Seasonal changes in reproductive condition and plasma levels of sex steroids in the blue cod,Parapercis colias (Bloch and Schneider) (Mugiloididae)

Gonad and plasma samples were taken from blue cod captured throughout the reproductive cycle, gonad condition was assessed, and plasma levels of 17-hydroxyprogesterone (17OHP), 17,20-dihydroxy-4-pregnen-3-one (17,20P), testosterone (T), 17-estradiol (E₂) and estrone (E₁) were measured by radioimmunoassay. It was confirmed that spawning occurred over an extended period in late winter and spring, with individual fish being involved in multiple spawning events. Plasma levels of T were bimodal in both sexes with peaks (maximum of 6.0 ng.ml^–1) occurring 2 months prior to, and also during the early part of the spawning period. 17,20P was elevated in males (2.1 ng.ml^–1) in mid-spermatogenesis coinciding with the first T peak (4.9 ng.m.^–1). 17,20P was detectable but not significantly elevated (0.6–1.2 ng.ml^–1) at any sample time in females. E₂ was elevated in mature females (1.0 ng.ml^–1) early in the spawning period but remained at assay detection limits (0.3 ng.ml^–1) at all other sample times. Neither 17OHP nor E₁ were detectable in the plasma of either sex. It is suggested that bimodal increases in sex steroids prior to spawning may be a feature of species with rapid recrudescence.     

Biochemical composition of eggsin relation to egg quality in the Japanese eel, Anguilla japonica

This paper presents the relationship between egg quality and egg biochemical composition of cultured and wild Japanese eel, Anguilla japonica. Eggs were obtained by artificialinduction of maturation. Fertilization and hatching rates were used as characteristics of egg quality. Egg quality characteristics showed large variation; fertilization rate, 0–96; hatching rate, 0–84%. The biochemical composition also showed a large variation. There was no marked relationship between egg quality and fatty acid contents of eggs, except for n-6 highly unsaturated fatty acids (HUFA). Both the fertilization and hatching ratesincreased proportionally withincreases of the -tocopherol g(-Toc) contentin eggs. A more significant correlation was found between the amount of -Toc relative to the amount of HUFA and egg quality. The results of this study show that the egg quality of Japanese eel is affected by the –Toc level, andin particular, the ratio of -Toc to HUFAin the eggs. Abbreviations: BHT – butylhydroxytoluene; EFA – essential fatty acids; FAME – fatty acid methyl esters; HPLC – high performance liquid chromatography; HUFA – highly unsaturated fatty acids; NADH – nicotinamide adenine dinucleotide; NADPH - nicotinamide adenine dinucleotide phosphate; ROS – reactive oxygen species; -Toc –-tocopherol.     

Oxygen consumption and feeding rates of gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>) reveal lack of acclimation to cold

Low temperature has been implicated in inducing outbreaks of winter syndrome or winter disease in farmed gilthead sea bream (Sparus aurata). The responses of gilthead sea bream to reduced temperature followed by maintenance at low temperaturewere studied. In a first experiment, oxygen consumptionwas measured when water temperature was reduced from 18°C to 8 °C at either a rate of 1 °C· day-1 or as two sharp drops (from 18 °C to 12 °C, and from 12 °C to 8 °C). In a second experiment, the water temperature was reduced from 16 °C to 8 °C or 12 °C and then maintained for 20 days to study the fish acclimation to these temperatures. In both experiments, fish stopped feeding below 13 °C and did not resume feeding when maintained at low temperatures. The decrease in metabolic activity, expressed by the oxygen consumption rate, was directly related to the fall in water temperature: the Q10(18 °C-8 °C) values were between 2.2–2.5, independently of the descend rate in water temperature. However, we observed a more reduced metabolic rate when the water temperature was below 12 °C. Fish maintained at low temperatures showed only a partial recovery in oxygen consumption (15% at 8 °C and 20% at 12 °C) after 20 days. A higher metabolic rate together with a fasting-temperature condition meant that maintenance at 12 °C was more aggressive than at 8 °C, as revealed by the condition factor and energy needs. Data suggest that 12 .C could be a threshold temperature for the metabolic activity of gilthead sea bream. The relationship between low temperatures and their possible implication in the appearance of winter disease in gilthead sea bream is also discussed.     

