The purpose of this series of study was to develop a non-fish meal diet for yellowtail, and feeding trials were conducted to examine the utilization of test diets formulated with soy protein concentrate, defatted soybean meal, corn gluten meal, and meat meal as protein sources. Juvenile (13g on average) and young (130g on average) yellowtail were fed experimental non-fish meal diets with these ingredients for 52 days and 75 days, respectively, and growth and feed performance parameters were compared with the fish meal-based control diet.
The experimental non-fish meal diets were of poor palatability to the juvenile fish, and this may be related to the inferior feed performance and growth compared to the control. Young fish fed the experimental diets showed active feeding, and normal growth was sustained for the first 46 days of feeding, but thereafter stagnant growth, poor feed gain ratio, and high mortality were observed irrespective of the dietary treatments. Moreover, at the end of the experiments, both juvenile and young fish fed the non-fish meal diets revealed the green liver symptom and poor blood characteristics, indicative of the abnormal physiological status.
Thus, these results proved that non-fish meal diets used in this study were not efficient in maintaining normal growth and health of juvenile and young yellowtail for a long rearing period. The poor feed performances is thought to be linked with the appearance of green liver, but its mechanism remains unknown.

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The seasonal change in breaking strength and proximate composition of the muscle of cultured adult red seabream (two to three years old) was investigated through a year from March in 1995 to February in 1996. In male fish, the breaking strength significantly changed parallel to gonad-somatic index (GSI) for five months from March to July in the periods of maturating and spawning. The positive correlation was noticed between the breaking strength of the muscle and GSI. In female fish, the breaking strength also tended to vary along with GSI for five months from March to July, although not significantly. These results indicate that the seasonal change in firmness mainly depends upon the sexual maturation and spawning. The significant change in breaking strength was unobservable after July in male and female fish. As to proximate composition of the muscle, in both male and female fish, the crude protein content significantly changed through a year. In both male and female fish, the positive correlation was noticed between the crude protein content and the body weight. The water content also changed through a year in both male and female fish. No seasonal change in crude lipid content was observed in male and female fish. These results suggest that the seasonal change in meat quality of red seabream mainly depends on the change in quality and/or quantity of the muscle firmness caused by the sexual maturation and spawning.

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Ontogenic change patterns in otolith Sr:Ca ratios were examined in amphidromous, landlocked (Lake Biwa population) and hatchery-reared ayu, so as to determine the usefulness of otolith microchemistry for discrimination between amphidromous and stocked non-amphidromous individuals. Otolith Sr:Ca ratios of amphidromous ayu fluctuated strongly along the life history transect in accordance with the migration pattern from sea to freshwater, via brackish water in the river mouth. On the contrary, the Sr:Ca ratios of Lake Biwa and hatchery-reared ayu remained at consistently low levels throughout the otolith. The higher ratios in amphidromous ayu, in the otolith region from the center to 220-400μm equivalent to the first 90-110 days from hatching, corresponded to the initial sea-going period, suggesting that otolith Sr:Ca ratios are affected by ambient water salinity or the seawater-freshwater gradient in Sr concentration. No relationship between changes in the Sr:Ca ratio and water temperature was found. The findings clearly indicated that otolith Sr:Ca ratios reflected individual life histories, enabling amphidromous ayu to be distinguished from stocked non-amphidromous ayu.

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Ayu larvae, amphidromous salmonidei fish, were collected at discrete depths in the center of the main current and the waters adjacent to the banks in the Shimanto Estuary during November 1992-February 1993. Yolk-sac larvae smaller than 7.0mm BL were dispersed throughout the estuary. After absorption of yolk, larvae aggregated near the bottom of the estuary, and remained there until reaching 10mm BL. Larvae of 10mm BL and larger were observed to aggregate in the waters adjacent to the banks. Thus, larvae migrate from the near bottom waters in the central channel to the waters adjacent to the banks of the estuary. We suggest that an increase in the specific gravity of larvae after yolk absorption facilitates the aggregation near the bottom. The larval characteristic of sinking to the bottom of the estuary may enhance their retention in the estuary.

