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Histological Assessment of Organs in Sexually Mature and Post-spawning Steelhead Trout
and Insights into Iteroparity
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Zachary L. Penney1 and Christine M. Moffitt1,2 *
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penn4282@vandals.uidaho.edu; 208-885-7139 (phone)
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Department of Fish and Wildlife Sciences, University of Idaho, Moscow, Idaho, USA;
US Geologic Survey, Idaho Cooperative Fish and Wildlife Research Unit, Department of Fish
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and Wildlife Sciences, University of Idaho, Moscow, Idaho, USA, cmoffitt@uidaho.edu; 208-
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885-7047 (phone); 208-885-9080 (fax)
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* Corresponding author, Christine M. Moffitt
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For Reviews in Fish Biology and Fisheries
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Special issue: Teaming up Atlantic and Pacific Salmonid Biologists to Enhance Recovery of
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Endangered Salmon in North America
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This draft manuscript is distributed solely for purposes of scientific peer review. Its content is
deliberative and predecisional, so it must not be disclosed or released by reviewers. Because the
manuscript has not yet been approved for publication by the U.S. Geological Survey (USGS), it does
not represent any official USGS finding or policy."
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Abstract
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Steelhead (Oncorhynchus mykiss) are anadromous and iteroparous, but repeat-spawning rates are
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generally low. Like other anadromous salmonids, steelhead fast during freshwater spawning
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migrations, but little is known about the changes that occur in vital organs and tissues.
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Intuitively, it would seem that any fish capable of repeat spawning would not undergo the same
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irreversible cellular degeneration as occurs in semelparous salmon and begin replacing energy
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soon after spawning. Using Snake River steelhead as a model we examined the gastrointestinal
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tract of post-spawn steelhead (kelts) to document if freshwater feeding was occurring, and used
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histological analysis to assess the cellular architecture in the pyloric stomach, ovary, liver, and
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spleen in sexually mature and kelt steelhead. We found 38% of the kelts contained food or fecal
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material in the gastrointestinal tract and that feeding was related to external condition. We found
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a significant renewal of villi in the pyloric stomachs of kelts was associated with feeding. No
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vitellogenic oocytes were observed in sections of kelt ovaries, but perinucleolar and early/late
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stage cortical alveolus stages were present suggesting iteroparity was possible. We documented a
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negative correlation between the quantity of perinucleolar oocytes in ovarian tissues and fork
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length suggesting that larger steelhead may invest more into a single spawning event. Liver and
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spleen tissues of kelts were found to show minimal cellular deterioration. Together, these
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findings indicate that the physiological processes causing rapid senescence and death in
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semelparous salmon are not the same in steelhead, and recovery begins in freshwater.
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Keywords: Iteroparity, Histology, Steelhead, Fasting
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Introduction
Within the salmonidae family two reproductive life history strategies exist: semelparity and
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iteroparity. Semelparity is exclusive to the Pacific salmon (Oncorhynchus sp) that spawn once
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and die, although exceptions to this have occasionally been documented (Tsiger 1994; Unwin et
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al. 1999). All other salmonids are iteroparous, but the degree of inter and intra-specific post-
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spawning survival is highly variable (Crespi and Teo 2002; Quinn and Meyers 2004). Steelhead
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O. mykiss, the anadromous form of rainbow trout, and Atlantic salmon Salmo salar are
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considered iteroparous, but show complex polytypic life history strategies that involve
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residualism (Viola and Schuck 1995, Christie et al. 2011), anadromy (Schaffer and Elson 1975;
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McDowall 1987; Pascual et al. 2001), and combinations of life history strategies (Docker and
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Heath 2003; Thrower et al. 2004; Pearse et al. 2009; Null et al. 2013). Iteroparity can increase
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genetic diversity of stocks and individual lifetime fitness (Seamon and Quinn 2010), as well as
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provide a safeguard against complete brood year failures (Narum et al. 2008). Consequently,
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anadromy can complicate many of the benefits afforded by iteroparity. Foremost, the energetic
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and physiological costs of migrations between seawater and freshwater can be high and repeat
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spawning rates among steelhead and Atlantic salmon are generally low (<10%) (Fleming and
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Reynolds 2004).
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Within steelhead, a range of life history strategies can exist, including stream-maturing
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(summer/fall run) and ocean-maturing (winter/spring run) reproductive strategies (Behnke 1992).
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Stream-maturing steelhead return to freshwater early (June-November) and undergo the final
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stages of gonadal maturation while in freshwater, whereas ocean-maturing steelhead return to
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freshwater (December-April) much closer to the time of spawning. As a spring spawner, low
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rates of iteroparity in stream-maturing steelhead have been commonly attributed to energy
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limitations due to longer migration distances and the extended time in freshwater compared to
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ocean-maturing steelhead (Burgner et al. 1992). However, high post-spawning mortalities in
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ocean-maturing steelhead (Busby et al. 1996) has not been well explained. Considering the
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differences in migration distance and time spent in freshwater between the two ecotypes, it
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appears that factors other than simple energetic depletion may contribute to post-spawning
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survival.
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A defining characteristic expressed by both semelparous and iteroparous anadromous
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salmonids, including steelhead, is the prolonged fasting that accompanies freshwater re-entry to
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spawn. Most anadromous salmonids enter a period of voluntary anorexia during spawning
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migrations and rely on lipids and protein stored in somatic and visceral tissues. It has been
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theorized that by discontinuing active consumption in freshwater during spawning, Pacific
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salmonids and Atlantic salmon can curtail the energy required for digestion, which can account
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for as much as 40% of basal metabolism in feeding fish (Wang et al. 2006). Through fasting,
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anadromous salmonids may able to conserve energy by significantly lowering the energetic
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demands of their basal metabolism, thus allocating the bulk of their stored energy to support
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upstream migration, completion of gonadal maturation, the physical exertion of spawning, and in
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the case of iteroparous species, emigration back to the ocean.
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Post-spawn steelhead and Atlantic salmon, known as kelts (Allan and Ritter 1976), are
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known to re-initiate feeding activity in freshwater following spawning (Quinn and Myers 2004),
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but it is presumed that majority of somatic energy is replaced in the ocean. However, the low
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proportion of repeat-spawners in steelhead and Atlantic salmon populations suggests that most
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kelts do not survive despite attempts at feeding. In some cases, such as stream-maturing
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Columbia River steelhead the rates of repeat-spawning are so low that the stocks have often been
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regarded, as functionally semelparous (Burgner et al. 1992). Little is known about the effects that
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prolonged fasting and catabolism have on organ systems in steelhead or Atlantic salmon,
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especially those involved in digestion, energy storage, and vitellogensis. Studies with other non-
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mammalian vertebrates has shown that there is a gradual reduction of intestinal epithelium
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(atrophy) and nutrient transport capacities of the gastrointestinal tract during fasting, but that
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digestive processes are rapidly restored at the onset of feeding (Wang et al. 2006; Secor et al.