Inhibition of HPI axis response to stress in gilthead sea bream (Sparus aurata) with physiological plasma levels of cortisol

This study investigates the effect of corticosteroid (cortisol) administration on the stress response of the gilthead sea bream Sparus aurata subjected to a 48 h confinement. The effect of (in-vitro and in-vivo ) cortisol administration on the in-vitro ACTH sensitivity of the interrenal tissue; the plasma levels and tissue concentration of cortisol; and the plasma levels of ACTH, -MSH, -endorphin and glucose were determined. Confinement caused a transient and concomitant increase in plasma cortisol and ACTH levels. However, in cortisol-fed fish the plasma ACTH levels were lower, indicating a suppresion of the ACTH release from the corticotropes by cortisol. In contrast to the activation of the corticotropes, the levels of plasma melanotrope derived peptides were not affected. In spite of the fact that interrenal cells of cortisol-fed gilthead sea bream released less cortisol than controls, the interrenal sensitivity to ACTH was not affected by in-vivo and in-vitro cortisol administration. This suggests that the interrenal sensitivity to ACTH in stressed (confinement) sea bream is probably not regulated by -MSH, N-ac--END, or by cortisol. Thus, in gilthead sea bream the interrenal sensitivity to ACTH could be regulated at the hypothalamus and/or pituitary and communicated via circulating ACTH levels.     

Fatty acid compositions of the major phosphoglycerides from fish neural tissues; (n−3) and (n−6) polyunsaturated fatty acids in rainbow trout (Salmo gairdneri) and cod (Gadus morhua) brains and retinas

The fatty acid compositions of brain phosphoglycerides from a freshwater fish, the rainbow trout (Salmo gairdneri), and a marine fish, the cod (Gadus morhua), were determined and compared with those from a terrestrial mammal, the rat. Fish brain lipids were characterized by a higher degree of unsaturation encompassing increased percentages of (n–3)PUFA (226 and 205) and lower percentages of (n–6)PUFA (204 and 224). However the distribution of fatty acids and specific PUFA between different phosphoglycerides was essentially similar in rat and fish brain tissue. PE and PS contained the highest percentages of 226(n–3), PI was characterized by higher 180 and 204(n–6)/205(n–3), and PC had higher 160 and the lowest percentage of PUFA in all species. A generally similar pattern was found in the fish retinal phosphoglycerides except that PC was also rich in 226(n–3). Overall trout brain phosphoglycerides were slightly more unsaturated than the cod lipids but with lower (n–3)/(n–6) ratios whereas cod retinal lipids were more unsaturated than the trout retinal lipids.     

The nature of hematological response in fish

Hematological status was examined in rainbow trout,Oncorhynchus mykiss, held for 3–4 weeks under temperature, photoperiod and P_{O 2} conditions approximating those of their winter, spring and summer habitats. The most striking change observed was in red cell population composition. In winter fish mature cells were predominant; juvenile and developing erythrocytes characterized spring and summer animals. Hemoglobin, hematocrit and both mean erythrocytic volume and hemoglobin were modestly lower in spring and summer than in winter fish. Red cell numbers were not significantly affected. These observations suggest that avoidance of viscosity-based increases in circulatory work cost is more advantageous than elevation of blood O₂-carrying capacity. Although hemoglobin isomorph profiles were significantly altered, there is little evidence that such changes are of critical adaptive importance. Given presumed age-based reduction in gas transport effectiveness, the replacement of mature and senescent cells by more metabolically-competent juvenile cells appears to be the pivotal event in hematological response. Leucocyte counts were significantly elevated in spring and summer as compared to winter fish. Lymphocyte/heterophil ratios declined from 8.27 in winter fish to 3.13 in summer trout. Thrombocyte, monocyte, eosinophil and basophil abundances were little changed.     