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Fourteen enzymes or protein were assayed in four natural populations of Clarias macrocephalus collected from different parts of Thailand; north (Chiangrai), central (Prachinburi) and south (Pattani and Yala). Starch-gel electrophoresis followed by histochemical staining were conducted and resulted in 19 loci resolved. Twelve loci (sAAT^*, GAPDH-1^*, 2^*, IDHP^*, LDH-1^*, 2^*, sMDH-1^*, ME-1, 2^*, GPI^*, MPI^* and PROT^*) were monomorphic while seven of them were polymorphic which included EST^*, FH^*, sMDH-2^*, ME-3^*, PGM^*, PGDH^* and sSOD^*. Observed heterozygosity were 0.080, 0.076, 0.038 and 0.050 respective to Chiangrai, Prachinburi, Pattani and Yala population. Genetic distance revealed two population groups, which were Chiangrai-Prachinburi and Pattani-Yala group and genetic distance was larger within the first group than those within the second group.

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Induction of the ovarian development was investigated in three groups of eels with respect to the importance of the pretreatment reproductive stage. Initially, the developmental stages of oocytes varied widely among these groups; silver eels had the most advanced oocytes which were in the early stage of vitellogenic growth, whereas oocytes from feminized eels were in the oil droplet or early vitellogenic stage and those from cultivated eels were in the oil droplet stage.
Eels received weekly intramuscular injections of salmon pituitary homogenate (SPH) at 20μg per g body weight (low-dose treatment). The ovarian follicles of silver eels developed the fastest and reached the final maturational stage after, on average, 10 weeks of treatment. The oocytes of feminized eels and cultivated eels developed more slowly, requiring approximetely 17 and 19 weeks of treatment, respectively. All silver eels were induced to the final maturational phase (100%), but lower ratios were seen for feminized and cultivated eels (64 and 29%, respectively).
Feminized eels were also subjected to a high-dose treatment of SPH (40μg per g body weight). High-dose treated feminized eels were induced to the final maturational phase in less time (11 weeks) and with greater efficiency (71 %) than low dose-treated feminized eels. It was shown that eels whose follicle diameter was over about 180μm could be stimulated to develop through the final maturational phase. This result indicates that the majority of feminized eels may be expected to reach the final maturational phase after SPH treatment. However, a 100% rate to reach the final maturational phase could not be attained, possibly due to immunoreaction to SPH, the stress of injections, or other yet unidentified factors.
We conclude that silver eels are the best candidates for artificial maturation. Even though feminized eels were not the most responsive group, their abundance makes them an important resource.Moreover, we recommend that eels should be induced to the final maturational phase during a relatively short period of SPH treatment (about two to three months) for artificial maturation to be successful.

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The genetic variation in populations of the diamond-shaped squid Thysanoteuthis rhombus in the Pacific was examined through mitochondrial DNA analysis. Part of the cytochrome oxidase subunit I gene was amplified with the polymerase chain reaction (PCR) and sequenced for 23 individuals, five from each of three samples collected off Kyoto/Fukui, Okinawa Island, and the Ogasawara Islands in the western Pacific, and also for eight individuals from a sample caught off Galapagos Islands in the eastern Pacific. Resultant sequence data showed that there were seven variable nucleotide positions in the 399 base-pair segment of the gene and that all 23 sequences were grouped into six haplotypes. No marked genetic difference was observed between populations on both sides of the Pacific. The PCR-mediated SSCP (single-strand conformation polymorphism) analysis applied to a larger number of individuals of more than 200 from adjacent waters around Japan (off Kyoto/Fukui and Okinawa Island) revealed no divergence in haplotype frequency between these two regional samples.