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2002). It therefore seems apparent that for steelhead or Atlantic salmon kelts to recover
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energetically, their digestive functions must also be restored. Additionally, little is known about
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how other organ systems involved in energy storage, blood filtration, waste removal, and red
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blood cell production respond in recovering kelts.
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In the following study, Snake River steelhead were used as a model to qualitatively and
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quantitatively evaluate the histological architecture of liver, spleen, gastrointestinal, and ovary
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tissues to provide insight on the physiological effects of prolonged fasting in an iteroparous
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salmonid. Histological assessments provide information about tissue function at the cellular level
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and can reveal physiological information that may not be apparent via external condition and
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non-lethal measures of nutrition (i.e. blood chemistry, fat-meter). The primary objectives of this
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study were to (1) determine of Snake River kelts were attempting to replace energy via during
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freshwater emigration, and (2) describe the gross changes in cellular architecture in pre-
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spawning and post-spawning fish and evaluate the capacity for repeat-spawning based on the
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functionality of tissues.
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Methods
Sample locations and procedures
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Sections of the stomach, ovarian, liver, and spleen tissues of Snake River steelhead were
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sampled in 2009 and 2010 at two phase of the reproductive cycle, first at or near sexual maturity
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(mature) and secondly in migrating kelts (Table 1). Samples collected at maturity were obtained
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from hatchery stocks returning to Dworshak National Fish Hatchery (DNFH) (46°30’N, -
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116°19’W) or intercepted at Nez Perce Tribal Hatchery on the Clearwater River, ID (46°30’N, -
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116°39’W ). Samples from kelts were collected from mixed stocks of Snake River steelhead at
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the Lower Granite Dam juvenile bypass facility (46°39’N, -117°26’W), WA (Fig 1). We
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sampled to select representative proportions of female and male steelhead at both phases for
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comparison, but few male kelts were ever captured (Table 1).
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Before sampling, steelhead were euthanized with a lethal dose (200mg/L) of MS-222
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(Finquel, Argent Laboratories, Redmond, WA) buffered with NaHCO3 or in some cases CO2 was
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used at DNFH. Fish were measured for fork length (nearest 0.5 cm). All mature steelhead were
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considered to be in good external condition, whereas the condition of kelts was rated as good,
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fair, or poor. Kelts in good condition exhibited no or only minor injuries, minimal fungal
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infection, minor fin deterioration, silver or bright coloration, firm tissue texture, and were active.
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Kelts in fair condition exhibited moderate injuries (non-life threatening), fungal infections over
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1-10% of the body, moderate fin deterioration, pale coloration, and moderate activity. Kelts in
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poor condition exhibited severe injuries (life threatening), fungal infections greater than 10% of
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the body, severe fin deterioration, pale coloration, spongy tissue texture, and were listless. The
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gastrointestinal tract of kelts was examined at the time of necropsy for the presence of
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identifiable food or evidence of digestion via the presence of fecal material. Sections of the
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stomach, liver, spleen, pyloric stomach, and ovarian tissues (kelts only) were removed using a
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scalpel and transferred to jars containing 10% buffered neutral formalin (pH 6.8) at a 10:1 ratio
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of formalin for tissue fixation. After fixation the stomach was sagittally bisected to provide a
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cross section of the pyloric stomach. Fixed tissues were shipped to Colorado Histo-Prep (Fort
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Collins, Colorado) for processing, where tissues were dehydrated, embedded in paraffin,
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sectioned at ~ 4 to 6 μm, mounted on glass microscope slides, and stained with hematoxylin and
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eosin (H&E) (Luna 1968).
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Histological analysis
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Histological analyses and scoring were accomplished with a compound light microscope
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(Leitz Laborlux) fitted with a Leica EC3 camera and photographic software (LAS EZ 1.8.0;
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Leica Microsystems, Cambridge, Ltd). Evaluations were made at magnifications of 4X, 10X, and
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40X, which provided fields of view at 9.6 mm2, 1.5 mm2, and 0.1 mm2, respectively. We
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evaluated tissue architecture with a categorical scoring system (Table 2). All scoring was
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blinded.
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Pyloric stomach
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We evaluated and scored cross sections of the pyloric stomach using six metrics. We
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quantified the detachment of the submucosa from the surrounding muscularis and stratum
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compactum as severe, moderate, and minor to no detachment. The density of villi, which are
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comprised of columnar epithelial cells and goblet cells lining the stomach lumen was scored as
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low, moderate, and high. The extent of villiar invagination was estimated based on depth or
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distance of the tip of villi (lamina epithelialis) to the stratum compactum. Villi invagination was
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then scored as <1/4 the distance to the stratum compactum, 1/4 to 1/2 the distance to the stratum
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compactum, or > 1/2 the distance to the stratum compactum. The cellular integrity of columnar
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epithelial cells comprising the villi was evaluated and ranked by observing the severity of
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deterioration of the cell membrane, cytoplasm and nucleus. The results were scored as severe,
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moderate, and minor to no (absent) deterioration. The length to width ratio of columnar epithelial
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cells was assessed to examine changes in overall cell size. As the name suggests, it was assumed
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that columnar epithelial cells would exhibit length to width ratios similar to a rectangle (length >
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width) rather than square (length = width). The length to width ratio of columnar epithelial cells
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was ranked either as < 1/2 length to width ratio or > 1/2 length to width ratio. These measures
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were validated via micrometer-calibrated measurements of photographs. Finally, we evaluated
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the lamina epithelialis for the presence or absence of goblet or mucous cells to provide an
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indication of mucous secretion. All scores for the pyloric stomach were assessed over the entire
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tissue, regardless of magnification.
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Ovary
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We enumerated the quantity of perinucleolar oocytes (pre-lipid deposition), the quantity of
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early/late cortical alveolus stage oocytes (lipid deposition present), and the quantity of
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vitellogenic oocytes ( lipid and yolk deposition) in three to four fields for each ovary section.
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Only oocytes sectioned through the nucleus or exhibiting clear lipid or yolk deposition were
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enumerated in each field and it was assumed that all oocytes were randomly distributed within
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the ovary. To validate this assumption, we compared the frequency of oocyte counts by category
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and field. We found 92% of the ovarian tissues (N = 51) showed no significant variations across
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fields and concluded pre and post-lipid oocyte distribution was random and could be averaged
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across all fields by dividing the total sum of pre and post-lipid oocytes by the total number of
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fields examined.
