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Whole body and tissue blood volumes of two strains of rainbow trout (Oncorhynchus mykiss)
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Camp. Biochem. Physiol. Vol. 97A, No. 4, pp. 615-620, 1990 Printed in Great Britain
WHOLE BODY AND TISSUE BLOOD VOLUMES OF TWO STRAINS OF RAINBOW TROUT (ONCORHYNCHUS W. H. U.S. Fish
GINGERICH,*
MYIUSS)
R. A. FITYER~
and J. J.
RACH
and Wildlife Service, National Fisheries Research Center, P.O. Box 818, La Crosse, WI 54602, USA (Received 8 May 1990)
Abstract-l.
Estimates of apparent packed cell, plasma and total blood volumes for the whole body and for 13 selected tissues were compared between Kamloops and Wytheville strains of rainbow trout (Oncorhynchus mykiss) by the simultaneous injection of two vascular tracers, radiolabeled trout erythrocytes (-%-RBC) and radioiodated bovine serum albumin (‘*51-BSA). 2. Whole body total blood volume, plasma volume and packed cell volume were slightly, but not significantly greater in the Wytheville trout, whereas, the apparent plasma volumes and total blood volumes in 4 of 13 tissues were significantly greater in the Kamloops strain. 3. Differences were most pronounced in highly perfused organs, such as the liver and kidney and in organs of digestion such as the stomach and intestines. 4. Differences in blood volumes between the two strains may be related to the greater permeability of the vascular membranes in the Kamloops strain fish.
INTRODUCTlON Growth, stamina, survival, reproduction and body composition are known to differ among genetic strains of the same fish species. These differences have been relatively well documented between and among domesticated and wild strains of rainbow trout, Oncorhynchus mykiss (Bamhart, 1969; Northtote et al., 1970; Ayles et al., 1975; Reisenbichler and McIntyre, 1977; Gall and Cross, 1978; Reinitz et al., 1979; Scott and Sumpter, 1983; Leider et al., 1986); however, little is understood of the physiological basis that underlies them. Physiological differences that have been identified have generally been attributed to specific biochemical adaptations (Iuchui, 1973; Tsuyuki and Williscroft, 1973; Klar et al., 1979a,b; Redding and Schreck, 1979). Few solely physiological differences have been identified. Distinct physiological differences have been observed between hatchery-reared and wild rainbow trout (Barnhart, 1969); some of which were direct responses to stressing agents (Wydoski et al., 1976; Casillas and Smith, 1977; Woodward and Strange, 1987). The basis of response to stressing agents probably results from different physiological performance capabilities between the strains or from different capacities for metabolic activities (Dickson and Kramer, 1971). Differences in total tissue blood volumes and in the relative degree of vascularization of tissues may confer selective advantages to genetically distinct races of fish and could contribute directly to the physiological performance capability of the animal. For example, enhanced vascularization
*To whom correspondence should be addressed. TPresent address: Department of Physiological Chemistry, School of Medicine, University of Wisconsin-Madison. WI 57106, USA.
or perfusion of certain tissues or organs, such as swimming muscle or heart, might aid the “fight or flight” type responses in races of wild fish, whereas enhanced vascularization of organs of the gastrointestinal tract might improve food conversion efficiency in domesticated stocks of fish. To our knowledge, differences in relative whole body blood volumes and tissue blood volumes in different strains of the same species of fish have not been examined. The objective of the present study was to compare apparent plasma, packed cell, and total blood volumes in the whole body and in selected tissues of two divergent strains of rainbow trout. The strains were chosen because of known differences in their performance characteristics under hatchery conditions and in the wild. Wytheville strain (WS) rainbow trout exhibit robust growth under hatchery conditions and are generally amenable to conditions of intensive culture (R. Simon, personal communication National Fisheries Research Center, Leetown, West Virginia). Kamloops strain (KAMS) rainbow trout were selected because they represent a strain of trout that has not been extensively cross-bred in hatcheries and because of their enhanced growth and survival in the wild (Ayles, 1975; Linder et al., 1983). MATERIALS AND METHODS Fish culture and holding
Adult (3 year old), mixed-sex, gonadally immature fish of both strains were used in the study. Adult KAMS trout were obtained from Peterson Trout Farms, Peterson, Minnesota, and cultured in outside raceways for at least 3 months before they were used in our experiments. Eyed eggs of WS rainbow trout were obtained from the Erwin (Tennessee) National Fish Hatchery and raised to adults at the La Crosse (Wisconsin) National Fisheries Research Center,
615
616
W. H. GINGERICH er al. Table I. Comparison of tissue and organ weights as a per cent of body weight in two strains of rainbow trout. Values represent the mean and SE of the number of individuals shown in parentheses Strain Tissue Posterior kidney Anterior kidney Liver Branchial basket Small intestine Large intestine Pyloric caecum Heart Eyes Stomach Brain Perivisceral fat Red muscle White muscle Skin Head Fins Swim bladder Spleen Bone
Wytheville (8)
Kamloops (7)
0.57 * 0.03 0.24 + 0.02 0.97 + 0.09 2.73 + 0.07 0.27 * 0.03 0.39 + 0.03 1.01 & 0.08 0.14 t 0.01 0.71 * 0.04 1.33 * 0.07 0.12 * 0.01 0.89+0.17 3.43 f 0.29 46.7 i 1.18 7.48 f 0.21 9.02 i_ 0.42 2.86 + 0.37 0.18 f 0.03
0.71 + 0.04* 0.27 + 0.04
0.08 i 0.01
9.97 f 0.37
I.18& 0.06 2.69+0.08 0.43+-0.02* 0.48kO.05 1.28kO.18 0.1350.00 0.53*o.oLx* 1.39& 0.04 0.07f 0.00' 1.35+ 0.41 4.02f 0.28 50.5k l.24* 6.13+0.24* 8.18&O.SO I.81* 0.09* 0.19+ 0.02 0.14* 0.021 11.6+ 0.37'
r value (two-tailed) 0.019 0.564 0.885 0.723 0.001 0.152 0.170 0.195 0.005 0.505 0.000 0.499 0.203 0.047
0.001 0.270
0.030 0.704 0.008 0.012
‘Denotes values significantly different (P < 0.05) from Wytheville strain.