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Gamma-ray detection methods show diffusion of ²²²Rn out of otoliths of orange roughy Hoplostethus atlanticus and variable uptake of ²²⁶Ra into otoliths of H. atlanticus both of which invalidate the assumptions of closure to ²²²Rn diffusion and the constant uptake of ²²⁶Ra that are critical to the ²¹⁰Pb: ²²⁶Ra method of age estimation from otoliths of H. atlanticus.

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Effects of cations and anions on the maintenance of motility during 24-hour cool storage of spermatozoa from the common carp, Cyprinus carpio L., were examined. When spermatozoa were stored in artificial seminal plasma (ASP), which consisted of 55mM NaCl, 82.4mM KCl, 2.0mM CaCl₂, 0.8mM MgCl₂, and 20mM NaHCO₃ buffered with 20mM TAPS-NaOH at pH 8.0, the percentage motility index (percentage motility/initial percentage motility × 100) remained high for seven hours, then fell significantly by 24 hours. The motility of stored spermatozoa is strongly influenced by the pH of ASP. Samples of spermatozoa stored for 24 hours in ASP at pH 7.0-8.0 included significantly higher percentages of motile spermatozoa than those at pH 6.0, pH 8.5, and pH 9.0. Absence of K⁺, Na+⁺, Mg²⁺ or HCO^-₃ ions from ASP at pH 8.0 did not result in noticeable changes in motility within three or seven hours. Only the spermatozoa stored in ASP without Ca²⁺ ions showed remarkably decreased motility at seven and 24 hours. These results indicate that Ca²⁺ ions and maintenance of the pH between 7.0 and 8.0 are essential features of a suitable isotonic diluent for short-term storage of the carp spermatozoa.

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The relationship between the distribution of Japanese common squid beneath a boat and under water irradiance of fishing lights was investigated during nighttime jigging operations off the Oki Islands, Shimane Prefecture in November 1995. The downward spectral irradiance under fishing lights of a power of 300kW (metal-halide type; white) was continuously measured with an underwater radiometer. The spatial distributions of squid attracted by the fishing lights were simultaneously detected with color sector scanning sonar.
Sonar images of squid from a vertical-section across the boat were approximately triangular-shaped. The squid were distributed in the water layer between 30 and 70 meters deep, the densest part of the image occurring in the layer between 55 and 65 meters deep. Values of the downward spectral irradiance, when schools were distributed from 30 to 70 meters, ranged 1.8×10^-2 to 5.4×10^-4μW•cm^-2•nm^-1 at 510nm, and 5.8×10^-2 to 6.4×10^-4μmol•m^-2•s^-1 at PAR. Further, vertical profiles of the squid corresponded closely to the distribution of underwater irradiance intensity. We also determined that the upper limit of the squid distribution approximated the 2×10^-2μW•cm^-2•nm^-1 contour at 510nm.

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Antibody production against orally administered human γ globulin (HGG) was examined in carp, to elucidate the immunological significance of the transport of proteins into blood circulation, observed in agastric fish species. Weekly administration of HGG via oral route into fish induced serum antibody against the protein, in a dose dependent manner. The antibody titer, however, decreased sharply from 4 weeks to 6 weeks after initial administration of the antigen, even with continued treatment. The antibody titer elevated again by intramuscular injection of the antigen, indicating the immune suppression is not owing to oral tolerance.