Utilization of yolk fatty acids by goldfish embryos and larvae

In each of two separate experiments, eggs from a single female goldfish were fertilized, incubated at 22°C and sampled regularly up to day 6 when the larvae could be expected to commence feeding. Hatching normally occurred on Day 4. Lipids were extracted from the eggs and larvae and the neutral lipid and neutral phospholipids were isolated on aminopropyl columns. Fatty acid analysis of the eggs revealed the typical situation in fish where the phospholipids were rich in polyunsaturated fatty acids (PUFA) and the neutral lipids were rich in monounsaturated fatty acids (MUFA). Assay of lipid masses revealed that little depletion of lipid occurred until after hatch and that the neutral phospholipids were the principal fraction consumed. Although the neutral lipid mass did not change substantially during development, its fatty acid profile did. The proportions of several PUFA in the neutral lipids, especially 226(n–3), 205(n–3) and 204(n–6), increased substantially during development while proportions of MUFA and 182(n–6) declined. This appears to be a mechanism by which the larva can retain essential fatty acid released on hydrolysis of phospholipid while deriving the benefits of catabolism of phospholipid as fuel, namely the provision of phosphate and choline for intermediary metabolism and for the synthesis of macromolecules and neurotransmitter.Abbreviations AA arachidonic acid (204(n–6)) - DHA docosahexaenoic acid (226(n–3)) - EPA eicosapentaenoic acid (205(n–3)) - MUFA monounsaturated fatty acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PUFA polyunsaturated fatty acid - SFA saturated fatty acid     

Optimal dietary carbohydrate to lipid ratio in African catfish Clarias gariepinus (Burchell 1822)

An 8-week feeding trial was conducted in a recycling water system at 28±1°C to investigate carbohydrate to lipid ratio (CHO:L ratio) in African catfish Clarias gariepinus (12.32±0.04g). Five isonitrogenous (40% crude protein) and isoenergetic (20kJg^–1 gross energy (GE)) fishmeal based diets with varying carbohydrate to lipid (CHO:L g/g) ratios of 0.74, 1.13, 1.66, 2.47 and 3.42 for diets 1–5, were tested, respectively. The diets containing a fixed protein to energy ratio (P:E ratio) of 20-mg proteinkJ^–1 GE were fed to triplicate groups of 20 fish (per 30-L tank). Fish were fed 5% of their body weight per day adjusted fortnightly. Diet 1, containing 14% carbohydrate and 21% lipids with a CHO:L ratio of 0.74 produced the poorest (P<0.05) growth rates, feed and protein efficiency. Increasing carbohydrate content in the diets to 27% concomitant with a reduction in lipid content to 16% with a CHO:L ration of 1.66 of diet 3 significantly improved (P<0.05) growth rates, feed and protein efficiency. A further increase in dietary carbohydrate up to 38% and a decrease in lipids levels to 11% with a CHO:L ratio ranging from 1.66 to 3.42 (diet 3 – 5) did not significantly improve the fish performance. Apparent net protein utilisation (ANPU) of fish fed diet 4 was higher (P<0.05) than for diets 1–3 but did not differ from diet 5. Higher lipid deposition (P<0.05) in whole body and liver were observed with decreasing dietary CHO:L ratios as increasing lipid levels. Whole body protein and liver glycogen content, digestive enzyme activities (protease and lipase) and histological examination of intestine and liver of fish fed varying CHO:L diets did not show any discernible changes among the dietary treatments. However intestinal -amylase activity increased (P<0.05) with increasing dietary carbohydrate levels. This study revealed that African catfish can perform equally well on diets containing carbohydrate ranging from 27 to 38% of the diet, with lipid content ranging from 16 to 11% or at CHO:Lg/g ratio of 1.7–3.4.     