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Liver
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Five metrics were assessed (Table 2): The density of melanomacrophage aggregates
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dispersed in the parenchyma was scored as none visible, low (< 5 aggregates), and high (> 5
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aggregates). The separation of hepatocytes within their respective cords was evaluated for the
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amount of detachment and scored as light, moderate, and minor or none. The overall separation
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between heptatocyte cords (sinusoid spaces) was estimated and classified as less than 10μm or
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greater than 10μm. The proportion of vacuolization within parenchyma was observed and ranked
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as absent, between 1-10% in the field of view, and greater than 10% in the field of view.
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Hepatocyte cellular structure was scored to consider integrity of the cell membrane, cytoplasm,
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and nucleus and ranked as severe, moderate, or minor to no (absent) cellular deterioration. The
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density of melanomacrophage aggregates, the apparent hepatocyte detachment, and sinusoid
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spacing were scored over the entire liver tissue area at 10X. Scores for the degree of
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vacuolization and hepatocyte integrity from each microscopic field assessed at 40X were
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averaged and rounded up.
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Spleen
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Spleen tissues were evaluated to consider the distribution of white pulp nodules within the
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red pulp as discrete, or diffuse wherein no clear separation of the white and red pulp was visible.
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The percent of fields occupied by red pulp was estimated and categorized as 0-39%, 40-60%, or
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61-100%. We evaluated the presence or absence of maturing erythrocytes and leukocytes by
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noting the presence of differentiating cells or cell types. All scores for the spleen were derived
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from assessments of the entire tissue, regardless of the magnification used.
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Statistical analysis
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Statistical comparisons of the frequency of scoring metrics by category within each tissue
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were made to explore the differences within and between both mature and kelt phases. Within
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mature steelhead we evaluated differences between sexes. Within the sample of female kelts we
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evaluated metrics by condition, and also to evaluated differences between kelts with and without
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presence of food at necropsy. Because sample sizes for male kelts were small (poor = 3, fair = 5,
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and good = 5) we did not make statistical comparisons between males and female kelts. We
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compared metrics between mature and kelt steelhead for good condition females only.
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Statistical comparisons of the frequency of scores by category were made using exact Chi-
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square analyses with Monte Carlo estimates derived from 20,000 permutations. These estimates
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were used to account for occasional low frequencies (< 5 per cell) in which case the use of an
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asymptotic Chi-Square test was not valid. Comparisons of oocyte counts in kelts by condition
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were tested using Wilcoxon rank-sum tests. The relationship of fish length to several scoring
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metrics transformed to progressive numerical scores was examined using Spearman correlations.
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All statistical tests were conducted using SAS 9.3 (SAS Institute, Carey, North Carolina), and
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statistical significance against a null hypotheses was reported as a P value.
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Results
Evidence of feeding
Of the 65 migrating kelts examined, 25 had food or fecal material in the gastrointestinal tract.
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Food items included salmon smolts, other small unidentified fish, fish eggs, and various
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terrestrial and aquatic invertebrates. The proportion of feeding kelts significantly decreased with
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decreasing condition (58% of good condition versus 7% of poor condition; P < 0.01, Table 3). In
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male kelts, we detected no significant relationship between the proportion of feeding fish and
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fish condition.
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Pyloric stomach
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We found no significant variation between male and female mature steelhead in any of the
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metrics scored in the pyloric stomach, and all fish were considered in good condition. However,
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we found significant differences attributed to fish condition in female kelt scores of submucosa
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integrity, villi density, villi invagination, and columnar epithelial cell integrity (P < 0.02; Fig 2).
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We found 23 of the 38 female kelts in good or fair condition had minor to no detachment of the
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submucosa from the muscularis of the pyloric stomach, whereas the majority of poor condition
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kelts (12/14) exhibited severe and moderate detachment of the submucosa. The villi density was
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highest in good condition female kelts (Figs 3 and 4). The invagination of villi to the stratum
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compactum was highly variable between good, fair, and poor condition kelts. Invaginations in
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good condition kelts were all > 1/4 the distance to the stratum compactum and 60% of those
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evaluated were > 1/2 the distance. Fair condition kelts exhibited a wide range of villi
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invagination, and nearly all poor condition kelts showed villi invagination < 1/2 the distance to
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the stratum compactum (Fig 2). Regardless of condition, the majority of kelts showed minor to
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no signs of columnar epithelial cell deterioration.
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Comparisons between stomach tissues of mature and kelt steelhead showed several
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differences in structural patterns likely related to feeding recovery. Over 60% of mature
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steelhead had low densities of villi but over 70% of kelts had significantly higher villi density (P
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< 0.0001; Fig 5). We found significant differences between the extent of invagination of villi
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between mature and kelt steelhead (exact P < 0.0001). The depth of villi invagination in mature
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steelhead was very shallow, and nearly 80% of villi measured were <1/4 the distance to the
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stratum compactum. In kelts, villi invagination was more pronounced and all were > 1/4 the
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distance to the stratum compactum. No significant variations in submucosa integrity, columnar
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epithelial cell integrity, length to width ratio of columnar epithelial cells, or presence of goblet
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cells were found between mature or kelt steelhead.
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We found a significant negative correlation of submucosa integrity with fish size in mature
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steelhead (r = -0.4654; P <0.007; N = 33). However, compared to kelts, the range in length of
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mature fish sampled (72 to 82 cm) was different from that of kelts (52 to 82 cm) sampled. We
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found no such relationship with fork length in kelts. No mature steelhead exhibited severe
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submucosa deterioration and very few (< 5) showed moderate deterioration, and the lack of
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correlation could be influenced by sample sizes in each scoring category.
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Ovary
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No vitellogenic oocytes were observed in any of the ovarian tissues from kelts. However,
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perinucleolar and early/late stage cortical alveolus stage oocytes were observed indicating that
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Snake River steelhead are capable of repeat-spawning. Oocyte atresia was infrequent. We found
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no variations in perinucleolar and early/late stage cortical alveolus oocytes by fish condition (Fig
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6). However, we detected a negative correlation between fish fork length and perinucleolar
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oocytes (r = -0.606 ; P < 0.002; N = 24). There was no significant correlation with length and
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counts of early/late stage cortical alveolus oocytes.
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Liver
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We detected a significantly higher proportion of vacuolization in liver parenchyma of mature
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steelhead males compared to that observed in mature females (P = 0.043; Fig 7). Mature female
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steelhead had significantly lower proportions of cellular deterioration in hepatocytes than
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proportions observed in males (P <0.001). We found no significant differences in the density of
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melanomacrophage aggregates, the amount of hepatocyte detachment, or sinusoid spacing
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between male and female mature steelhead.