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Patterns of food and microhabitat utilization by two dominant species, the filefish, Rudarius ercodes, and the surfperch, Ditrema temmincki, in a Zostera bed were investigated from May through December in 1993 and 1994. In May, the filefish took mostly caprellid amphipods, detritus and polychaetes, while the surfperch consumed mainly cyclopoid copepods, detritus and gammaridean amphipods. In and after July, however, the contribution of detritus to the diets of both species increased greatly. Thus, the dietary overlap was lowest in May, but subsequently increased until November. Throughout the study period, both species were uniformly distributed horizontally from the margin to the center of the seagrass bed, but not vertically relative to the substrate and seagrass canopy. In June 1993 and 1994, vertical (i. e., microhabitat) distributions of both species were similar. After June, surfperch occurred more frequently in the upper parts of the water column, while filefish were abundant in the lower parts. In contrast to the dietary overlap, the microhabitat overlap was at a maximum in June, subsequently decreasing to a minimum in December. In 1994, when the surfperch density was lower than that in 1993, the filefish significantly increased in number and tended to occur more often in the upper parts of the water column, where it had been infrequently seen the previous year. These results suggested that resource partitioning resulting from interspecific competition plays a major role in the coexistence of R. ercodes and D. temmincki in the seagrass bed.

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A methanol extract was prepared from the ordinary muscles of nine Amazonian fish specimens caught in May, the end of the rainy season and 11 fish specimens in August, the middle of the dry season of the same year at Manáus where about 1, 200km from the estuary of the Amazon River. Free amino acids and other components of the extract were analyzed. The muscle extracts from Amazonian fish specimens caught in the rainy and dry seasons were generally rich in taurine, creatine, 5'-inosinic acid or inosine, and several inorganic ions. The muscle extract of Pellona castelnaena was rich in taurine and several acidic and neutral amino acids. Relatively higher amounts of trimethylamine oxide were detected in the muscle extracts of Astronotus crassipinnis, Cichla sp., Pellona castelnaena, and Prochilodus nigricans caught in the rainy season and A. crassipinnis, Psectrogester amazonica, and Pellona castelnaena caught in the dry season. A large peak that was presumed to be γ-aminobutyric acid appeared in the chromatogram of amino acid analysis for Metynnis hypsauchen. Amounts of dry matter of the muscle extracts from fish specimens caught in the rainy season were slightly higher than those of the muscle extracts from fish specimens in the dry season.

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The autoxidation rate of oxymyoglobin (MbO₂) from sardine, saury, carp and sheep holoMb was measured as a function of temperature after heating and cooling. The observed first-order rate constant (k_obs) is found to be temperature dependent. The k_obs values for MbO₂ are not so different among these fish species at the same given temperature, but found to be higher, by 2.5_??_4.2 times as a whole, than those of sheep Mb. In connection with a structural perturbation, the autoxidation rate of a reconstituted MbO₂ was measured directly while heating. In the same way, the assigned k'_obs value at normal conditions for bonito reconstituted MbO₂ is also found to be higher than that of the sperm whale one, but their kobs values are not different under heat denaturation. The autoxidation reaction has been thermally mediated through structural unfolding/refolding. These results suggest that according to an origin of the structure looseness associated with the heme pocket perturbation, thus fish myoglobins show cooperatively higher susceptibility to the autoxidation of MbO₂ than mammalian myoglobins.

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Marine microalgae, Pavlova lutheri, Tetraselmis tetrathele, and Chaetoceros gracilis, were cultured for some time during the stationary growth phases, respectively, although they are generally harvested as live feed around the early stationary growth phases. Their fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were periodically analyzed during the culture periods, and thus evaluated the effect of the prolonged culture period on the accumulation of fatty acids in each microalga. By culturing P. lutheri longer than the ordinary harvest time, a slight increase was observed in the combined contents of EPA and DHA after reaching the stationary growth phase, while the prolongation of the culture period was not effective for T. tetrathele. On the other hand, C. gracilis accumulated the fatty acids rapidly and largely during the stationary growth phase and thus increased the combined contents of EPA and DHA, although their relative percentages decreased with the culture time. At later stationary growth phase of C. gracilis, the total fatty acid content and the combined contents of EPA and DHA were about eight and four times larger than those at the late exponential growth phase, respectively, and were the largest among all the samples examined. Therefore, prolonging the culture period after reaching the stationary growth phase will be advantageous at least for the harvest of C. gracilis enriched in fatty acids including EPA and DHA.