under culture conditions described by Gingerich (1986). The mean weight of the KAMS fish used in the study was
541.6 + 17.2 g (X f SD; n = 16) and ranged from 407.4 to 622.4g; the weight of the WS fish used in the study was 433.1 & 72.1 g (X&SD; n = 13) and ranged from 357.3 to 600.8 g. The fish were reared in outside raceways and transferred to rectangular 50001 fiberglass tanks in the laboratory at least 4 weeks before an experiment to enable them to adapt to conditions of confinement and photoperiod (12 hr L: 12 hr D). Chemicals %hromium (sodium chromate, 350-600 mCi/mg Cr) was purchased from Amersham Chemical Company* (Arlington Heights, IL). Radioiodated bovine serum albumin (lz51BSA) was obtained from New England Nuclear (Boston, MA). Phosphate buffered saline (PBS) used to wash red cell suspensions and to dilute the blood-protein suspensions was prepared according to Gingerich et al. (1987). Bovine serum albumin (Fraction V) was obtained from the Sigma Chemical Co. (St. Louis, MO). All other chemicals were of reagent quality. Experimental Fish were allowed to adapt to plexiglass restraining tubes (Gingerich, 1986) for 24 hr before a dorsal aorta cannula was implanted (Beyenbach and Kirschner, 1975). Fish were then allowed to recover an additional 24 hr before an experiment was started. Preparation of %r radiolabeled erythrocytes (“Cr-RBC) and of the S’Cr-RBC and 12sI-BSA suspension was as described by Gingerich and Pityer (1988). The red cell suspension that was injected into the fish had the following characteristics: albuminated PBS (4.0 g/l00 ml), mean “Cr activity 1,772,238 f 57,036 cpm/ml (X If: SE; n = 7), mean lzsI activity 12,428,867 * 1,293,OOO cpm/ml, and final hematocrit 30%. The activity of 5’Cr in the plasma portion of the red cell suspension was consistently less than 0.05% of the activity in whole blood, indicating that little hemolysis of the erythrocytes had occurred. Whole body blood volumes and tissue blood volumes were determined in two separate experiments according to methods described by Gingerich and Pityer (1988). Whole
*References to trade names do not constitute endorsement by the U.S. Government.
body plasma and packed cell volumes were determined in five WS fish and nine KAMS fish by constructing a tracerdilution curve from data derived from four serial blood samples (0.4 ml/sample) taken at hourly intervals after the injection of the red cell suspension. Plasma and packed cell volumes in individual tissues were determined in a second experiment with nine WS and seven KAMS. Fish were dissected 4 hr after a single dose (1 .Oml/kg) of red cell suspension was injected into each fish; tissues were prepared and counted as described by Gingerich and Pityer (1988). Total blood volume estimates for both whole body and tissues were calculated as the sum of the apparent plasma and packed cell volumes. Tissue hematocrit values were determined by dividing the relative packed cell volume of the tissue by its relative total blood volume. The hematocrit ratio for each tissue was calculated as the ratio of the tissue hematocrit to that of the dorsal aorta hematocrit, which was determined by centrifugation. Data analysis Wet tissue and organ weights were divided by the body weight to convert each value to a relative percentage of body weight. Relative tissue and organ weights were compared between the two strains by unpaired t-test, after the data had been transformed to arc-sine per cent values. Relative vascular volumes (i.e. packed cell volume, plasma volume, and total blood volume) were correlated with body weight within each strain by simple linear regression analysis. Sex-related differences in relative vascular volumes of the same strain were evaluated by ANOVA. Differences in apparent packed cell, plasma and total blood volumes and hematocrit ratios between the two strains of fish were tested for significance by ANOVA. The level for statistical significance was set at P < 0.05. Variances in the data sets between strains of fish were tested for homogeneity by the method of Bartlett (Sokal and Rolf, 1969) before data analysis. All statistical tests were performed with PC-SAS (SAS Institute, Cary, NC). RESULTS
Tissue and organ weights Relative tissue and organ weights differed significantly between fish of the WS and KAMS strains (Table 1). The mean relative brain weight of KAMS was half that of the WS and relative eye weight was
Blood volumes in two strains of rainbow trout
less than 60%. Similarly, the skin and fins of KAMS comprised a significantly smaller proportion of total body weight than those of WS. Conversely, the relative weight of the posterior kidney, small intestine, spleen and the white muscle mass were greater in KAMS. Whole body and tissue blood volumes
Apparent whole body plasma, packed cell, and total blood volumes were not significantly different between the two strains (Table 2). Whole body blood volumes were not correlated with body weight in either strain; rather, blood volume in both was a relatively constant percentage of body weight. No sex-related differences in whole body blood volumes were noted in either strain of trout. Tissue plasma volumes and total blood volumes were consistently greater in KAMS than in WS (Table 3). Inter-strain differences were most pronounced in highly perfused organs and organs of digestion, but were not limited to those tissues. Significant differences between the two strains of trout were noted in the apparent plasma volumes and total blood volumes of the liver, anterior and posterior kidney and stomach. A slight, but not statistically significant, difference was observed in the small intestine. Packed cell volumes in all tissues were consistently greater in the tissues of WS, but the only significant inter-strain difference was found in the white muscle. No sex-related differences were detected for any of the tissue blood volumes in either strain. Small, but statistically significant, correlations of body weight with tissue blood volume were observed in both strains of fish. In KAMS fish, the packed cell volumes of the small intestine (R2 = 0.57) and stomach (R2 = 0.60) were positively correlated with body weight. In the WS, body weight was positively correlated with total blood volume in red muscle (R2 = 0.56), plasma volume in eye (R2 = 0.61), heart (R2 = 0.49), and red muscle (R2 = 0.65), and with packed cell volume in gill filaments (R2 = 0.65), pyloric caecum (A2 = 0.46), and white muscle (R2 = 0.57). No other correlations of body weight with total, plasma, or packed cell tissue volumes were observed. The highest hematocrit ratios (93-110%) were noted in the brain and the gill filaments of both species (Table 3); in all other tissues this ratio was less than 75%. This ratio provides a reliable estimate of the relative volumes of the plasma and blood spaces in tissues relative to the distribution of each tracer in the general circulation (Gingerich and Pityer, 1988). The lower hematocrit ratios in tissues of KAMS fish are the result of relatively greater apparent plasma volumes and smaller packed cell volumes in the tissues. A significant inter-strain difference in Table 2. Whole packed red cell volume, plasma volume, and total blood volume (ml/l00 g) in two strains of rainbow trout determined by the simultaneous injection of “Cr-RBC and 12j1-BSA tracers. Values represent the mean + SE of the number in parentheses Strain Wytheville (5) Kamloops (9)