Effects of Varying Dietary Fatty Acid Composition on Growth and Survival of Seahorse, Hippocampus Sp., Juveniles

Three commercially available fatty acid enrichment emulsions (DC Selco, DC DHA Selco and DC Super Selco) were used to enrich Artemia nauplii fed to seahorse, Hippocampus sp. fry. The emulsions varied in their n-3 highly unsaturated fatty acid (HUFA) composition. Total n-3 HUFA content ranged from 200 to 450mgg^-1 between the three emulsions while levels of eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) ranged between 47–220 and 80–190mgg^-1, respectively. Survival and growth of seahorses at the end of the 30 day growth trial were greater in treatments receiving enriched Artemia. Seahorses receiving Artemia enriched with DC DHA Selco and DC Super Selco showed significantly (p < 0.05) greater mean survival (71.6 ± 6.0% and 78.3 ± 6.0%, respectively) than those receiving unenriched Artemia (48.3 ± 6.0%). Mean standard length was also significantly greater (p < 0.05) in fry fed DC DHA Selco and DC Super Selco enriched Artemia (20.2 ± 0.3 and 19.7 ± 0.3mm, respectively) compared to those fed unenriched Artemia (18.1 ± 0.3mm). The results show that dietary n-3 HUFA are essential for optimal growth and survival of Hippocampus sp. and, based on the fatty acid compositions of the enriched Artemia used in this study, indicate that the level of dietary DHA supporting optimal growth and survival is greater than 9.3mgDHAg^-1 dry weight.     

Ionoregulatory and respiratory responses of brown trout,Salmo trutta,exposed to lethal and sublethal aluminium in acidic soft waters

Soft water acclimated (Ca²⁺ 0.02 mM; Na⁺ 0.03 mM; K⁺ 0.01 mM; pH 7.0), cannulated brown trout (Salmo trutta) were exposed to various pH and aluminium (Al) regimes (pH 7.0, pH 5.0, pH 5.0 plus Al: 50, 25, and 12.5 g l^–1) for up to 5 days in order to determine (i) the sublethal concentration of Al at pH 5.0 for this species (ii) their ionoregulatory and respiratory status. No mortality or physiological disturbances were evident at pH 7.0 or pH 5.0. All trout died within 48 h at pH 5.0 in the presence of Al at 50 g l^–1 and 67% died over the 5 day period at pH 5.0 in the presence of Al at 25 g l^–1. Fish at these lethal Al concentrations showed significant decreases in arterial blood oxygen content (C_aO₂) but no changes in plasma osmolarity or the concentrations of plasma Na⁺, K⁺ and Cl^–. Physiological disturbance was more marked at the 50 g l^–1 Al concentration. The surviving fish at 25 g l^–1 showed few signs of physiological recovery while continually exposed to this regime. No fish died during the exposure to water of pH 5.0 containing 12.5 g l^–1 Al, but physiological disturbance was still apparent. These sublethally-stressed trout showed a transient decline in the plasma concentrations of Na⁺ and Cl^–1. Although C_aO₂ decreased, recovery was evident. The data suggest that in the brown trout, environmental Al concentration is as important as pH and calcium concentration in determining the physiological status of the fish.     

Quantitation of inert feed ingestion in larval silver sea bream (Sparus sarba) using auto-fluorescence of alginate-based microparticulate diets