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The content of dietary fibers, laminaran and alginate, in 22 dried products of brown algae and their effects on cecal microflora and levels of plasma lipid in rats were surveyed. After washing with methanol, 0.1 _N hydrochloride fractions (F1: crude laminaran and fucoidan), 1% sodium carbonate fractions (F2: crude alginate) and insoluble fractions (F3) were extracted from the algae using the ethanol precipitation method. F1, F2 and F3 ranged from about 1 to 22%, 10 to 67% and 3 to 59%, respectively, of dry matter. Laminaran was detected in only three products by bioassay using Clostridium ramosum. Laminaran content was the largest in arame Eicenia bicyclis. Fucoidan was not detected in arame by TLC. Diets containing 5% arame powder (AP) (whose content is too high for normal intake), 0.75% Fl, 2.2% F2 and 1.5% F3 of arame, which are respectively dose equivalents to 5% of AP, were administered to male rats for seven days. AP and F2 increased fecal weight. AP, F1 and F2 increased cecal weight. F1 lowered cecal pH. Plasma triglyceride was suppressed by AP, F1 and F3. Although Fl, F2 and F3 increased cecal bifidobacteria, AP did not change bifidobacteria and decreased lactobacilli.

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This study was carried out to survey the effects of laminaran and depolymerised sodium alginate (AG5), which are water soluble polysaccharides of brown algae and are not highly viscous, on the intestinal microflora and blood lipids in vitro and/or in vivo. Laminaran was fermented by human (two adults and an infant) fecal and rat cecal microflora in vitro. AG5 was fermented by these microflora besides infant fecal microflora. Laminaran was a more strongly fermented and supressed ammonia production than AG5. In vivo, the diets containing 0.4, 2 and 8% of laminaran or 0.4, 2 and 10% of AG5 were administered to male rats for one week. Eight and 10% of these polysaccharides lead to diarrhea and a decreased body weight gain. Cecal weights increased depending on the dose of the polysaccharides. Liver weights decreased depending on the dose of AG5. Cecal pH values were the lowest at 2% polysaccharides. Though 2% of laminaran increased cecal propionate and n-butyrate, 2% of AG5 decreased cecal short-chain fatty acids. AG5 decreased plasma triglycerides and cholesterol. High density lipoprotein cholesterol of plasma increased in the case of the diets containing 0.4% AG5. The ratio of cecal bifidobacteria to total bacteria was the highest, 0.4% at both polysaccharides. These results indicate that laminaran and AG5 have a profitable and different effect on intestinal microflora and levels of blood lipid depending on their concentrations.

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The relationship between the denaturation of fish myofibrils by the addition of chitin, derived from crustaceans and cephalopods, and the state of water by dehydration was studied. The changes in the state of water in myofibrils associated with dehydration were analyzed on the basis of the sorption isotherm curve. The involvement of these changes in the denaturation of myofibrils was investigated with respect to ATPase activity as a parameter.
The addition of chitin resulted in a decrease in water activity and suppressive effects of chitin on the denaturation of myofibrils in the intermediate moisture content of about 15% or more. However, in both cases chitin had no effect below such moisture content. These results suggest that the suppressive effects of chitin occur by the stabilization of the hydrated water that is incorporated between chitin and water in high moisture content surrounding the myofibrils, and such effects are likely to be associated with the physicochemical properties of chitin.

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Vitelline envelope-Related proteins (VERPs) were immunologically detected in serum and liver of female Sakhalin taimen Hucho perryi, but not in males, using an antiserum against vitelline envelope. Two VERPs were observed in serum as female-specific proteins with molecular weights of 48 kDa and 42 kDa on SDS-PAGE under non-reduced conditions. VERPs were induced in serum and the liver of immature fish by injection of 10 μg estradiol-17β (E₂) per kg of body weight. These results suggest that the origin of VERPs is the liver. During vitellogenesis, one VERP band first appeared in July in the serum of five-year-old fish. Two VERP bands were observed during winter and became undetectable in June after ovulation. The changes in VERPs were parallel to those in vitellogenin.