Packed red cell
Plasma
Total
1.53 f 0.08 I .39 + 0.04
3.74 f 0.34 3.24 k 0.29
5.27 +_0.30 4.63 F 0.31
617
W. H.
618
GINGERICH er
the ratio was found only for the white muscle. No significant sex-related differences were observed for hematocrit ratios in either strain, The sum of weights of tissues examined in this study accounted for about 93% of total body weight in KAMS and 89% in WS. When estimated as the sum of the products of apparent tissue blood volume and per cent of body weight for all of the tissues examined, the total volume of blood was 1.44 ml/lOOg in WS and 1.99ml/lOOg in KAMS. The values represented 27.3% of the mean estimated total blood volume (5.27 ml/100 g) in WS and 42.9% in KAMS (4.63 ml/100 g). The values do not include blood in regions considered to be in general circulation, such as the major arteries and veins, the branchial basket, and the venae cavae, or in structures that we did not examine for blood volumes (head and fins). DISCUSSION It is likely that WS and KAMS are two of the most divergent strains of rainbow trout currently produced in the United States federal hatchery system. The WS originated in northern California, spawns in the fall, and has been widely inbred in the United States because of its excellent growth performance under conditions of intensive culture (R. Simon, personal communication). The strain is considered to be one of the most domesticated of all rainbow trout strains in the United States. Conversely, the KAMS strain originated in the Kamloop Lakes Region of western British Columbia, spawns in spring, and is considered to be relatively wild. Eggs are routinely collected from wild brood stock. Kamloops trout are a frequent choice of fishery managers for release in the wild because of their tendency to reach large size in natural environments where forage is not limited (Ayles, 1975; Linder et al., 1983). The significant differences we observed between the two strains in relative tissue-to-body and organ-to-body weight ratios and in apparent tissue blood volumes may reflect differences in their genotypes. Significant differences between the two strains were observed in tissue to body weight ratios for 9 of the 20 tissues or organs we examined. These differences may be partly the result of disparities in the absolute body weights of the two groups of fish. The KAMS were about 1OOg heavier in absolute mean body weight than the WS. Because body weights of fish increase exponentially as fish grow, disproportionately greater percentages of relative tissue weights in larger fish are found in tissues that compose the greater proportions of body mass (Denton and Yousef, 1976). Differences in body weights between strains would tend to distort the percentage composition of minor body tissues and organs and partly explain why tissues, such as the heart, eyes and fins, constituted a significantly smaller proportion of the body weight in the larger KAMS. This was not true for all tissues. Relative brain weights were proportionately smaller and organs of digestion were larger in KAMS than in WS, despite the overall larger size of the KAMS. Absolute weights of the brains of KAMS were less than those of the WS and resulted
al.
in mean relative brain weights that were only 58% of those in WS. Conversely, the organs of digestion generally represented a greater proportion of body weight in KAMS than in WS. However, because the relative visceral weight in trout declines with age (Denton and Yousef, 1976) the heavier KAMS should have had proportionately smaller visceral masses than the lighter WS. Such differences in relative body weights are probably related to genetic differences between strains. Phylogenetically, smaller blood volumes are thought to represent evidence of a more efficient circulatory system, since a smaller volume of transport fluid per unit of mass is required to maintain respiratory and ionoregulatory functions and water balance (Conte et al., 1963). As a group, fish have perhaps the smallest blood volumes of all vertebrates (Thorson, 1961). Their highly efficient respiratory system and relatively low basal metabolism reduce the requirements for a large blood volume. Whole body blood volume estimates for the two strains of rainbow trout used in this study were well within the range of values previously reported for the species (Conte et al., 1963; Stevens and Randall, 1968; Duff et al., 1987; Gingerich et al., 1987; Gingerich and Pityer, 1988). Apparent packed cell volumes, plasma volumes, and total blood volumes estimated for the whole body were not different between the two strains, even though these values were greater in the WS. The proportions of plasma volume to total blood volume between the two species were nearly identical. Apparent tissue blood volumes were significantly greater in four of the 13 tissues examined in the KAMS. The greatest differences in tissue blood volumes were noted in highly perfused tissues such as the kidney, liver and intestine, or in tissues that receive a substantial portion of cardiac output, such as red and white muscle (Barron et al., 1987). With the exceptions of gill filaments, brain, white muscle and kidney, estimated packed cell volumes for all tissues were virtually identical in the two strains. Thus. inter-strain differences in the total blood volumes of tissues were probably a direct result of greater apparent plasma volumes in KAMS. Apparent volumes of distribution for the tracers used in this study were calculated under the assumption that the tracer remained within the vascular tree. This may not be valid for the “‘1-BSA in trout. Vascular-extravascular exchange of plasma proteins has been extensively documented in mammals studied with radiolabeled plasma protein and erythrocyte tracers (Swan and Nelson, 1971). The rates of exchange are related to the molecular weights of the proteins; the rates of movement across vascular membranes are faster for smaller proteins than for larger ones (Dewey, 1959). Thus, the apparent tissue plasma volumes that we report may be overestimated for two reasons. First, capillary membranes in fish appear to be more permeable to plasma proteins than those of terrestrial animals (Hargens et al., 1974); and second, the mass of the principal plasma protein in trout is greater than that of bovine serum albumin (Ohkawa et al., 1987). As a result, the ‘251-BSA tracer would yield an estimate of volume that was greater than that of the vascular tree.
Blood volumes in two strains of rainbow trout
619
Gingerich and Pityer (1988) compared the total Friedman J. (1971) Microcirculation. In Physiology (Edited by Selkurt E. E.). pp. 259-273. Little, Brown and Co., volumes in a number of tssues from WS Boston, MA, USA. rainbow trout with both YTr-RBC and 12’I-BSA
blood
tracers and concluded that the greater volume of distribution for ‘251-BSA resulted from its diffusion into an artificiaily expanded extravascular space within the tissues. If that conclusion is correct, it is likely that the differences we observed between selected tissues in the two strains were the result of a more rapid movement of ‘2SI-BSA into the extravascular space of the KAMS. Differences between the two strains in the rate of ‘*%BSA diffusion into the interstitial space suggests a disparity in factors that control the trans-~apiIia~ exchange of ffuids and plasma proteins. Possible factors include variations in hydrostatic pressure between the capillary and interstitial fluids, differences in the plasma and interstitial colloidal osmotic pressures, or differences in the permeabilities of the membranes lining the capillary walls of the tissues ~F~edman, 1971). Movement of plasma proteins into the interstitial space of tissues is an important factor
in the co-transport of water-insoluble substances into the tissues (i.e. non-esterified fatty acids and some hormones) and in tissue distribution of defensive immunoproteins (Renkin, 1986). The adaptive advantage for enhanced or suppressed movement of proteins through tissue membranes of fish is not clear. Acknowledgements-‘The
authors thank MS Linda Gardner for technical assistance, MS Georginia Ardinger for typing
the manuscript and Dr Reginald Reisenbickler for reviewing it. REFERENCES
Ayles G. B. (1975) Influence of the genotype and the environment on growth and survival of rainbow trout (Salvo gairdneri) in central Canadian aquaculture lakes. Aquaculture 6, 181-188. Barnhart R. A. (1969) Effects of certain variables on hematological characteristics of rainbow trout. Trans. Am. Fish. Sot. 9% 41 I-418. Barron M. G., Tarr B. D. and Hayton W. L. (1987) Temperature-dependence of cardiac output and regional blood Sow in rainbow trout (Salmo gairdneri) Richardson. J. Fish Biol. 31, 735-744. Beyenbach K. W. and Kirschner L. B. (1975) Kidney and urinary bladder function of the rainbow trout in Mg and Na excretion. Am. J. Pnysiot. 229, 389-393. Casillas E. and Smith L. S. (1977) Effects of stress on blood coagulation and hematology in rainbow trout (Sulmo gairdneri). J. Fish Biol. 10, 481-491. Conte F. P., Wagner H. H. and Harris H. 0. (1963) Measurement of blood volume in the fish (Salmo gairdnet?). Am. J. Physiot. 205, 533-540.