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In kelts, we observed that hepatocyte integrity varied among good, fair, and poor condition
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females. Over 60% of good condition (N=24) and poor condition (N=14) kelts had minor to no
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hepatocyte deterioration, whereas approximately 80% of fair condition kelts (N=13) showed
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moderate deterioration (Fig 9). In samples with deterioration, we noted disintegration or loss of
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the cellular membrane and nucleation within hepatocytes. Hypertrophy of hepatocytes was
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occasionally observed and some exhibited vacuolization within the cytoplasm (Fig 9). We found
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no significant variation in the proportion of melanomacrophage aggregates, attachment of
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hepatocytes, spacing of sinusoids, or vacuolization among good, fair, and poor female kelts.
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Our comparisons of liver tissues between good condition mature and kelt steelhead
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determined that the proportion of mature females with minor to no hepatocyte cellular
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deterioration was significantly higher than that of kelts (P = 0.006; Figs 8 and 9). Good condition
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female kelts exhibited moderate hepatocyte detachment within cords (P = 0.002), in contrast
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with samples from mature fish that had little to none. Variations in the degree of vacuolization in
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livers between mature and kelt steelhead were noted (P = 0.05). We observed approximately
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60% of livers from mature steelhead with vacuolization scores between 1 to 10%, and 20% with
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scores > 10%, while nearly 50% of kelts showed a complete absence of liver vacuolization.
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Small areas of melanomacrophage aggregates were present in all mature and kelt livers and
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clustered near bile ducts and veins. Sinusoid spacing was consistent between phases and rarely
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exceeded 10 μm between hepatocyte cords.
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Spleen
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We found significant differences in the proportion of red pulp in male versus female mature
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steelhead (P = 0.001; Fig 10). Proportionally, 70% of male steelhead exhibited red pulp ratios
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greater than 61%. In migrating female kelts, differences in the proportions of cellular
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differentiation in the spleen were associated with fish condition. We observed some evidence of
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red blood cell differentiation as well as leukocyte production or infiltration in 75% of kelt
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spleens. Only fair and poor condition kelt spleens were observed without cell production or
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monocyte activity (Fig 11). No other metrics were associated with fish condition. Regardless of
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reproductive phase, we observed that spleens with higher proportions of white pulp had greater
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amounts of cell differentiation in the white pulp. In spleens with elevated proportions of red
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pulp, mature erythrocytes were observed as the dominant cell type.
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We found a statistically significant relationship between fish length and the proportion of red
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pulp in kelts (r = 0.4347; P = 0.034; N = 24). Although this trend suggested that larger kelts had
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higher proportions of red pulp in spleens, these relationships were driven by two large kelts (73
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and 75 cm) with red pulp ratios > 61%.
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Discussion
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The energetic and physiological deterioration of semelparous Pacific salmon during
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spawning has been documented extensively (Green 1916; Robertson and Wexler 1960;
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Robertson et al. 1961; Hane et al. 1959; Brett 1995; McPhee and Quinn 1998; Barry et al. 2010;
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Morbey et al. 2005), but has not been well described in anadromous iteroparous salmonids
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(Belding 1934). Intuitively, it would seem that any fish capable of repeat spawning would not
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undergo the same irreversible cellular degeneration as occurs in semelparous salmon. We found
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little evidence of extensive cellular deterioration in the pyloric stomach, ovary, liver or spleen in
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mature or kelt steelhead suggesting that the physiological processes causing rapid senescence
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and death in semelparous salmon are likely not the same in steelhead. Furthermore, the
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observation of active feeding in migrating kelts indicates that these fish are attempting to replace
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energy even while still in freshwater.
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Gastrointestinal response
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Steelhead are not believed to feed actively during freshwater migrations prior to spawning,
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but occasionally freshwater feeding has been documented in mature Pacific (Garner et al. 2009)
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and Atlantic salmon (Johansen 2001). Reductions or loss of villi and microvilli in the
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gastrointestinal tract is a common characteristic found in fasting or starving poikilothermic
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organisms (i.e. fish, snakes) (Ehrlich et al. 1976; Secor et. al. 2002; Krogdahl and Bakke-
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McKellep 2005; Wang et al. 2006). Considering that the majority of mature steelhead at DNFH
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were held on site for 2-3 months and not provided any opportunities to feed prior to sampling,
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we are confident these fish were in a fasting state. The pyloric stomach of mature steelhead had
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low densities of villi and almost little villiar invagination. These observation were similar to the
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histological assessments of post-spawn sockeye O. nerka and Chinook O. tshawytscha salmon
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(Robertson and Wexler 1960). The reduction of villi in fasting fish has also been documented in
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non-salmonid species. Zeng et al. (2012) showed that the gastrointestinal tract and liver in food
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deprived juvenile catfish (Siluris meridionalis) were down-regulated as a physiological response
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to endure unfavorable environmental conditions. However, it is important to note that cellular
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deterioration of the columnar epithelial cells rarely occurred in mature steelhead suggesting that
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an irreversible degeneration was not occurring.
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From an evolutionary perspective, the volitional anorexia in anadromous semelparous and
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some iteroparous salmonids is likely the result of returning to a less productive environment
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(freshwater) at a generally much larger size (McDowell 1987; Gross 1987; Fleming 1998). Wang
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et al. (2006) hypothesized that reductions in organ size and enzymatic activity, specifically in the
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gastrointestinal tract, could provide considerable energy savings to fasting or starving organisms
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via lowering basal metabolic demands. This hypothesis is especially relevant to fasting
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anadromous salmonids preparing to spawn, which primarily rely on somatic energy stores for
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migration and spawning. Bioenergetically, it is conceivable that anadromous salmon returning to
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spawn in a less productive environment at a larger size would save energy by fasting for three
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primary reasons: (1) they would not actively pursue food with little energetic gain, (2) lower
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basal metabolic costs by not operating the gastrointestinal tract, and (3) the use of stored somatic
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energy forgoes the costs of specific dynamic action (loss of energy during digestion). Therefore
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we agree with Wang et al (2006) hypothesis and further hypothesize that the gastrointestinal tract
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of iteroparous salminds enters a cellular stasis or hibernation during reproduction, which is
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supported by the observed renewal of villi and invagination found in kelts.
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It is unclear if steelhead kelts must first ingest food to begin gastrointestinal recovery. Secor
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et al. (2002) found that following prolonged periods of fasting in pythons that intestinal mass and
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microvilli density rapidly increased following the ingestion of specific amino acids and peptides.