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The distribution of D-amino acids was examined on the tissues of seven species of marine bivalve mollusks belonging to two different subclasses, Pterimorphia and Heterodonta. High concentration of D-alanine was found in all tissues of only Heterodonta and not in the tissues of Pterimorphia except in gills and midgut gland. Several other D-amino acids including D-arginine also occurred in the several tissues of both groups of bivalves.
Along with the salinity stress on the hard clam Meretrix lusoria in 150% seawater, only D- and L-alanine increased in adductor muscle, gills, and midgut gland. The incorporation of D-alanine from external seawater was found only in gills, hemolymph, and midgut gland of hard clam, and the levels of D- and L-alanine in adductor and foot muscles decreased in the seawater containing 50mM D-alanine and returned to the control level after recovery in normal seawater.
During the starvation of hard clam for 22 days, D- and L-alanine as well as other free amino acids decreased considerably in most tissues. The anoxia of hard clam for a week duration gave no large effect on the levels of free amino acids but resulted in the slight increases of D- and L-alanine levels after seven days.

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Mummichog Fundulus heteroclitus Linnaeus were reared during the spawning season with a vitamin K free diet for 11 weeks. Most of the male fish died during the experiment, while female fish seldom died. The menaquinone-4 (MK-4) content in the tissues markedly decreased during the experimental feeding. In contrast, none of the male or female fish (control fish) which were fed a diet supplemented with menadione sodium bisulfite (MSB) died. After 11 weeks, MK-4 was found to have accumulated in various tissues of the control fish, especially in the gonadal tissue, except for the liver tissue. In the kidney tissue of the female fish fed a vitamin K free diet, a large number of immature erythrocytes were observed. This suggests that the female fish had been affected by hematoporia and/or hematopoiesis brought about by a deficiency in vitamin K. However, larval mortality was not affected by the vitamin K deficiency. These results suggest that vitamin K intake is necessary for mummichog, particularly for the male fish during the spawning season even though they can continue to grow without any vitamin K supplement.

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Steady-state levels of lipid peroxide (LPO) in lateral muscles and liver of normal live fish, their elevation induced by xenobiotics and the distribution in lipid class were studied using yellowtail, red sea bream, carp, sweet fish, etc. LPO levels expressed in terms of meq/kg lipid, so to speak peroxide value (PV), vary in inverse proportion to the lipid contents; the higher the lipid content the lower the PV. Thus, PVs in live fish fluctuate with lipid contents. On the other hand, LPO levels expressed in terms of nmol/g tissue showed a steady-state reflecting physiological activity of tissue; 100-200nmol/g tissue in liver and dark muscle, and lower than 50nmol/g tissue in ordinary muscle. The levels was elevated by abdominal injection of CCl₄ and a bacterial culture. In lipids of wild yellowtail tissues, nearly the same total amounts of LPO were distributed in neutral lipid and phospholipid fractions.

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The activities of glutamate oxaloacetate transaminase (GOT) were measured spectrophotometrically from five tissues (liver, heart, kidney, brain, and ovary) of a non air-breathing, herbivorous fresh water fish Labeo rohita. The activities were measured separately for cytosolic (c-) GOT and mitochondria) (m-) GOT. For all tissues examined, the maximum activities were observed in the spawning phase, followed by the pre- and post-spawning phases for both c- and m-GOTs. The native agarose gel electrophoretic pattern revealed two GOT forms of different charges throughout the pre- to post-spawning phases. However, the spawning phase was marked by the expression of an additional anionic form of GOT in all tissues studied. These results indicate a distinct utilization of aspartate particularly in the spawning phase in fish via transamination reactions.

