differences in gonad echogeneity were detected between prespawn males and kelts, providing further evidence of maturational differences. Following spermiation, remnant testicular mass may be absorbed or engulfed by macrophages, which ultimately results in both smaller testes and a denser testicular mass (Billard 1983; Cauty and Loir 1995). Although differences in testis density as displayed by ultrasound echogeneity are subjective and not quantifiable because measurements are on the nominal scale, additional research may eventually lead to the development of a quantifiable criterion for this observable characteristic. In the present study, prespawn female ovaries and prespawn male testes examined via ultrasound were at the final stages of development and dominated the body cavity, maximizing differences between mature fish and kelts. However, the degree of distinction between prespawn steelhead and kelt gonads will depend upon the magnitude of gonad development differences present at the time of ultrasound appraisals. In the case of female steelhead, this may only be problematic if the oocytes and ovary are in the very early stages of development (e.g., months prior to spawning). In rainbow trout, the ovaries undergo a pronounced increase in weight 4 5 months prior to spawning as oocytes accumulate exogenous vitellogenin. For example, Sumpter et al. (1984) reported an increase in rainbow trout ovary weight from 9.59 g to over 200 g in the 5-month period prior to spawning. Thus, egg and gonad size in maturing females are likely to be large enough to be readily detected via ultrasound months prior to spawning. Conversely, the presence of few large, overripe eggs in the body cavity is a clear indication of postspawn maturational status. If the variation in testis size among the two maturational types we examined is applicable to other summer-run steelhead populations in the region, then individuals with a maximum testis area of 1.25 cm 2 or greater can accurately be classified as

9 MATURATIONAL STATUS IN STEELHEAD 975 prespawners. The probability of an erroneous classification will diminish considerably with increasing deviation from the classification rule. We found a greater than 95% probability of correct identification for male kelts with testes 1.02 cm 2 or smaller and for prespawn males with testes larger than 1.41 cm 2. Conversely, if the variation in testis area among these samples does not persist in other populations, or if immature, prespawn males are encountered during ultrasound exams, misclassification of maturational type is more likely to occur. Unlike developing eggs, individual spermatozoa cannot be distinguished within the gonad via 7.5-MHz ultrasound examination, and the entire gonad must be at least 0.20 cm 2 to be distinguishable from other anatomical structures in the visceral region. Billard (1983) demonstrated that testicular mass of brown trout Salmo trutta was elevated and relatively consistent 3 months prior to spermiation. Mean testicular mass was only 0.79 g in immature brown trout prior to spermatogenesis but was g during spermatogenesis. Ninety-five percent of the testicular mass was generated during the first 4 weeks of development. In the 2 months following spawning, mean testicular mass dropped by 85% as compared to mature mass (Billard 1983). In rainbow trout, a 10-fold increase in the gonadosomatic index (GSI) was documented over the course of 2 months; GSI values peaked 1 2 months prior to spawning (Scott and Sumpter 1989). Once testicular mass and size had peaked, they remained fairly constant until the conclusion of spawning, at which time dramatic decreases were noted (Scott and Sumpter 1989). Based on the data presented here and those of other researchers, differences in gonad size between prespawn and postspawn males should remain elevated for sufficient periods to allow accurate determination of maturational status via the ultrasound approach. To minimize erroneous classification in ultrasound measurements, researchers should possess knowledge of the reproductive cycle of the species under examination, should have an indication of potential migration overlaps between prespawners and postspawners, and should conduct sufficient preliminary ultrasound studies to describe gonad size variation in the populations of interest. Furthermore, the cost of misclassification should also be considered, depending on the particular research goals and objectives. For example, there may be circumstances in which it is not permissible to misclassify a prespawn individual as a postspawner (e.g., steelhead kelts collected for reconditioning or quantification of spawning success). If previous data exist on gonad size variation, then conservative classification criteria can be adopted to minimize the probability of misclassifying a specific maturational category. Various modeling exercises (e.g., DFA) can be utilized to both describe the variation in gonad size and to estimate classification error rates. The use of ultrasound images to identify mature, prespawn males and male kelts in this study was corroborated by blood plasma steroid data. Miltproducing specimens not only had a significantly larger maximum testis area but also had significantly elevated levels of T, 11-KT, and DHP relative to kelts. The concentrations of T, 11-KT, and DHP observed among prespawn male steelhead in the present study are similar to those reported by other researchers. During the spawning period (i.e., in mature fish), Campbell et al. (1980) and Scott et al. (1980) reported mean T levels of approximately 60 and 70 ng/ml, respectively, in resident male rainbow trout. Campbell et al. (1980) measured a mean 11-KT level of 98 ng/ml, and Baynes and Scott (1985) reported 11-KT levels ranging from 80 to 100 ng/ml in male rainbow trout. Baynes and Scott (1985) also observed mean DHP concentrations between 20 and 30 ng/ml in mature males. In the present study, all male kelts had T concentrations below 1.2 ng/ml, and only 8 of 13 kelts had measurable concentrations of DHP ( ng/ml). Male kelts did have measurable concentrations of 11-KT ( ng/ml), but the values were significantly lower than those found in prespawn fish. In a study linking rainbow trout gonad regression with changes in hormone concentrations, Baynes and Scott (1985) observed values of T, 11-KT, and DHP in postspawn males similar to the values we report here. In the 3 months following spermiation, hormone concentrations dramatically declined to approximately 10 ng/ml for T, 4 ng/ml for 11-KT, and 3 ng/ml for DHP in non-milt-producing males (Baynes and Scott 1985). Profiles of steroids in resident rainbow trout have linked peak T levels with spermatogenesis (Billard 1978; Scott et al. 1980) and peak 11-KT levels with spermiation (Campbell et al. 1980; Baynes and Scott 1985). Numerous studies have demonstrated that T peaks prior to 11-KT, and both steroids continue to decline throughout the spawning period. Similar cyclical patterns in T and 11- KT have also been reported in male coho salmon O. kisutch (Fitzpatrick et al. 1986), arctic char Salvelinus alpinus (Elofosson et al. 2000), and Atlan-