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The columnar epithelial cells and goblet cells comprising the villi are important in the secretion
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of enzymes, mucous, as well as the uptake of nutrients in the gastrointestinal tract (Mader 2001).
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Presumably, the observed increases in villi density and invagination provide more surface area
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for the digestion and absorption of food. Significant variations in villi density and invagination
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did occur between good, fair, and poor kelts, which is possibly due to the finding that good
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condition kelts were more likely to feed than fair or poor condition kelts. However, comparisons
313
between feeding and non-feeding good condition female kelts found no significant variations in
314
any of the pyloric scoring metrics. In reconditioning experiments with Atlantic salmon kelts,
315
Johnston et al. (1987) found that many kelts began feeding soon after spawning, but that many
316
had difficulties in swallowing feed and only after swallowing food several times did the rejection
317
of feed become less prevalent. This suggests that gastrointestinal regeneration may not occur at
318
the same rate or may be more difficult for some kelts than others. One possibility is that non-
319
feeding kelts had simply evacuated food from the gastrointestinal tract prior to sampling.
320
Carnivorous species like steelhead have short gastrointestinal tracts and the residence period of
321
food in the gut is relatively short (Arrington et al. 2002), thus it is possible feeding was
322
undetected in some kelts. Additionally, it has been shown that acute stress can alter the cellular
323
composition and reduce the permeability of the gastrointestinal tract (Olsen et al. 2005; Olsen et
324
al. 2008). Therefore, it is possible that stresses not detected via external examination could also
325
potentially explain the variations in cellular microstructure between feeding and non-feeding
326
kelts.
327
In addition to feeding, the gastrointestinal tract is also important in osmoregulation in
328
migrating kelts. As in smolts, the migration of kelts from a hypo- to hyper-tonic environment
329
requires a change in the osmoregulation of ions from the gills to the gastrointestinal tract. The
330
process of the re-acclimation to seawater has been shown to be greatly accelerated in kelts.
331
Talbot et al. (1992) found that Atlantic salmon kelts were able to re-adapt to seawater within 48
332
hours, thus indicating that the gut and all organs responsible for the salt and water balance had
16
333
recovered some or all function 4 to 6 weeks after spawning. This quick acclimation is likely
334
beneficial to kelts on two levels. Not only does it reduce the stress and time required to re-
335
acclimate in the estuary allowing for quicker access to high energy food in the ocean, but it likely
336
aids in the recovery from many freshwater-specific external infections such as (Saprolegnia sp.)
337
and parasitic copepods (Salmincola sp).
338
Ovarian composition
339
We observed perinucleolar (pre-lipid) and early/late stages of cortical alveolus oocytes (post-
340
lipid) in Snake River kelts, suggesting that these populations are capable of re-maturation. Like
341
their freshwater resident conspecifics, steelhead exhibit group synchronous ovarian development
342
characterized by the development of one primary group of oocytes at a time, which are
343
accompanied by numerous “resting” previtellogenic oocyte stages (Blazer 2002). This type of
344
ovarian development pattern has also been defined as determinant fecundity, where the
345
recruitment of oocytes in relation to secondary growth is arrested prior to spawning (Rideout and
346
Tomkiewicz 2011). Interestingly, Robertson and Wexler (1960) found small nucleated oocytes
347
present in the ovaries in spawning sockeye salmon, but not in the ovaries of Chinook salmon.
348
Exceptions to repeat spawning have been found for some salmonids that were previously
349
considered to follow strict semelparous life histories. Tsiger et al. (1994) found that male masu
350
salmon O. masou maturing and spawning in freshwater could later adopt anadromous life
351
histories and return to spawn again. Unwin et al. (1999) found that precocious male Chinook
352
salmon could be spawn and remature to spawn again under experimental conditions.
353
All oocytes found in Snake River steelhead ovaries were in previtellogenic stages indicating
354
that vitellogenesis was not occurring. This finding is not surprising considering that these fish
355
had recently ovulated the primary group of oocytes during spawning (<1 – 2 months earlier).
17
356
Oogenesis is an energetically expensive process for all fish species (Rideout and Tomkiewicz
357
2011), thus immediate gonadal recrudescence would not be expected to occur in a post-spawning
358
fish with depleted somatic energy. Johnson et al. (1987) found oogenesis and vitellogenesis
359
proceeded in Altantic salmon kelts only after restoration of body energy reserves. In our studies,
360
the exact date and location of spawning were unknown for kelts, but it is unlikely that any of
361
these fish had adequate time to restore their total body energy to re-initiate oogenesis and
362
vitellogenesis, even with the re-initiation of feeding.
363
Recent studies by Null et al. (2013) on acoustically tagged steelhead kelts in the Sacramento
364
documented that 10% of kelts rematured or residualized in freshwater. Although residualized
365
kelts grew less than those that returned to the ocean, their survival was higher. Steelhead that re-
366
mature can remain in the ocean a year or more before return migration (Burgner et al.1992).
367
Variations in spawning periodicity, known as skip-spawning, are common and can occur in
368
mature fish due to low somatic energy, poor physical health, or poor environmental conditions
369
(Rideout et al. 2005; Rideout and Tomkiewicz 2011). In anadromous species, migrating long
370
distances, skip spawning is common. We found no female kelts in our study that had retained
371
ripe eggs, and some follicular atresia was observed. The absence of vitellogenisis in Snake River
372
kelt ovaries in our studies suggests that the pre-vitellogenic oocytes were in a resting stage,
373
presumably until re-feeding was initiated and somatic energy reserves restored.
374
With the extended migration distance to return to the ocean it is likely that Snake River
375
steelhead would exhibit skip-spawning to re-build energy stores. Jonsson et al. (1991) found
376
smaller Atlantic salmon kelts (< 60cm) were more likely to exhibit consecutive spawning,
377
whereas larger kelts were more likely to exhibit skip-spawning (> 90 cm), and smaller kelts had
378
higher survival. The residualized and anadromous steelhead kelts tagged by Null et al. (2013)
18
379
were observed as consecutive spawners, and were <60 cm. It has been shown that larger
380
iteroparous salmonids generally exhibit lower post-spawning survival over smaller salmonids
381
likely due to difficulty in replacing energy stores (Fleming 1998).