Dewey W. C. (19.59)Vascular-extravascular exchange of i3’I plasma proteins in the rat. Am. J. Physiol. 197,423-431. Denton J. E. and Yousef M. K. (1976) Body composition and organ weights of rainbow trout (Salmo gairdneri). J. Fish Biol. 8, 489-499.
Dickson I. W. and Kramer R. H. (1971) Factors influencing scope for activity and active and standard metabolism in rainbow trout (~~I~0 gairdneri). J. Fish. Res. Board Can. 28, 587-596.
Duff D. W., Fitzgerald D., Kullman D., Lipke D. W., Ward J. and Olson K. R. (1987) Blood volume and red cell space in tissues of the rainbow trout (Salmo gairdneri). Camp. Biochem. Physiol. glA, 393-398.
Gall G. E. A. and Cross S. J. (1978) A genetic analysis of the performance of three rainbow trout broodstocks. Aqu&ulture
IS, 113~127.
Ginaerich W. H. (1986) Tissue distribution and eliminacon of rotenonk in rainbow trout. Aquat. Toxicol. 8, 27-40. Gingerich W. H., Pityer R. A. and Rach J. J. (1987) Estimates of plasma, packed cell and total blood volume in tissues of the rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. WA, 251-256. Gingerich W. H. and Pityer R. A. (1988) Comparison of whole body and tissue blood volumes in rainbow trout (Salmo gairdneri) with ‘*%bovine serum albumin and 5’Cr-ervthrocvte tracers. Fish Phvsiol. Biochem. 6. 39-47. Hargens A. R.,Millard R. W. and Johansen K. (1974) High capillary permeability in fishes. Comp. Biochem. Physiol. 48A, 657-670. Iuchui I. (1973) Chemical and physiological properties of the larval and adult hemoglobins in rainbow trout, Salmo gairdneri irideus. Comp. 3iochem. 1087-I 101.
Fhys~oI. 44&
Klar G., Stalkner C. and Farley T. (1979a) Comparative physical and physiological performance of rainbow trout, Salmo gairdneri, of distinct lactate dehydrogenase B2 phenotypes. Comp. Biochem. Physiol. 63A, 229-235. Klar G., Stalkner C. and Farley T. (1979b) Comparative blood lactate response to low oxygen concentrations in rainbow trout, Salmo gairdneri, LDH B’ phenotypes. Comp. Biochem. Physiol. 63A, 236-240.
Leider S. A., Chilcote M. W. and Loch J. J. (1986) Comparative life history characteristics of hatchery and wild steelhead trout (Salmo gairdneri) of summer and winter races in the Kalama River, Washington. Can. J. Fish. Aquat. Sci. 43, 1389-1409.
Linder D., Sumari 0.. Nyholm K. and Sirkomaa S. (1983) Genetic and phenotypic variation in production traits in rainbow trout strains and strain crosses in Finland. Aquaculture 33, 129-134. Northcote T., Williscroft S. and Tsuyuki H. (1970) Meristic and lactic dehydrogenase genotype differences in stream populations of rainbow trout below and above a water fail. J. Fish. Res. Board Can. 27, 1987-1995. Ohkawa K., Tsukada Y., Nunomura W., Ando M., Kimura I., Hara A., Hibi N. and Hirai H. (1987) Main serum protein of rainbow trout (Sulmo gairdneri): its biological properties and significance. Comp. Biochem. Physiol. SSB, 497-50 1, Redding J. M. and Schreck C. B. (1979) Possible adaptive significance of certain enzyme polymorphisms in steelhead trout (Salmo gairdneri). J. Fish. Res. Board Can. 36, 544-551,
Reisenbichler R. R. and McIntyre J. D. (1977) Genetic differences in growth and survival of juvenile hatchery and wild steelhead trout. Salmo gairdneri. J. Fish. Res. Board Can. 34, 123-128.
Reinitz G. L., Ohrme L. and Hitzel F. (1979) Variations of body composition and growth among strains of rainbow trout, T+uns. Am. Fish. Sot. 108, 2044207. Renkin E. M. (1986) Some consequences of capillary permeability to macromolecules: Starling’s hypothesis reconsidered. Am. J. Physiol. 250, H706-H710. Scott A. P. and Sumpter J. P. (1983) A comparison of the female reproductive cycles of autumn-spawning and winter-spawning strains of rainbow trout (Salmo gctirdneri Richardson). Gen. Comp. Endocrinol. 52, 79-85. Sokal R. R. and Rohlf F. J. (1969) BiQmetry.W. H. Freeman and Company, San Francisco, USA. Stevens E. D. and Randall D. J. (1968) The effect of exercise on the distribution of blood to various organs in rainbow trout. Comp. Biochem. Physiol. 25, 615-625.
620
W. H. GINGERICHet al.
Swan H. and Nelson A. W. (1971) Blood volume I: Critique: Spun vs isotope hematocrit: ‘*%HSA vs 5’Cr-RBC. Surg. Annu. 173, 481-495. Thorson T. B. (1961) The partitioning of body water in Osteichthyes: Phylogenetic and ecological variations in aquatic vertebrates. Viol. BUN. (Woods Hole) 120, 238-254. Tsuyuki H. and Williscroft S. N. (1973) The pH activity relations of two LDH homotetramers from liver and their
physiological significance. J. Fish. Res. Board Can. 30, 996-1003. Woodward C. C. and Strange R. J. (1987) Physiological stress response in wild and hatchery-reared rainbow trout. Trans. Am. Fish. Sot. 116, 574-579. Wydoski R. S., Wedemeyer G. A. and Nelson N. C. (1976) Physiological response to hooking stress in hatchery and wild rainbow trout (Salmo gairdneri). Trans. Am. Fish. sot. 105, 601-606.