The ingestion of an inert feed as a sole food source was investigated in larval silver sea bream (Sparus sarba) fed an alginate-based microparticulate diet. Using the auto-fluorescent properties of pigments associated with the alginate base, ingestion and gut content were investigated over a 6 h experimental period in fed and unfed larvae. By extracting and measuring chlorophyll a (Chl a) and phaeopigment content of feeding larval fish and relating this to standardized Chl a and phaeopigment content of the diet, relative to diet weight, it was determined that individual fed 7-day old larvae had a maximum gut content of 1.05±0.09 g diet while 14-day old fed fish had a maximum gut content of 3.17±0.90 g diet. On average, the gut content of 14-day old fish was 2.89 times greater than the gut content of 7-day old fish. The dry weight of larval sea bream increased from 43±4.2 g at day 7 to 134.3±20.4 g at day 14 indicating that growth of fish fed this inert feed was substantial. Gut pigment dynamics suggested that Chl a was degraded to phaeopigments by 7-day but not 14-day old larvae and the individual gut dietary content varied considerably in 14-day old fish. The maximum Chl a and phaeopigment content in larval sea bream was 0.4 ng ind^–1 and 0.55 ng ind^–1 for 7-day old fish and 1.54 ng ind^–1 and 2.81 ng ind^–1 for 14-day old fish respectively. The present method may potentially allow simple and direct assessment of larval fish feed ingestion in both an experimental and commercial setting.     

Antioxidant effect of dietary tocopherol and ascorbic acid on growth and survival of Litopenaeus vannamei postlarvae

The nutritional effect of vitamin E in dietsfor Litopenaeus vannamei postlarve (PL19)was investigated. Four formulated diets withdifferent combinations of -tocopherylacetate (-TA), ascorbic acid (AA) andhighly unsaturated fatty acids (HUFA) weretested, using four replicates.No significant differences in survival wereobserved among treatments after 34 days offeeding. However, shrimp fed with a dietcontaining 2% fish oil (low n-3 HUFA content),200 mg.kg^–1 -TA and100 mg.kg^–1 AA (diet H/E/C) showedsignificantly better growth than those fed adiet supplemented with 5% fish oil (high n-3HUFA content), 200 mg.kg^–1 -TA and100 mg.kg^–1 AA (diet H+/E/C). Shrimp fedwith a diet containing 5% fish oil,900 mg.kg^–1 -TA and100 mg.kg^–1 AA (diet H+/E+/C) showed a significantly higher tissue level of n-6 PUFAthan postlarvae fed diet H+/E/C. No definiteconclusion could be drawn about a possibleinteraction between -TA and AA, since acomparison of the diet containing 5% fish oil,200 mg.kg^–1 -TA and700 mg.kg^–1 AA (H+/E+/C+) and the dietH+/E/C did not show any significant differencesin any of the measured parameters. Theantioxidative status of the shrimp tissue(measured by means of the thiobarbituric acid(TBA) assay and expressed as nM malonaldehyde(MA) per gramme dry weight) was equal for alltreatments. Nevertheless, there was a slightlylower MA value with the diet H+/E/C+,indicating that AA may be an effectiveantioxidant in the aqueous phase and at thewater/lipid interface of the tissue. The tissuelevels of -T and AA were highlydependent on the amounts in diets and nocorrelation between -T and AAincorporation could be observed.     

A drop in ambient temperature results in a transient reduction of interrenal ACTH responsiveness in the gilthead sea bream (Sparus aurata, L.)

This study investigated the effects of a drop in water temperature (18 °C to 9 °C in 24 h) on the pituitary and interrenal hormones of the gilthead sea bream Sparus aurata. The in vitro sensitivity of the interrenal tissue to ACTH, plasma levels of cortisol, ACTH, -MSH, GH, glucose, lactate and ions were determined. In vitro ACTH, stimulated the release of cortisol from isolated interrenal glands from control gilthead sea bream in a concentration dependent fashion. However, the interrenal cells were less sensitive to ACTH as soon as 24 h following the onset of the temperature drop. At this time, plasma cortisol and ACTH levels were raised, and plasma GH concentrations were decreased, whereas no significant changes were found in plasma -MSH. After 96 h plasma ACTH levels had recovered whereas plasma cortisol levels were still higher than controls after 8 days of the beginning of the experiment. Interrenal sensitivity had recovered after 8 days. The results may help to clarify the relationship between the stress response and the aetiology of the winter syndrome in sea bream.     