382
We found the number of pre-lipid oocytes was negatively correlated with fork length. Our
383
finding is of particular interest as stocks in the Snake River basin that contain two steelhead run-
384
types, known as A and B-run (Brannon et al. 2004). The A-run steelhead return to freshwater
385
earlier (July to August), have shorter marine residences (1-2 years), are smaller in size (<70 cm),
386
and generally spawn earlier (March-April) than B-run fish. The B-run steelhead return later
387
(August to October), have longer marine residence (2-3 years), are generally larger (>70 cm),
388
and spawn later (April-June). During our sampling of kelts in 2009 and 2010, we observed lower
389
proportions of large kelts in the juvenile bypass system at the Lower Granite Dam (unpublished
390
data).
391
We hypothesize that the lower number of perinucleolar oocytes in larger B-run kelts may
392
relate to greater energetic investments in size to increase fecundity, egg size, and juvenile
393
survival, thus exhibit a life history similar to semelparity (Fleming 1998; Crespi and Teo 2002;
394
Fleming and Reynolds 2004). Observations of acoustically tagged Snake River steelhead by
395
Jones (2013) do not support a higher mortality of large B-run kelts following spawning. Of the
396
30 B-run kelts tagged at Fish Creek on the Lochsa River, ID, 29 were detected at the Lower
397
Granite Dam forebay indicating they successfully migrated from the spawning system.
398
Unfortunately, estimates of the quantity of B-runs steelhead passing the Lower Granite Dam via
399
routes other than the bypass facility are unavailable. Regardless, the successful migration of
400
large B-run kelts does not suggest that size is as important as restoring energy.
19
401
Liver and spleen responses
402
Our assessments of mature and kelt steelhead livers showed little evidence of severe liver
403
deterioration or loss of function at the cellular level. Of the two phases examined, tissues in kelts
404
had the most hepatocyte detachment and deterioration, although samples from < 10% of kelts
405
showed severe hepatocyte deterioration. Robertson and Wexler (1960) reported pronounced
406
hepatocyte degeneration, that included diminishments in cytoplasm and occasionally a complete
407
loss of nucleation in spawning semelparous salmon.
408
Compared to white muscle tissue the liver is not a primary energy storage tissue in
409
anadromous salmonids (Brett 1995), but it does serve as a readily accessible energy source
410
during fasting or starvation. In semelparous pink salmon O. gorbuscha, Phleger (1971) found
411
that the liver lost the ability to synthesize triglycerides following freshwater re-entry and
412
spawning. Mommsen et al. (1980) examined the enzymatic activity of Fraser River sockeye
413
salmon O. nerka livers during spawning migrations and found a gradual decrease in metabolic
414
enzymes and protein content as fish progressed toward spawning. In salmonids, carbohydrates
415
comprise only a very small component of white muscle tissue composition and are a not a
416
significant source of energy (Brett 1995), however the liver does store glycogen as a small
417
source of energy for immediate use when needed. French et al. (1983) found that that the liver of
418
post-spawn sockeye salmon increased in its capacity to synthesize glucose following complete
419
exhaustion of somatic lipids, perhaps providing a final energy source for nest guarding. Thus, it
420
appears that a complete loss of liver function is rare even among spawning semelparous species
421
and may explain the overall lack of hepatocyte deterioration found in Snake River steelhead
422
livers.
20
423
The effects of starvation and prolonged fasting on the cellular architecture of the liver have
424
been documented for a variety of fish species (Hochachka 1961; Connor et al. 1964; Vijayan and
425
Moon 1992; Hur et al. 2006; Rios et al. 2007; Kullgren et al. 2010). Among the noticeable
426
histological changes in the livers of starving fish are losses of glycogen or lipid vacuolization
427
(Wolf and Wolfe 2005; Hur et al. 2006). In our study, the only significant variation in liver
428
vacuolization was observed between male and female pre-spawn steelhead, where males had a
429
higher proportion of vacuolization. French et al. (1983) found that liver glycogen reserves in
430
Fraser River sockeye were highest in females just before spawning, but did not examine males.
431
As livers are important to vitellogenin synthesis, it is possible that glycogen reserves in female
432
were used to complete oogenesis before spawning. This difference between sexes may also
433
partially explain the variations in hepatocyte integrity between male and females. Wolf and
434
Wolfe (2005) note that it is not uncommon to observe hepatocye hypertrophy in the livers of
435
sexually maturing females due increases in estrogen vitellogenin. Therefore, it is possible that
436
the variations in vacuolization and hepatocyte integrity between mature male and female
437
steelhead reflected these differences in energy allocation for oogenesis.
438
Regardless of condition, sex, or phase all steelhead livers had > 5 melanomacrophage
439
aggregates. Agius and Roberts (2003) noted the melanomacrophage aggregates are generally
440
prolific in the kidney, spleens, and livers in starving fish. We did not observe large numbers of
441
aggregates, but perhaps a more refined method of scoring aggregates would have resolved
442
potential variation among factors of condition, sex, and mature and kelt phases.
443
We found little evidence of cellular deterioration or loss of spleen function in either mature
444
or kelt steelhead. Only fair and poor condition kelts showed a complete loss of cellular activity in
445
the spleen suggesting other potential pathological problems. Robertson and Wexler (1960)
21
446
detected a reduction in the quantity or complete disappearance of lymphoid cells and increase in
447
fibrous tissue in the spleen of spawning semelparous Chinook and sockeye salmon, but found
448
only 1 of 16 spleens examined with cellular necrosis.
449
We found a higher proportion of red pulp in males over females in comparisons between
450
mature steelhead. Rebok et al. (2011) found that white pulp increased in the spleens of female
451
Ohrid trout Salmo letnica during spawning, but did not examine males. Rebok et al. (2011)
452
postulated that gonadal maturation likely reduced the proportion of circulating lymphocytes
453
thereby potentially increasing the white pulp in the spleen of females. Variations in red pulp can
454
be caused by the use of erythrocytes for exercise and activity. Strenuous exercise influences the
455
proportion of erythrocytes stored in the spleens of O. mykiss, and other teleosts (Kita and Itazawa
456
1989; Franklin et al.1993). It is possible that the spleens of mature male steelhead in our study
457
contained higher proportions of erythrocytes in red pulp in preparation for competition on the
458
spawning grounds.
459
We found no variations in the arrangement of white pulp nodes by sex, condition, or phase in
460
our study, but red and white pulp nodes in teleosts are reported to be more diffuse than in
461
mammals (Mumford et al. 2012). Although not scored, melanomacrophage aggregates were
462
present in all steelhead spleens. Like the liver and kidney, prolonged fasting and starvation
463
generally increases the number of melanomacrophage aggregates (Agius and Roberts 1981,
464
Mizuno et al. 2002), thus future evaluations of spleen in response to prolonged fasting during
465
spawning may be warranted.