Camp. Biochem. Physiol. Vol. 97A, No. 4, pp. 615-620, 1990 Printed in Great Britain
WHOLE BODY AND TISSUE BLOOD VOLUMES OF TWO STRAINS OF RAINBOW TROUT (ONCORHYNCHUS W. H. U.S. Fish
GINGERICH,*
MYIUSS)
R. A. FITYER~
and J. J.
RACH
and Wildlife Service, National Fisheries Research Center, P.O. Box 818, La Crosse, WI 54602, USA (Received 8 May 1990)
Abstract-l.
Estimates of apparent packed cell, plasma and total blood volumes for the whole body and for 13 selected tissues were compared between Kamloops and Wytheville strains of rainbow trout (Oncorhynchus mykiss) by the simultaneous injection of two vascular tracers, radiolabeled trout erythrocytes (-%-RBC) and radioiodated bovine serum albumin (‘*51-BSA). 2. Whole body total blood volume, plasma volume and packed cell volume were slightly, but not significantly greater in the Wytheville trout, whereas, the apparent plasma volumes and total blood volumes in 4 of 13 tissues were significantly greater in the Kamloops strain. 3. Differences were most pronounced in highly perfused organs, such as the liver and kidney and in organs of digestion such as the stomach and intestines. 4. Differences in blood volumes between the two strains may be related to the greater permeability of the vascular membranes in the Kamloops strain fish.
INTRODUCTlON Growth, stamina, survival, reproduction and body composition are known to differ among genetic strains of the same fish species. These differences have been relatively well documented between and among domesticated and wild strains of rainbow trout, Oncorhynchus mykiss (Bamhart, 1969; Northtote et al., 1970; Ayles et al., 1975; Reisenbichler and McIntyre, 1977; Gall and Cross, 1978; Reinitz et al., 1979; Scott and Sumpter, 1983; Leider et al., 1986); however, little is understood of the physiological basis that underlies them. Physiological differences that have been identified have generally been attributed to specific biochemical adaptations (Iuchui, 1973; Tsuyuki and Williscroft, 1973; Klar et al., 1979a,b; Redding and Schreck, 1979). Few solely physiological differences have been identified. Distinct physiological differences have been observed between hatchery-reared and wild rainbow trout (Barnhart, 1969); some of which were direct responses to stressing agents (Wydoski et al., 1976; Casillas and Smith, 1977; Woodward and Strange, 1987). The basis of response to stressing agents probably results from different physiological performance capabilities between the strains or from different capacities for metabolic activities (Dickson and Kramer, 1971). Differences in total tissue blood volumes and in the relative degree of vascularization of tissues may confer selective advantages to genetically distinct races of fish and could contribute directly to the physiological performance capability of the animal. For example, enhanced vascularization
*To whom correspondence should be addressed. TPresent address: Department of Physiological Chemistry, School of Medicine, University of Wisconsin-Madison. WI 57106, USA.
or perfusion of certain tissues or organs, such as swimming muscle or heart, might aid the “fight or flight” type responses in races of wild fish, whereas enhanced vascularization of organs of the gastrointestinal tract might improve food conversion efficiency in domesticated stocks of fish. To our knowledge, differences in relative whole body blood volumes and tissue blood volumes in different strains of the same species of fish have not been examined. The objective of the present study was to compare apparent plasma, packed cell, and total blood volumes in the whole body and in selected tissues of two divergent strains of rainbow trout. The strains were chosen because of known differences in their performance characteristics under hatchery conditions and in the wild. Wytheville strain (WS) rainbow trout exhibit robust growth under hatchery conditions and are generally amenable to conditions of intensive culture (R. Simon, personal communication National Fisheries Research Center, Leetown, West Virginia). Kamloops strain (KAMS) rainbow trout were selected because they represent a strain of trout that has not been extensively cross-bred in hatcheries and because of their enhanced growth and survival in the wild (Ayles, 1975; Linder et al., 1983). MATERIALS AND METHODS Fish culture and holding
Adult (3 year old), mixed-sex, gonadally immature fish of both strains were used in the study. Adult KAMS trout were obtained from Peterson Trout Farms, Peterson, Minnesota, and cultured in outside raceways for at least 3 months before they were used in our experiments. Eyed eggs of WS rainbow trout were obtained from the Erwin (Tennessee) National Fish Hatchery and raised to adults at the La Crosse (Wisconsin) National Fisheries Research Center,
615
616
W. H. GINGERICH er al. Table I. Comparison of tissue and organ weights as a per cent of body weight in two strains of rainbow trout. Values represent the mean and SE of the number of individuals shown in parentheses Strain Tissue Posterior kidney Anterior kidney Liver Branchial basket Small intestine Large intestine Pyloric caecum Heart Eyes Stomach Brain Perivisceral fat Red muscle White muscle Skin Head Fins Swim bladder Spleen Bone
Wytheville (8)
Kamloops (7)
0.57 * 0.03 0.24 + 0.02 0.97 + 0.09 2.73 + 0.07 0.27 * 0.03 0.39 + 0.03 1.01 & 0.08 0.14 t 0.01 0.71 * 0.04 1.33 * 0.07 0.12 * 0.01 0.89+0.17 3.43 f 0.29 46.7 i 1.18 7.48 f 0.21 9.02 i_ 0.42 2.86 + 0.37 0.18 f 0.03
0.71 + 0.04* 0.27 + 0.04
0.08 i 0.01
9.97 f 0.37
I.18& 0.06 2.69+0.08 0.43+-0.02* 0.48kO.05 1.28kO.18 0.1350.00 0.53*o.oLx* 1.39& 0.04 0.07f 0.00' 1.35+ 0.41 4.02f 0.28 50.5k l.24* 6.13+0.24* 8.18&O.SO I.81* 0.09* 0.19+ 0.02 0.14* 0.021 11.6+ 0.37'
r value (two-tailed) 0.019 0.564 0.885 0.723 0.001 0.152 0.170 0.195 0.005 0.505 0.000 0.499 0.203 0.047
0.001 0.270
0.030 0.704 0.008 0.012
‘Denotes values significantly different (P < 0.05) from Wytheville strain.