10 976 EVANS ET AL. tic salmon Salmo salar (Hunt et al. 1981). The level of DHP has been positively correlated with milt volume, spermatocrit, and the total number of spermatozoa produced in the testes, and is suspected to control the ionic composition of seminal fluids in male rainbow trout (Baynes and Scott 1985; Vizziano et al. 1996). The mature steelhead examined here had steroid levels similar to those of resident, captive stocks of rainbow trout, suggesting that similar cyclical changes in hormone concentration took place prior to and following spawning. Although both ultrasound gonad measurements and steroid concentrations could be used to discriminate between prespawn male steelhead and kelts, the ultrasound method has numerous advantages over radioimmunoassay measurements of steroid concentrations. Unlike the radioimmunoassay technique, ultrasound is rapid and noninvasive, and can be conducted both in the field and in a laboratory environment. Furthermore, the training, labor, and costs associated with ultrasound use are minimal compared to the expertise, time, and laboratory equipment cost required to conduct radioimmunoassays. An ultrasound operator must simply be familiar with the equipment, the specimen s reproductive cycle, and general visceral anatomy in order to interpret diagnostic images. Once anesthetized, individual fish can be examined with ultrasound in less than 30 s, and images can be stored on a computer for future viewing. Although the primary objective of this study was to develop ultrasound classification criteria to distinguish prespawn from postspawn steelhead encountered at main-stem hydroelectric facilities in the Snake River basin (see Evans 2003; Evans et al., in press), ultrasound has far-reaching potential for other fisheries research applications. In the aquaculture of salmonids, ultrasound can be used to distinguish maturing broodfish from immature individuals, allowing mature fish to ripen in isolation and immature fish to continue somatic growth and reproductive development via feeding. Because ultrasound can identify sex in captive salmonids up to 5 months prior to spawning (Martin et al. 1983; Arkush and Petervary 1998), a single ultrasound examination can substantially reduce handling of broodstock and may ultimately reduce the stress and disease transmission associated with multiple handling events. Ultrasound has also recently been used to diagnose structural abnormalities in fish (Poppe et al. 1998). Lastly, ultrasound may provide an alternative to the surgical biopsies used for determining reproductive development in white sturgeon Acipenser transmontanus (Webb et al. 2002). Webb et al. (2002) recently used profiles of reproductive hormones to develop a lessinvasive method of determining both the sex and reproductive development of white sturgeon. Classification success in their study ranged from 96% in mature males to 98% in maturing females. Ultrasound may provide a more rapid and noninvasive way to diagnose maturational status of white sturgeon while achieving accuracy levels similar to those of reproductive hormone concentrations. Acknowledgments We thank Alfred Caudle, University of Georgia, College of Veterinary Medicine, for helping to select the appropriate ultrasound equipment and for providing instructions for its initial use. Our thanks go to Gerry Rowan, Greg Davis, and Joe Blodgett for allowing us to sample adult steelhead at their hatcheries. We are grateful to Roy Beaty for valuable contributions to the design and initial review of the study. We thank Clifford Pereira for providing advice regarding statistical analysis. Staff members of the Columbia River Inter-Tribal Fish Commission provided equipment, services, and logistical support, for which we are grateful. The U.S. Army Corps of Engineers provided partial funding for this research, and we would like to thank Rebecca Kalamasz, Rex Baxter, and Mike Halter for their assistance. Doug Markle, Darius Adams, and Ken Collis provided helpful comments that improved earlier drafts of this manuscript. References Arkush, K. D., and N. A. Petervary The use of ultrasonography to predict reproductive development in salmonid fish. International Association for Aquatic Animal Medicine 29: Baynes, S. M., and A. P. Scott Seasonal variations in parameters of milt production and in plasma concentration of sex steroids of male rainbow trout (Salmo gairdneri). General and Comparative Endocrinology 75: Billard, R A quantitative analysis of spermatogenesis in the trout Salmo trutta fario. Cell and Tissue Research 230: Blythe, B., L. A. Helfrich, W. E. Beal, B. Boswarth, and G. S. Libey Determination of sex and maturational status of striped bass (Morone saxatilis) using ultrasonic imaging. Aquaculture 125: Bonar, S. A., G. L. Thomas, and G. B. Pauley Use of ultrasonic images for rapid nonlethal determination of sex and maturity of Pacific herring.

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