466
Summary
22
467
This study was the first to examine in detail the histological architecture of organs of
468
steelhead at maturity and as migrating kelts. Although many changes can be detected between
469
these two stages, the majority of steelhead showed little cellular atrophy that would be indicative
470
of loss of cellular function. Moreover, our measures of tissue histology have been paired with
471
assessment of plasma blood profiles (unpublished data) that indicate kelts are in homeostasis.
472
The evidence of developing oocytes, changes in the epithelial structures of the pyloric stomach
473
between maturity and kelt migration, and observations of feeding, all provide strong support for
474
potential iteroparity.
475
476
23
477
Acknowledgments
478
Funding for this study was provided by the Columbia Inter-Tribal Fish Commission, Doug
479
Hatch, project manager. We thank the staff at Dworshak National Fish hatchery for their
480
cooperation with sampling. The US Army Corps of Engineers, and Nez Perce Tribe provided
481
assistance with logistics of sampling at Lower Granite Dam juvenile fish bypass facility. We
482
thank Dr. Rajan Bawa and staff at Colorado Histo-prep for processing tissues samples for
483
microscopic analysis. Ann Norton, Boling Sun, James Nagler, Chris Williams, Andrew Pierce,
484
Victor Cajas of the University of Idaho provided invaluable assistance in interpretation of
485
samples, laboratory and field assistance. We thank Amy Long and Vicki Blazer for their reviews
486
of earlier drafts. Additional sampling support was provided by Aaron Penney, Jessica Buelow,
487
Kala Hamilton, Andrew Paper, William Schrader, Martin Perales, Veatasha Dorsey, and Bryan
488
Jones. This study was performed under the auspices of University of Idaho protocol # 2009-10.
489
Any use of trade, firm, or product names is for descriptive purposes only and does not imply
490
endorsement by the U.S. Government.
491
24
492
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Secor SM, Lane JS, Whang EE, Ashley SW, Diamond J, (2002) Luminal nutrient signals
665
for intestinal adaptation in pythons. American Journal of Gastrointestinal Physiology
666
283: G1298-G1309
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667
Talbot C, Stagg RM, Eddy FB (1992) Renal, respiratory and ionic regulation in Atlantic
668
salmon (Salmo salar L.) kelts following transfer from freshwater to seawater. Journal
669
of Comparative Physiology 162(4): 358-364.
670
Thrower,FP, Hard JJ, Joyce JE (2004) Genetic architecture of growth and early life-
671
history transitions in anadromous and derived freshwater populations of steelhead.
672
Journal of Fish Biology 65 (Supplement A): 286-307
673
Tsiger VV, Skirin VI, Krupyanko NI, Kashlin KA, Semenchenko AY (1994) Life history
674
form of male masu salmon (Oncorhynchus masou) in South Primore, Russia.
675
Canadian Journal of Fisheries and Aquatic Sciences 51: 197-208.
676
Unwin MJ, Kinnison MT,Quinn, TP (1999) Exceptions to semelparity: Postmaturation
677
survival, morphology, and energetic of male Chinook salmon (Oncorhynchus
678
tshawytscha). Canadian Journal of Fisheries and Aquatic Sciences 56: 1172-1181/
679
Vijayan MM, Moon TW (1992) Acute handling stress alters hepatic glycogen
680
metabolism in food-deprived rainbow trout (Oncorhynchus mykiss). Canadian Journal
681
of Fisheries and Aquatic Sciences 49: 2260-2266
682
Viola A E, Schuck ML, (1995) A method to reduce the abundance of residual hatchery
683
steelhead in rivers. North American Journal of Fisheries Management 15:488–493
684
Wang T, Hung CY, Randall DJ (2006) The comparative physiology of food deprivation:
685
686
687
688
689
From feast to famine. Annual Review of Physiology 68:223-251
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Northwest Fish Culture Conference 27: 63-64
Wolf JC, Wolfe MJ (2005) A brief overview of nonneoplastic hepatic toxicity in fish.
Toxilogical Pathology 33: 75-85
33
690
Zeng LQ, Li FJ, Li XM, Cao AD, Fu SJ,Zhang YG (2012) The effects of starvation on
691
digestive tract function and structure in juvenile southern catfish (Siluris meridionalis
692
Chen). Comparative Biochemistry and Physiology, Part A 162: 200-211
34
693
Table 1. Number of fish sampled and tissues examined, separated by spawning year, and
694
reproductive stage. All but four sexually mature males and one female in 2010 were sampled at
695
Dworshak National Fish Hatchery. All kelts were sampled at Lower Granite Dam.
696
697
Phase
Sex
698
699
2010
Total
Liver, spleen and pyloric stomach
Sexually mature
700
701
2009
Kelt
702
F
16
17
33
M
0
14
14
F
24*
29*
0
M
3
10
13
703
Ovary
704
Sexually mature
F
0
11
11
705
Kelt
F
24
29
53
706
* Indicates one sample was missing from analysis of pyloric stomach and liver.
707
708
35
709
Table 2. Summary of histological evaluations by tissue type and magnification in pyloric
710
stomach, liver, spleen, and ovarian tissues. Numerical scores and criteria are presented for each
711
metric.
Tissue
Metric (magnification)
Scoring criteria
Pyloric stomach
Submucosa integrity (4X)
1 = Severe detachment from
muscularis ; 2 = Moderate
detachment from muscularis; 3 =
Light or no detachment from
muscularis
Villi density (4X)
1 = Low ; 2 = Moderate; 3 = High
Villi invagination (10X)
1 = <1/4 distance to stratum
compactum; 2 = 1/4 ≤ 1/2
distance to stratum compactum;
3 = 1/2 distance to stratum
compactum
Columnar epithelial cell integrity
1 = Severe cell deterioration,
(40X)
poor nucleation; 2 = Moderate
cell deterioration, nucleation
present ; 3 = Light to no cell
deterioration, nucleus present
L:W ratio of columnar epithelial cells
1 = ≤1/2 length to width ratio; 2
(40X)
= >1/2 length to width ratio;
Presence of goblet cells (40X)
712
1 = Absent; 2 = Present
36
Table 2, continued.
