doi:10.1006/jfbi.2001.1798, available online at http://www.idealibrary.com on. Free and total cortisol levels in semelparous and iteroparous chinook salmon.
Journal of Fish Biology (2001) 59, 1673–1676 doi:10.1006/jfbi.2001.1798, available online at http://www.idealibrary.com on
Free and total cortisol levels in semelparous and iteroparous chinook salmon T. P. B*¶, M. J. U†, J. A. M* T. P. Q‡ *University of Wisconsin Aquaculture Program, 1605 Linden Drive, Madison, WI 53706, U.S.A., †National Institute of Water and Atmospheric Research Ltd, Box 8602, Christchurch, New Zealand and ‡University of Washington, School of Aquatic and Fishery Sciences, Box 355020, Seattle, WA 98195, U.S.A. (Received 23 March 2001, Accepted 17 September 2001) Total cortisol, free cortisol and percent free cortisol were all significantly higher in semelparous male chinook salmon Oncorhynchus tshawytscha than in iteroparous and immature fish. The findings suggest that the regulation of both total and free cortisol concentrations may play key roles in mediating the post-spawning death of semelparous Pacific salmon. 2001 The Fisheries Society of the British Isles
Key words: cortisol; iteroparous; reproduction; salmon; semelparous.
It is generally accepted that highly elevated cortisol levels mediate the post-spawning death of semelparous Oncorhynchus Pacific salmon by causing tissue degeneration, suppressing the immune system, and impairing various homeostatic mechanisms (Dickhoff, 1989; Stein-Behrens & Sapolsky, 1992). However, hypercortisolism alone may not fully explain the mechanism of programmed death because total serum cortisol levels can be elevated in semelparous fishes at other life cycle stages without causing significant mortality (e.g. following stress, Barton & Iwama, 1991). Moreover, hypercortisolism may also occur at spawning in rainbow trout Oncorhynchus mykiss (Walbaum), an iteroparous species (Bry, 1985). Inasmuch as only the non-protein bound or free cortisol is thought to have biological activity, it was hypothesized that differences in the regulation of free cortisol concentrations may explain the differential post-spawning survival of semelparous and iteroparous salmonids. As far as is known, there are only two reports describing changes in corticosteroid binding associated with reproductive maturation in salmonid fishes, and both studies support this hypothesis. In semelparous sockeye salmon Oncorhynchus nerka (Walbaum), Idler & Freeman (1968) found that corticosteroid binding levels were low in conjunction with elevated total corticosteroid levels in spawning females (i.e. free cortisol levels were presumably high in mature fish). In contrast, in iteroparous rainbow trout, Caldwell et al. (1991) found that the percentage of protein-bound cortisol was significantly greater in reproductively mature females compared to immature fish (i.e. free cortisol was low in mature fish). Such data from separate studies and species, however, may not be directly comparable. To better clarify the role of hypercortisolism in regulating the programmed senescence and death of Pacific salmon it would be ideal to compare cortisol levels between semelparous and iteroparous fish of the same species. In this regard, Unwin et al. (1999) recently reported that between 2·1 and 6·8% of autumn-run male chinook salmon Oncorhynchus tshawytscha (Walbaum) from New Zealand matured as yearling parr, and c. 80% of those fish survived to spawn in subsequent years. The availability of these fish afforded a unique opportunity to test the present hypothesis by measuring total and free ¶Author to whom correspondence should be addressed. Tel.: +1 608 263 2087; fax: +1 608 262 6872; email: tpbarry@facstaff.wisc.edu 1673 0022–1112/01/121673+04 $35.00/0
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cortisol levels in reproductively mature iteroparous and semelparous chinook salmon of the same species and population. Three groups of male fish were used in the studies: immature, mature semelparous fish, and mature male parr. For the purposes of this study, it was assumed that all mature parr were iteroparous (Unwin et al., 1999). The immature males (n=22, mean mass 75 g, 1 year old) were collected in May 1998 at the National Institute of Water and Atmospheric Research’s Silverstream Research Station on the lower Waimakariri River, New Zealand. The mature semelparous and iteroparous fish were all prespawning, spermiating males at the same stage of reproductive development. The mature males (n=17, mean mass 4600 g, 3 and 4 years old) were collected at the Silverstream Research Station. These salmon had been reared for 12 months in freshwater hatcheries, released to the sea, and had returned in May 1998 to a counting fence at the release point. The mature male parr (n=47, mean mass 32 g, 1 year old) were collected in May 1998 from hatchery production stock at the Glenariffe Salmon Research Station on the upper Rakaia River, New Zealand. Mature parr remain in fresh water throughout their first year of life, which distinguishes them from ‘ jacks ’ that go to sea and return (Unwin et al., 1999). The mature male parr, sampled by hand-sorting c. 4000 freshwater-reared fish, were easily distinguished morphologically from immature fish, and all freely expressed milt (Unwin et al., 1999). All fish were anesthetized with 2-phenoxyethanol during sorting to minimize handling stress, and held for 6 days before blood sampling to allow recovery from stress. Care was taken to minimize stress-induced elevations in cortisol. Fish were removed from the holding tanks in groups of five to eight for sampling, sacrificed with a lethal dose of 2-phenoxyethanol (mature parr and immature males) or a sharp blow to the head (mature fish), blotted dry, weighed and measured. Blood was collected directly into microcentrifuge tubes after severing the tail with a sharp scalpel. For all fish sampled, the interval between the commencement of handling and blood collection did not exceed 5 min. Blood samples were refrigerated for 24 h and centrifuged at 5000 g for 15 min. The serum was collected, stored at 75 C, and then shipped on dry ice to the University of Wisconsin-Madison for analysis. Because of low serum volumes from the mature male parr, serum from three to four fish was randomly pooled for analysis. Serum cortisol levels were measured using an enzyme-linked immunoassay (ELISA) that had been validated for use in chinook salmon (Barry et al., 1993). Free and protein-bound cortisol were separated using a commercial ultrafiltration device (model MPS-1, Amicon Corp., Beverly, MA, U.S.A.) according to the methods of MacMahon et al. (1983) and Lentjes et al. (1997), except that the samples were filtered at 15 C. Because of the reported isotope effect in the binding of [3H]-cortisol to serum binding proteins, free cortisol concentrations were measured in the protein-free filtrates by ELISA rather than by radiometry (Lentjes et al., 1997). Results are reported as mean... Percent data were arcsin transformed before analysis. Means were compared by analysis of variance (ANOVA) followed by Fishers LSD test (Steel & Torrie, 1960), with a significance level of P