Brackishwater carp culture in potentially waterlogged areas using animal wastes as pond fertilizers

In order to assess the possibilities of utilizing drainage effluents (salinity range 5.0–12.5), fish culture experiments were carried out. Experiments on polyculture using cow dung (24 000 kg ha^–1 y^–1) as pond fertilizer were conducted at five different salinity levels (0.3–8.5). Studies have revealed that carp perform well in salinities up to 7.5 and reasonably high fish production has been obtained. Even though the ponds had a high trophic status, higher salinities ( > 7.5) appear to repress fish growth probably due to low dissolved oxygen (DO), high BOD and high NH₄-N. Experiments on monoculture of common carp (Cyprinus carpio) conducted at two different salinity levels (0.3–0.9 and 6.0–7.0) using four different organic fertilizers (cow dung at 24 000 kg and 20 000 kg, poultry at 1500 kg, duck at 6000 kg and sheep/goat at 1500 kg ha^–1 y^–1) have revealed the highest fish growth to be in poultry-treated ponds, followed in decreasing order by duck and sheep/goat wastes. Similar trends in fish production were observed both in fresh- and salt-water ponds. However, fish production was lower in ponds having higher salinities ( > 7.5). Nevertheless, these studies indicated that inland saline waters can be utilized for fish culture. With minor modifications in the existing technology of fish culture in stagnant freshwater fish ponds, animal wastes could be used to fertilize brackishwater fish ponds.     

Changes in the content of total lipid,lipid classes and their fatty acids of developing eggs and unfed larvae of the Senegal sole,Solea senegalensis Kaup

Total lipids, lipid classes and their associated fatty acids were quantified in developing eggs, yolk-sac larvae and starving larvae (from day 1 to day 5 after hatching) of the Senegal sole,Solea senegalensis Kaup. Larvae during early development and starvation consumed about 0.6% of its dry weight per day, mainly due to lipid catabolism. There was a net consumption of approximately 1.7% total lipid per day, and a net energy utilization of 1.3 kcal g^–1 dry weight biomass day^–1, mostly derived from lipid depletion. The overall decrease of total neutral lipids (mainly triacylglycerols and sterol esters) was 3.4 faster than that of total polar lipids (primarily phosphatidylcholine), with rates of 29.2 and 8.7 g mg^–1 dry weight biomass day^–1, respectively. There was a concomitant increase in PE, PS and phosphatidic acid during the period under study. Total saturated and total monounsaturated fatty acids were catabolized (primarily 160 and 161 (n-7)) as energy substrates at rates of 7.4 and 10.9 g mg^–1 total lipid day^–1, whereas total PUFAs were conserved. DHA was specifically retained in PE, whereas EPA and DHA were catabolized in PC and triacylglycerol. Total DMA and AA contents in total lipid increased during early development and starvation. The data denote a pattern of lipid metabolism during early development of Senegal sole similar to that of other marine larval fish, with eggs containing high amounts of total lipids (presence of oil globule/s), from temperate waters and with short developmental periods; the pattern contrasts with fish larvae from eggs of cold water fish species that contain low levels of total lipids (lack of oil globule/s) and have long developmental periods.Abbreviations AA all-cis-5,8,11,14-eicosatetraenoic acid (arachidonic acid, 204(n-6)) - C free cholesterol - DHA all-cis-4,7,10,13,16,19-docosahexaenoic acid (226(n-3)) - DMA dimethyl acetal - EPA all-cis-5,8,11,14,17-eicosapentaenoic acid (205(n-3)) - HUFA highly unsaturated fatty acids (C₂₀ with 3 double bonds) - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - PUFA polyunsaturated fatty acid(s) - SE sterol ester - TAG triacylglycerol     