Tissue
Liver
Metric (magnification)
Melanomacrophage aggregates (10X)
Scoring criteria
1 = Absent; 2 = < 5; 3 = > 5
Hepatocyte dettachment from rosette
1 = Severe detachment
(10X)
2 = Moderate;
3 = Light to no detachment
Hepatocyte rosette spacing (10X)
1 = <10μm; 2 = >10μm
Vacuolization in field of view (40X)
1 = absent; 2 = 1 to 10%
3 = > 10%
Hepatocyte cell integrity (40X)
1 = Severe cell deterioration, poor
nucleation ; 2 = Moderate cell
deterioration, nucleation present;
3 = Light to no cell deterioration,
nucleation present
Spleen
Distribution of white pulp
1 = Discrete; 2 = Diffuse
Percentage of red pulp coverage (10X)
1 = 0-39%; 2 = 40-60% ; 3 = 61100%
Cell production/differentiation (40X)
713
1 = Absent ; 2 = Present
37
Tissue
Metric (magnification)
Scoring criteria
Ovary
Quantity of perinucleolar oocytes (4X)
Directly quantified and averaged
in 3 to 4 separate zones
Quantity of early/late cortical alveolus oocytes (4X) Directly quantified and averaged
in 3 to 4 separate zones
Quantity of vitellogenic oocytes (4X)
Directly quantified and averaged
in 3 to 4 separate zones
714
38
715
Table 3. Summary of proportion of kelts with food or evidence of feeding at time of necropsy at
716
Lower Granite Dam, by sex and fish condition. Monte Carlo permutation exact P values are
717
provided for comparisons by sex and condition.
Sex
Female
Male
718
719
720
721
722
Condition
Good
N
24
Food (%)
14 (58)
P
Fair
14
6 (43)
0.0061
Poor
14
1 (7)
Good
5
2 (40)
Fair
5
2 (40)
Poor
3
0
Total
65
25 (38)
0.5809
39
723
724
Figure Captions
Fig 1 Map of Columbia/Snake River sub-basin with sampling sites indicated.
725
726
Fig 2 Significant scoring metrics for the pyloric stomach among good, fair, and poor condition
727
kelts for scores of A) submucosa integrity, B) villi density, C) villi invagination, and D)
728
columnar epithelial cell integrity.
729
730
Fig 3 Photomicrograph (4X) of pyloric stomach architecture in (a) mature steelhead (good
731
submucosa integrity, low villi density, <1/4 invagination), (b) poor condition kelt (poor
732
submucosa integrity, low villi density, <1/4 invagination), (c) fair condition kelt (fair submucosa
733
integrity, moderate villi density, and <1/2 invagination), and (d) good condition kelt (good
734
submucosa integrity, high villi density, >1/2 invagination).
735
736
Fig 4 Photomicrograph (40X) of columnar epithelial cell integrity in the pyloric stomach villi of
737
(a) poor condition kelt (severe to moderate cellular deterioration), (b) fair condition kelt
738
(moderate to minor cellular deterioration), and (c) good condition kelt (minor to no cellular
739
deterioration).
740
741
Fig 5 Significant scoring metrics for pyloric stomach histology between good condition female
742
sexually mature and kelt steelhead: A) villi density and B) villi invagination.
743
744
Fig 6 Photomicrograph (4X) of kelt ovaries (a) kelt ovary with high quantity of perinucleolar
745
(pre-lipid) and some early and late stage cortical alveolus (lipid deposition present) oocytes and
746
(b) ovary from a large kelt (> 70cm) with only early and late stage cortical alveolus oocytes.
40
747
748
Fig 7: Significant scoring metrics for the liver between male and female pre-spawn steelhead: A)
749
vacuolization and B) hepatocyte integrity.
750
751
Fig 8: Significant scoring metrics for liver histology between good condition female sexually
752
mature and kelt steelhead: A) hepatocyte detachment, B) vacuolization, and C) hepatocyte
753
integrity.
754
755
Fig 9. Photomicrograph (10X) of liver cellular architecture in (a) mature steelhead (high
756
vacuolization, minor to no hepatocyte detachment, minor to no hepatocyte deterioration), (b)
757
poor condition kelt (no vacuolization, severe hepatocyte detachment, severecellular
758
deterioration), (c) fair condition kelt (no vacuolization, minor to no hepatocyte deterioration),
759
and (d) good condition kelt (vacuolization present, minor to no hepatocyte attachment, minor to
760
no hepatocyte deterioration).
761
762
Fig 10 Comparison of red pulp coverage between the spleen of male and female pre-spawn
763
steelhead.
764
765
Fig 11 Photomicrograph (10X) of spleen cellular architecture in (a) mature steelhead (40-60%
766
red pulp, diffuse white pulp, and cellular activity present) (b) poor condition kelt (40-60% red
767
pulp, diffuse white pulp, loss of cellular activity), (c) fair condition kelt (61-100% red pulp,
768
white pulp discrete), cellular activity present), and (d) good condition kelt (0-39% red pulp,
769
white pulp diffuse, cellular activity present).
770
41
771
772
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
Fig 1.
42
795
796
100
797
A
Good
Fair
Poor
799
Percent of fish
80
798
800
B
60
40
20
0
801
Severe
Moderate
Minor/absent
Low
Submucosa detachment
802
100
High
Villi density
C
D
80
Percent of fish
803
804
Moderate
60
40
805
20
806
0
<1/4
808
809
811
>1/2
Invagination to stratum compactum
807
810
1/4 to 1/2
Fig 2.
Severe
Moderate
Minor/absent
Columnar epithelial cell deterioration
43
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
Fig 3.
44
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
Fig 4.
45
845
846
Mature
Kelt
847
100
A
848
80
849
850
60
851
40
852
20
Percent of fish
853
854
855
856
0
Low
Moderate
High
Villi density
100
B
857
80
858
60
859
860
40
861
20
862
863
0
<1/4
864
865
866
867
1/4 to 1/2
>1/2
Villi invagination to stratum compactum
Fig 5.
46
868
869
870
871
872
873
874
875
876
877
878
879
880
881
882
883
884
885
886
Fig 6.
47
100
Vacuolization
A
Male
Female
80
60
40
20
Percent of fish
0
Absent
1-10%
>10%
Hepatocyte deterioration
100
B
80
60
40
20
0
Severe
887
888
889
890
Fig 7.
Moderate
Minor/absent
48
Hepatocyte detachment
100
A
Mature
Kelt
80
60
40
20
0
Severe
Percent of fish
100
Moderate Minor/absent
Vacuolization
B
80
60
40
20
0
Absent
1-10%
>10%
Hepatocyte deterioration
100
C
80
60
40
20
0
Severe
891
892
893
Fig 8.
Moderate
Minor/absent
49
894
895
896
897
898
899
900
901
902
Fig 9.
50
903
904
905
100
Male
Female
906
80
Percent of fish
907
908
909
0
911
0-39%
913
916
917
918
919
920
921
40-60%
61-100%
Proportion of red pulp
912
915
40
20
910
914
60
Fig 10.
51
922
923
924
925
926
927
928
929
930
931
932
933
934
935
Fig 11.