under culture conditions described by Gingerich (1986). The mean weight of the KAMS fish used in the study was
541.6 + 17.2 g (X f SD; n = 16) and ranged from 407.4 to 622.4g; the weight of the WS fish used in the study was 433.1 & 72.1 g (X&SD; n = 13) and ranged from 357.3 to 600.8 g. The fish were reared in outside raceways and transferred to rectangular 50001 fiberglass tanks in the laboratory at least 4 weeks before an experiment to enable them to adapt to conditions of confinement and photoperiod (12 hr L: 12 hr D). Chemicals %hromium (sodium chromate, 350-600 mCi/mg Cr) was purchased from Amersham Chemical Company* (Arlington Heights, IL). Radioiodated bovine serum albumin (lz51BSA) was obtained from New England Nuclear (Boston, MA). Phosphate buffered saline (PBS) used to wash red cell suspensions and to dilute the blood-protein suspensions was prepared according to Gingerich et al. (1987). Bovine serum albumin (Fraction V) was obtained from the Sigma Chemical Co. (St. Louis, MO). All other chemicals were of reagent quality. Experimental Fish were allowed to adapt to plexiglass restraining tubes (Gingerich, 1986) for 24 hr before a dorsal aorta cannula was implanted (Beyenbach and Kirschner, 1975). Fish were then allowed to recover an additional 24 hr before an experiment was started. Preparation of %r radiolabeled erythrocytes (“Cr-RBC) and of the S’Cr-RBC and 12sI-BSA suspension was as described by Gingerich and Pityer (1988). The red cell suspension that was injected into the fish had the following characteristics: albuminated PBS (4.0 g/l00 ml), mean “Cr activity 1,772,238 f 57,036 cpm/ml (X If: SE; n = 7), mean lzsI activity 12,428,867 * 1,293,OOO cpm/ml, and final hematocrit 30%. The activity of 5’Cr in the plasma portion of the red cell suspension was consistently less than 0.05% of the activity in whole blood, indicating that little hemolysis of the erythrocytes had occurred. Whole body blood volumes and tissue blood volumes were determined in two separate experiments according to methods described by Gingerich and Pityer (1988). Whole
*References to trade names do not constitute endorsement by the U.S. Government.
body plasma and packed cell volumes were determined in five WS fish and nine KAMS fish by constructing a tracerdilution curve from data derived from four serial blood samples (0.4 ml/sample) taken at hourly intervals after the injection of the red cell suspension. Plasma and packed cell volumes in individual tissues were determined in a second experiment with nine WS and seven KAMS. Fish were dissected 4 hr after a single dose (1 .Oml/kg) of red cell suspension was injected into each fish; tissues were prepared and counted as described by Gingerich and Pityer (1988). Total blood volume estimates for both whole body and tissues were calculated as the sum of the apparent plasma and packed cell volumes. Tissue hematocrit values were determined by dividing the relative packed cell volume of the tissue by its relative total blood volume. The hematocrit ratio for each tissue was calculated as the ratio of the tissue hematocrit to that of the dorsal aorta hematocrit, which was determined by centrifugation. Data analysis Wet tissue and organ weights were divided by the body weight to convert each value to a relative percentage of body weight. Relative tissue and organ weights were compared between the two strains by unpaired t-test, after the data had been transformed to arc-sine per cent values. Relative vascular volumes (i.e. packed cell volume, plasma volume, and total blood volume) were correlated with body weight within each strain by simple linear regression analysis. Sex-related differences in relative vascular volumes of the same strain were evaluated by ANOVA. Differences in apparent packed cell, plasma and total blood volumes and hematocrit ratios between the two strains of fish were tested for significance by ANOVA. The level for statistical significance was set at P < 0.05. Variances in the data sets between strains of fish were tested for homogeneity by the method of Bartlett (Sokal and Rolf, 1969) before data analysis. All statistical tests were performed with PC-SAS (SAS Institute, Cary, NC). RESULTS
Tissue and organ weights Relative tissue and organ weights differed significantly between fish of the WS and KAMS strains (Table 1). The mean relative brain weight of KAMS was half that of the WS and relative eye weight was
Blood volumes in two strains of rainbow trout
less than 60%. Similarly, the skin and fins of KAMS comprised a significantly smaller proportion of total body weight than those of WS. Conversely, the relative weight of the posterior kidney, small intestine, spleen and the white muscle mass were greater in KAMS. Whole body and tissue blood volumes
Apparent whole body plasma, packed cell, and total blood volumes were not significantly different between the two strains (Table 2). Whole body blood volumes were not correlated with body weight in either strain; rather, blood volume in both was a relatively constant percentage of body weight. No sex-related differences in whole body blood volumes were noted in either strain of trout. Tissue plasma volumes and total blood volumes were consistently greater in KAMS than in WS (Table 3). Inter-strain differences were most pronounced in highly perfused organs and organs of digestion, but were not limited to those tissues. Significant differences between the two strains of trout were noted in the apparent plasma volumes and total blood volumes of the liver, anterior and posterior kidney and stomach. A slight, but not statistically significant, difference was observed in the small intestine. Packed cell volumes in all tissues were consistently greater in the tissues of WS, but the only significant inter-strain difference was found in the white muscle. No sex-related differences were detected for any of the tissue blood volumes in either strain. Small, but statistically significant, correlations of body weight with tissue blood volume were observed in both strains of fish. In KAMS fish, the packed cell volumes of the small intestine (R2 = 0.57) and stomach (R2 = 0.60) were positively correlated with body weight. In the WS, body weight was positively correlated with total blood volume in red muscle (R2 = 0.56), plasma volume in eye (R2 = 0.61), heart (R2 = 0.49), and red muscle (R2 = 0.65), and with packed cell volume in gill filaments (R2 = 0.65), pyloric caecum (A2 = 0.46), and white muscle (R2 = 0.57). No other correlations of body weight with total, plasma, or packed cell tissue volumes were observed. The highest hematocrit ratios (93-110%) were noted in the brain and the gill filaments of both species (Table 3); in all other tissues this ratio was less than 75%. This ratio provides a reliable estimate of the relative volumes of the plasma and blood spaces in tissues relative to the distribution of each tracer in the general circulation (Gingerich and Pityer, 1988). The lower hematocrit ratios in tissues of KAMS fish are the result of relatively greater apparent plasma volumes and smaller packed cell volumes in the tissues. A significant inter-strain difference in Table 2. Whole packed red cell volume, plasma volume, and total blood volume (ml/l00 g) in two strains of rainbow trout determined by the simultaneous injection of “Cr-RBC and 12j1-BSA tracers. Values represent the mean + SE of the number in parentheses Strain Wytheville (5) Kamloops (9)