Current forces on,and water flow through and around,floating fish farms

The wake generated by a screen or a net is analysed by the linear free-wake equations in conjunction with an eddy viscosity formulation. The behaviour of the near and far-field wake is investigated, and a relationship between the drag coefficient and the wake velocity is derived. A method for calculating the current forces experienced by the net structure and the resulting deformation is derived and compared with model tests. The effects of the wake behaviour and the deformation of the net cages, on the design and operation of floating fish farms are discussed and some recommendations are given.Nomenclature A Area of a net panel - dA Area of a portion of a net panel - A ^N Area of the net panel normal to the flow direction - A ^P Area of the net panel parallel to the flow direction - C_D,i Drag coefficient of thei ^th cylinder - C_D() Drag coefficient as function of the angle between the normal to the net and flow direction - C_L() Lift coefficient as function of the angle between the normal to the net and flow direction - D Diameter - D _i Diameter of thei ^th cylinder - DO Dissolved oxygen concentration - F _D Drag force - F _L Lift force - H Heaviside function - L Width of net panel - l _i Length of thei ^th cylinder - N Number of cylinders - N _i Number of cylinders in y-direction - N _j Number of cylinders in z-direction - N _N Number of cages in direction normal to the flow - N _C Number of cages in direction of the flow - r=u/U Velocity reduction factor - Rn=U*D/v Reynolds number - Sn Solidity ratio, defined as the ratio between the area covered by the threads and the total area of the net panel - t Time - U Current or free flow velocity - u ₁=U–u Velocity defect - u=U–u ₁ Wake velocity - x,y,z Cartesian coordinate system or field point for calculation of velocity - x _i ,y _i ,z _i Source point on screen element - Angle between the flow direction and the normal vector to the net, in the direction of the flow - Eddy viscosity - Mesh size - Kinematic viscosity - Density of water - Error function - Partial differential operator - Infinity     

Protein kinase C in the spleen of the turbot (Scophthalmus maximus L.)

PKC activity was detected in spleen extracts from the turbot, Scophthalmus maximus, a teleost flatfish that is farmed commercially in several countries, in assays with the substrate EGF- R651–658 as phosphate acceptor. The activity was purified about 700-fold by a three-step chromatographic procedure (DEAE-cellulose, phenyl-Sepharose and threonine-Sepharose). Maximal activity was obtained in the presence of the typical PKC cofactors Ca2+ (0.1 mM) PtdS (20 g ml–1) and either DAG (2 g ml–1) or PMA (2 g ml–1). Activity was dose-dependently inhibited by H7 and by the PKC-specific inhibitors PKC19–36 and N-myristoylated PKC19–31. The rate of phosphorylation was highest with the PKC-specific substrate MARCKS161–175. In immunoblotting, MC5 (a mouse monoclonal antibody raised against bovine PKC) recognized bands of 80 and 100 kDa. Immunoblotting with antibodies raised against mouse PKC isozymes (, , , , , , and ) indicated the presence of all these isozymes in turbot spleen.     

Stimulation of ureogenesis in the perfused liver of an Indian air-breathing catfish, Clarias batrachus, infused with different concentrations of ammonium chloride

The pattern of changes of activity of the urea cycle enzymes and the rate of urea-N excretion were studied in the perfused liver of an Indian air-breathing ureogenic walking catfish, Clarias batrachus. The liver was perfused with different concentrations of NH₄Cl for a period of 60 min to determine the role of ammonia for stimulation of hepatic ureogenesis and the threshold level of ammonia loading needed to cause such stimulation. Both the urea-N excretion and the ammonia uptake by the perfused liver were found to be a saturable process. Ammonia accumulated significantly in the liver infused with 1.25 moles g liver ^–1 min^–1 of NH₄Cl, followed by a maximum accumulation of about 28.5 moles g wet wt^–1 with the infusion of 5.08 moles g liver^–1 min^–1. The Vmax of the urea-N excretion (0.47 mol g liver^–1 min^–1) was obtained with the addition of 5.08 moles g liver^–1 min^–1 of NH₄Cl. Both the tissue and the specific activity of the urea cycle enzymes, except ornithine transcarbamylase and arginase, were stimulated significantly with the infusion of either 1.25 or 5.08 moles g liver^–1 min^–1 of NH₄Cl. Maximum stimulation of tissue activity of carbamoyl phosphate synthetase (about 120%) was seen with the infusion of 5.08 mol g liver^–1 min^–1, and for argininosuccinate synthetase (about 135%), and argininosuccinate lyase (about 50%) with the infusion of 10.81 mol g liver^–1 min^–1 of NH₄Cl. Higher accumulation of ammonia of about 10–15 mol g wet wt^–1 from the physiological level in the perfused liver while infusing with NH₄Cl was suggested to be one of the major causes of stimulation of ureogenesis. The presence of such physiological adaptive strategy is probably necessary in this unique group of air-breathing walking catfish to survive under hyper-ammonia stress in their normal habitat or while living outside water or while burrowing inside mud.     