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An endopeptidase which hydrolyzed myofibril strongly from the pyloric caeca of Pacific mackerel Scomber japonicus was purified to homogeneity electrophoretically. The molecular weight and the optimum pH of the endopeptidase was about 30k and around 9.5, respectively. Since the endopeptidase was not inhibited by serine protease inhibitors, e. g. PMSF and soybean trypsin inhibitor, but completely inhibited by E-64, a high specific cysteine protease inhibitor, it was considered to be a cysteine endopeptidase. This is the first report on the existence of the cysteine endopeptidase in the pyloric caeca. The endopeptidase showed a hydrolytic specificity for MCA-synthetic peptide substrates which had a basic amino acid at P1 position and a hydrophobic amino acid at P2 or P3 position. It hydrolyzed various proteins examined except for collagen.

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Various kinds of ethanol-producing marine yeasts from coastal water were isolated and characterized. Number of colony forming units of the marine yeasts on a medium were from several dozen to hundreds per 1 l of various coastal water. Ethanol productivity from glucose by 15 isolates of the marine yeasts widely ranged 9_??_77%. Above all, in five strains: TAB, 15, and 46 and TK5 and 12, the productivity was 58_??_77% which had the similar levels of those in industrial yeasts. Salt tolerance of the marine yeasts was measured and they grew and fermented in the presence of 2.0_??_3.0 M NaCl. Otherwise, the industrial yeasts were only in 1.0_??_2.0 M NaCl. In taxonomy of the marine yeasts, most of them were found to be genera Candida and Debaryomyces.

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The hemolymph of the acorn barnacle Balanus rostratus showed hemagglutinating activity and inhibition of calcium carbonate crystallization. A lectin having D-galactose-binding specificity was isolated from the hemolymph by a combination of affinity chromatography on acid-treated agarose gel and HPLC. The purified lectin was dependent on the presence of calcium ion for hemagglutinating activity. In SDS-PAGE, it gave one protein band (25kDa) under a reduced condition, while it gave two components (35 and 95kDa) without a reducing agent. The molecular mass of the intact lectin was estimated to be 120kDa by HPLC on TSK G-3000SW. Isolectric point was determined to be pH 4.4. The same amino-terminal sequence, Tyr-Val-Ser-Asn-Gln-Ser-Val-Glu-Pro-Asp-Ser-Ala-Asp-Thr-Ala, was obtained with the purified lectin as well as the components observed in SDS-PAGE. The purified lectin did not inhibit calcium carbonate crystallization at 1mg/30ml, although the hemolymph inhibited the crystallization at 0.1mg protein/30ml. The inhibitory factor(s) was an acidic and small molecular-weight compound(s).

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In order to quantify serum cortisol levels in the killer whale Orcinus orca and the bottlenose dolphin Tursiops truncatus, we have established a specific radioimmunoassay (RIA) system. Two kinds of antisera against cortisol (i. e., cortisol-3-(O-carboxymethyl)-oxime-BSA (FKA404) and cortisol-21-succinil-BSA (FKA402)) were used for RIA. High performance liquid chromatography/RIA analysis of serum extract has revealed that FKA404 reacts more specifically with cortisol in the sera without remarkable crossreaction with other corticoids.
The cortisol RIA system using FKA404 as an antiserum was validated. Intra and inter assay coefficient of variation were 1.9% and 3.2%, respectively. The minimum detectable quantity of cortisol was 0.052ng/ml. The competition curves for serum samples of the two cetacean species were parallel to the standard curve. These results indicate that this system is suitable for the measurement of cortisol in the killer whale and the bottlenose dolphin.
Serum cortisol levels (mean±SE) measured by the RIA with FKA404 was 3.8±1.8ng/ml in bottlenose dolphins. In killer whales, serum cortisol levels in the morning were significantly higher (2.5±0.8ng/ml) than those in the evening (1.5±0.5ng/ml). This result in killer whales coincided with studies in diurnal terrestrial mammals including humans.

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