Packed red cell
Plasma
Total
1.53 f 0.08 I .39 + 0.04
3.74 f 0.34 3.24 k 0.29
5.27 +_0.30 4.63 F 0.31
617
W. H.
618
GINGERICH er
the ratio was found only for the white muscle. No significant sex-related differences were observed for hematocrit ratios in either strain, The sum of weights of tissues examined in this study accounted for about 93% of total body weight in KAMS and 89% in WS. When estimated as the sum of the products of apparent tissue blood volume and per cent of body weight for all of the tissues examined, the total volume of blood was 1.44 ml/lOOg in WS and 1.99ml/lOOg in KAMS. The values represented 27.3% of the mean estimated total blood volume (5.27 ml/100 g) in WS and 42.9% in KAMS (4.63 ml/100 g). The values do not include blood in regions considered to be in general circulation, such as the major arteries and veins, the branchial basket, and the venae cavae, or in structures that we did not examine for blood volumes (head and fins). DISCUSSION It is likely that WS and KAMS are two of the most divergent strains of rainbow trout currently produced in the United States federal hatchery system. The WS originated in northern California, spawns in the fall, and has been widely inbred in the United States because of its excellent growth performance under conditions of intensive culture (R. Simon, personal communication). The strain is considered to be one of the most domesticated of all rainbow trout strains in the United States. Conversely, the KAMS strain originated in the Kamloop Lakes Region of western British Columbia, spawns in spring, and is considered to be relatively wild. Eggs are routinely collected from wild brood stock. Kamloops trout are a frequent choice of fishery managers for release in the wild because of their tendency to reach large size in natural environments where forage is not limited (Ayles, 1975; Linder et al., 1983). The significant differences we observed between the two strains in relative tissue-to-body and organ-to-body weight ratios and in apparent tissue blood volumes may reflect differences in their genotypes. Significant differences between the two strains were observed in tissue to body weight ratios for 9 of the 20 tissues or organs we examined. These differences may be partly the result of disparities in the absolute body weights of the two groups of fish. The KAMS were about 1OOg heavier in absolute mean body weight than the WS. Because body weights of fish increase exponentially as fish grow, disproportionately greater percentages of relative tissue weights in larger fish are found in tissues that compose the greater proportions of body mass (Denton and Yousef, 1976). Differences in body weights between strains would tend to distort the percentage composition of minor body tissues and organs and partly explain why tissues, such as the heart, eyes and fins, constituted a significantly smaller proportion of the body weight in the larger KAMS. This was not true for all tissues. Relative brain weights were proportionately smaller and organs of digestion were larger in KAMS than in WS, despite the overall larger size of the KAMS. Absolute weights of the brains of KAMS were less than those of the WS and resulted
al.
in mean relative brain weights that were only 58% of those in WS. Conversely, the organs of digestion generally represented a greater proportion of body weight in KAMS than in WS. However, because the relative visceral weight in trout declines with age (Denton and Yousef, 1976) the heavier KAMS should have had proportionately smaller visceral masses than the lighter WS. Such differences in relative body weights are probably related to genetic differences between strains. Phylogenetically, smaller blood volumes are thought to represent evidence of a more efficient circulatory system, since a smaller volume of transport fluid per unit of mass is required to maintain respiratory and ionoregulatory functions and water balance (Conte et al., 1963). As a group, fish have perhaps the smallest blood volumes of all vertebrates (Thorson, 1961). Their highly efficient respiratory system and relatively low basal metabolism reduce the requirements for a large blood volume. Whole body blood volume estimates for the two strains of rainbow trout used in this study were well within the range of values previously reported for the species (Conte et al., 1963; Stevens and Randall, 1968; Duff et al., 1987; Gingerich et al., 1987; Gingerich and Pityer, 1988). Apparent packed cell volumes, plasma volumes, and total blood volumes estimated for the whole body were not different between the two strains, even though these values were greater in the WS. The proportions of plasma volume to total blood volume between the two species were nearly identical. Apparent tissue blood volumes were significantly greater in four of the 13 tissues examined in the KAMS. The greatest differences in tissue blood volumes were noted in highly perfused tissues such as the kidney, liver and intestine, or in tissues that receive a substantial portion of cardiac output, such as red and white muscle (Barron et al., 1987). With the exceptions of gill filaments, brain, white muscle and kidney, estimated packed cell volumes for all tissues were virtually identical in the two strains. Thus. inter-strain differences in the total blood volumes of tissues were probably a direct result of greater apparent plasma volumes in KAMS. Apparent volumes of distribution for the tracers used in this study were calculated under the assumption that the tracer remained within the vascular tree. This may not be valid for the “‘1-BSA in trout. Vascular-extravascular exchange of plasma proteins has been extensively documented in mammals studied with radiolabeled plasma protein and erythrocyte tracers (Swan and Nelson, 1971). The rates of exchange are related to the molecular weights of the proteins; the rates of movement across vascular membranes are faster for smaller proteins than for larger ones (Dewey, 1959). Thus, the apparent tissue plasma volumes that we report may be overestimated for two reasons. First, capillary membranes in fish appear to be more permeable to plasma proteins than those of terrestrial animals (Hargens et al., 1974); and second, the mass of the principal plasma protein in trout is greater than that of bovine serum albumin (Ohkawa et al., 1987). As a result, the ‘251-BSA tracer would yield an estimate of volume that was greater than that of the vascular tree.
Blood volumes in two strains of rainbow trout
619
Gingerich and Pityer (1988) compared the total Friedman J. (1971) Microcirculation. In Physiology (Edited by Selkurt E. E.). pp. 259-273. Little, Brown and Co., volumes in a number of tssues from WS Boston, MA, USA. rainbow trout with both YTr-RBC and 12’I-BSA
blood
tracers and concluded that the greater volume of distribution for ‘251-BSA resulted from its diffusion into an artificiaily expanded extravascular space within the tissues. If that conclusion is correct, it is likely that the differences we observed between selected tissues in the two strains were the result of a more rapid movement of ‘2SI-BSA into the extravascular space of the KAMS. Differences between the two strains in the rate of ‘*%BSA diffusion into the interstitial space suggests a disparity in factors that control the trans-~apiIia~ exchange of ffuids and plasma proteins. Possible factors include variations in hydrostatic pressure between the capillary and interstitial fluids, differences in the plasma and interstitial colloidal osmotic pressures, or differences in the permeabilities of the membranes lining the capillary walls of the tissues ~F~edman, 1971). Movement of plasma proteins into the interstitial space of tissues is an important factor
in the co-transport of water-insoluble substances into the tissues (i.e. non-esterified fatty acids and some hormones) and in tissue distribution of defensive immunoproteins (Renkin, 1986). The adaptive advantage for enhanced or suppressed movement of proteins through tissue membranes of fish is not clear. Acknowledgements-‘The
authors thank MS Linda Gardner for technical assistance, MS Georginia Ardinger for typing
the manuscript and Dr Reginald Reisenbickler for reviewing it. REFERENCES
Ayles G. B. (1975) Influence of the genotype and the environment on growth and survival of rainbow trout (Salvo gairdneri) in central Canadian aquaculture lakes. Aquaculture 6, 181-188. Barnhart R. A. (1969) Effects of certain variables on hematological characteristics of rainbow trout. Trans. Am. Fish. Sot. 9% 41 I-418. Barron M. G., Tarr B. D. and Hayton W. L. (1987) Temperature-dependence of cardiac output and regional blood Sow in rainbow trout (Salmo gairdneri) Richardson. J. Fish Biol. 31, 735-744. Beyenbach K. W. and Kirschner L. B. (1975) Kidney and urinary bladder function of the rainbow trout in Mg and Na excretion. Am. J. Pnysiot. 229, 389-393. Casillas E. and Smith L. S. (1977) Effects of stress on blood coagulation and hematology in rainbow trout (Sulmo gairdneri). J. Fish Biol. 10, 481-491. Conte F. P., Wagner H. H. and Harris H. 0. (1963) Measurement of blood volume in the fish (Salmo gairdnet?). Am. J. Physiot. 205, 533-540.