Molecular cloning of the cDNA encoding the β subunit of thyrotropin and regulation of its gene expression by thyroid hormones in the goldfish, Carassius auratus

A cDNA encoding the subunit of thyrotropin (TSH) was isolated from a goldfish (Carassius auratus) pituitary gland cDNA library. By comparing the sequence with other teleost TSHs, a signal peptide of 19 amino acids and a mature hormone of 131 amino acids were predicted for goldfish TSH subunits. The resulting putative mature hormone of 131 amino acids had well-conserved cysteine positions and a putative N-linked glycosylation site; homology was 51–67% with TSHs from other teleosts, 38–43% with tetrapod TSHs, but only 27 and 29% with goldfish GTH-I and -II, respectively. We also examined the effects of thyroid hormones (TH) and thiourea (TU, an inhibitor of TH production) treatments on TSH and GTH subunit gene expressions in the goldfish pituitary gland. After thyroxine (T₄) treatment, circulating T₄ concentration increased and TSH mRNA level decreased. Supressing the amount of circulating T₄ and triiodothyronine (T₃) by TU treatment increased the TSH mRNA level. Moreover, T₄ replacement therapy (simultaneous treatment of both TU and T₄) caused a high level of circulating T₄ and a low level of circulating T₃, and a decrease in the TSH mRNA level. Thus, changing levels of circulating TH exert a negative feedback on the level of TSH subunit mRNA in goldfish in vivo. On the other hand, GTH subunit mRNA levels were not affected by changes in the levels of circulating TH.     

Effects of steroid hormones on in vitro oocyte maturation in white sturgeon (Acipenser transmontanus)

The in vitro effects of several steroids on the maturation of intact white sturgeon (Acipenser transmontanus) ovarian follicles were investigated. At the highest concentration (1024 ng ml^–1 for the C21 steroids and 1139 ng ml^–1 for the C19 steroids), all of the C21 steroids tested, progesterone (P4), 17-hydroxyprogesterone (17OHP), 17,20-dihydroxy-4-pregnen-3-one (17,20-P), 17,(20,21-trihydroxy-4-pregnen-3-one 20-S), 11-deoxycortisol (S) and cortisol (F), as well as testosterone (T) induced germinal vesicle breakdown (GVBD) at 14 and 22 h. At 6 h, only P4 and 17,20-P induced maturation at the highest concentration (1024 ng ml^–1). At 14 and 22 h, 11-deoxycortisol was the most potent steroid inducer of GVBD followed by P4, 17OHP, 17,20-P, and 20-S. The steroid 11-hydroxytestosterone (11OHT) was completely ineffective at all concentrations and exposure times. The C21 steroids induced oocyte maturation at concentrations ranging from 4 to 1024 ng ml^–1, whereas T induced GVBD at 225 to 1139 ng ml^–1. Calculation of the mean effective concentration that induced 50% GVBD (EC50) from the 22 h incubations revealed the following order of potencies: S > P4 > 17OHP > 17,20-P > 20-S >> F > T. These bioassay results, together with previous findings on the endogenous production of steroids by ovarian follicles from gonadotropin-primed females, indicate that more than one steroid has a biological role in the resumption of meiosis in sturgeon oocytes and provides empirical evidence for P4, 17OHP, S, 20-S, and 17,20-P as maturation-inducing steroids in white sturgeon.