Dewey W. C. (19.59)Vascular-extravascular exchange of i3’I plasma proteins in the rat. Am. J. Physiol. 197,423-431. Denton J. E. and Yousef M. K. (1976) Body composition and organ weights of rainbow trout (Salmo gairdneri). J. Fish Biol. 8, 489-499.
Dickson I. W. and Kramer R. H. (1971) Factors influencing scope for activity and active and standard metabolism in rainbow trout (~~I~0 gairdneri). J. Fish. Res. Board Can. 28, 587-596.
Duff D. W., Fitzgerald D., Kullman D., Lipke D. W., Ward J. and Olson K. R. (1987) Blood volume and red cell space in tissues of the rainbow trout (Salmo gairdneri). Camp. Biochem. Physiol. glA, 393-398.
Gall G. E. A. and Cross S. J. (1978) A genetic analysis of the performance of three rainbow trout broodstocks. Aqu&ulture
IS, 113~127.
Ginaerich W. H. (1986) Tissue distribution and eliminacon of rotenonk in rainbow trout. Aquat. Toxicol. 8, 27-40. Gingerich W. H., Pityer R. A. and Rach J. J. (1987) Estimates of plasma, packed cell and total blood volume in tissues of the rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. WA, 251-256. Gingerich W. H. and Pityer R. A. (1988) Comparison of whole body and tissue blood volumes in rainbow trout (Salmo gairdneri) with ‘*%bovine serum albumin and 5’Cr-ervthrocvte tracers. Fish Phvsiol. Biochem. 6. 39-47. Hargens A. R.,Millard R. W. and Johansen K. (1974) High capillary permeability in fishes. Comp. Biochem. Physiol. 48A, 657-670. Iuchui I. (1973) Chemical and physiological properties of the larval and adult hemoglobins in rainbow trout, Salmo gairdneri irideus. Comp. 3iochem. 1087-I 101.
Fhys~oI. 44&
Klar G., Stalkner C. and Farley T. (1979a) Comparative physical and physiological performance of rainbow trout, Salmo gairdneri, of distinct lactate dehydrogenase B2 phenotypes. Comp. Biochem. Physiol. 63A, 229-235. Klar G., Stalkner C. and Farley T. (1979b) Comparative blood lactate response to low oxygen concentrations in rainbow trout, Salmo gairdneri, LDH B’ phenotypes. Comp. Biochem. Physiol. 63A, 236-240.
Leider S. A., Chilcote M. W. and Loch J. J. (1986) Comparative life history characteristics of hatchery and wild steelhead trout (Salmo gairdneri) of summer and winter races in the Kalama River, Washington. Can. J. Fish. Aquat. Sci. 43, 1389-1409.
Linder D., Sumari 0.. Nyholm K. and Sirkomaa S. (1983) Genetic and phenotypic variation in production traits in rainbow trout strains and strain crosses in Finland. Aquaculture 33, 129-134. Northcote T., Williscroft S. and Tsuyuki H. (1970) Meristic and lactic dehydrogenase genotype differences in stream populations of rainbow trout below and above a water fail. J. Fish. Res. Board Can. 27, 1987-1995. Ohkawa K., Tsukada Y., Nunomura W., Ando M., Kimura I., Hara A., Hibi N. and Hirai H. (1987) Main serum protein of rainbow trout (Sulmo gairdneri): its biological properties and significance. Comp. Biochem. Physiol. SSB, 497-50 1, Redding J. M. and Schreck C. B. (1979) Possible adaptive significance of certain enzyme polymorphisms in steelhead trout (Salmo gairdneri). J. Fish. Res. Board Can. 36, 544-551,
Reisenbichler R. R. and McIntyre J. D. (1977) Genetic differences in growth and survival of juvenile hatchery and wild steelhead trout. Salmo gairdneri. J. Fish. Res. Board Can. 34, 123-128.
Reinitz G. L., Ohrme L. and Hitzel F. (1979) Variations of body composition and growth among strains of rainbow trout, T+uns. Am. Fish. Sot. 108, 2044207. Renkin E. M. (1986) Some consequences of capillary permeability to macromolecules: Starling’s hypothesis reconsidered. Am. J. Physiol. 250, H706-H710. Scott A. P. and Sumpter J. P. (1983) A comparison of the female reproductive cycles of autumn-spawning and winter-spawning strains of rainbow trout (Salmo gctirdneri Richardson). Gen. Comp. Endocrinol. 52, 79-85. Sokal R. R. and Rohlf F. J. (1969) BiQmetry.W. H. Freeman and Company, San Francisco, USA. Stevens E. D. and Randall D. J. (1968) The effect of exercise on the distribution of blood to various organs in rainbow trout. Comp. Biochem. Physiol. 25, 615-625.
620
W. H. GINGERICHet al.
Swan H. and Nelson A. W. (1971) Blood volume I: Critique: Spun vs isotope hematocrit: ‘*%HSA vs 5’Cr-RBC. Surg. Annu. 173, 481-495. Thorson T. B. (1961) The partitioning of body water in Osteichthyes: Phylogenetic and ecological variations in aquatic vertebrates. Viol. BUN. (Woods Hole) 120, 238-254. Tsuyuki H. and Williscroft S. N. (1973) The pH activity relations of two LDH homotetramers from liver and their
physiological significance. J. Fish. Res. Board Can. 30, 996-1003. Woodward C. C. and Strange R. J. (1987) Physiological stress response in wild and hatchery-reared rainbow trout. Trans. Am. Fish. Sot. 116, 574-579. Wydoski R. S., Wedemeyer G. A. and Nelson N. C. (1976) Physiological response to hooking stress in hatchery and wild rainbow trout (Salmo gairdneri). Trans. Am. Fish. sot. 105, 601-606.