Investigation of the Protective Effect of β-carotene in the Prevention of Lipid and Protein Oxidation in Carp Meat during Different Storage Times

ABSTRACT

This study was aimed to investigate the effects of dietary β-carotene supplement for 42 days on lipid and protein oxidation biomarkers in carp meat at different times of storage. Seventy-five fish were divided into three groups: group 1 as control group was fed with basic diet; groups 2 and 3 received 50 and 100 mg kg^?1 diet β-carotene, respectively. Based on the present study results, muscle malondialdehyde concentrations were significantly lower following β-carotene consumption at 0, 24, 72, and 96 h after harvest in group 3 compared to the control group (p < 0/05). Muscle protein carbonyl contents showed a significant decrease following β-carotene supplementation at 0, 48, and 96 h after harvest in group 3 compared to the control group. Ferric reducing antioxidant power values were significantly increased following β-carotene supplementation at 72 and 96 h after harvest in group 3 compared to the control group. The results indicated that β-carotene supplementation with a dose of 100 mg kg^?1 diet is largely effective to improve the oxidative status of carp meat by reducing lipid and protein oxidation.     

Surface Breaking Behaviour in a Population of Captive Rays Raja: The Expression of a Need to Forage?

Observations were made of the surface breaking behaviour of a population of captive rays Raja. The behaviour was found to have a temporal link with a scheduled feeding event, and to be most common in animals assumed to be hungry. It is suggested that the behaviour is appetitive and is a method of foraging appropriate to the captive situation. This revised version was published online in August 2006 with corrections to the Cover Date.     

PCR cloning and identification of the β-haemolysin gene of Aeromonas hydrophila from freshwater fishes in China

In this study, β-haemolysin gene (AHTPS30HEM) of Aeromonas hydrophila was cloned from diseased fish in mainland China. AHTPS30HEM gene (AB021152) resulted in a 1589 bp fragment which covers the open reading frame (ORF) in region 5–1486 coding for 493 amino acids. Multiple alignment of AHTPS30HEM with other β-haemolysin amino acid sequences showed 18 amino acid substitutions between AHTPS30HEM and A. hydrophila β-haemolysin. Although the ORF sizes between AHTPS30HEM and Aeromonas species are different, four cysteins and four potential N-glycosylation sites were conserved. To identify the β-haemolysin-producing virulent or pathogenic A. hydrophila, a specific PCR to amplify 208 bp target DNA of β-haemolysin gene was established. Twenty strains containing pathogenic A. hydrophila, Aeromonas sobria, Vibrio anguillarum, Cytophaga columnaris, Pseudomonas fluorescens, and Yersinia yuckeri were investigated by PCR. Based on the cloned β-haemolysin sequences, the specific PCR method for identification of the β-haemolysin gene of A. hydrophila was established, and surveyed on those samples. The results indicated that β-haemolysin-specific PCR might be useful in the detection of pathogenic A. hydrophila.     

The role of cAMP in regulating the β-adrenergic response of rainbow trout (Oncorhynchus mykiss) red blood cells

The -adrenergic response of teleost red blood cells (RBCs) enables the fish to maintain or even enhance the oxygen affinity of haemoglobin during various stress situations. The role of CAMP in the pronounced -adrenergic response of hypoxic rainbow trout RBCs was studied. Rainbow trout RBCs were incubated with three different -agonists (noradrenaline, adrenaline and isoproterenol, 10^–9 - 10^–4 M) at two oxygen tensions (P_{O 2}, 155 and 8 mmHg), and thereafter cAMP accumulation and cellular water content were measured.The cAMP concentration of non-stimulated trout RBCs was ca. 1200 nmol/kg dw. Of the three -agonists used, isoproterenol was the most effective in formation of cAMP, followed by noradrenaline and adrenaline. Oxygen tension affected the accumulation of cAMP in two ways. At physiological catecholamine levels (1–100 nM) there was either no difference between normoxic and hypoxic cells or a slight increase in the normoxic ones. At high catecholamine concentrations the accumulation of cAMP was greater in the hypoxic than in the normoxic cells. Oxygen tension also affected the magnitude of cell swelling but had no effect on the catecholamine concentrations causing half-maximal swelling (EC₅₀-values). The results indicate that, at physiological catecholamine levels, the -adrenergic response of rainbow trout RBCs is mainly regulated on the level of the Na⁺/H⁺ exchange.     

Complementary DNA cloning and functional analysis of lycopene β-cyclase in the brown alga Undaria pinnatifida

Fisheries Science - To date, there is no functional evidence of the enzymes catalyzing carotenogenesis in brown algae. In this study, we have investigated the activity of brown algal lycopene...     

The effects of hypoxia,in vivo,on red blood cell β-adrenoceptors in the rainbow trout,Oncorhynchus mykiss

Rainbow trout, Oncorhynchus mykiss, were exposed to 48h of environmental hypoxia (water partial pressure of oxygen = 8.0 kPa) at either 5 or 15°C. Blood was sampled during hypoxia via a dorsal aorta cannula to measure arterial blood partial pressure of oxygen and plasma catecholamine concentrations. After 48h, the number (B_max) and dissociation constant (K_d) of red blood cell surface -adrenoceptors were determined using a radioligand-displacement binding assay. At 5°C, plasma catecholamine levels were elevated at 24h whereas at 15°C, levels were elevated at 48h. At either temperature, following 48h of hypoxia, there was no change in B_max or K_d of red blood cell surface -adrenoceptors, compared to normoxic control fish. This study demonstrates that chronic exposure to moderate hypoxia does not affect the number or affinity of cell surface -adrenoceptors on trout red blood cells.     

The dynamics of in vitro 17 β-estradiol secretion by isolated ovarian follicles of the rainbow trout (Oncorhynchus mykiss)

This study investigated the effects of human chorionic gonadotropin (hCG), 17-hydroxyprogesterone (17P) and testosterone (T) on the in vitro production of 17-estradiol (E₂) by isolated ovarian follicles of the rainbow trout (Oncorhynchus mykiss). 17P at 100 ng ml^-1, and hCG at 100 IU ml^-1 stimulated E₂ production relative to controls, whereas lower doses were ineffective. T was the most effective in stimulating E₂ production, followed by 17P and hCG respectively. The timecourse of E₂ production was investigated for both static culture, and incubations with media replacement, with follicles being exposed to hormone treatment for 30 min, 1 or 3 h, or constantly. E₂ production was observed after 30 min, 3 and 3-6 h in response to T, 17P and hCG respectively. Under static culture, E₂ levels reached maximal levels in 6 h. Longer incubations resulted in further metabolism of E₂ to E₂-glucuronide, which resulted in the blurring of treatment effects after 18 h. Incubations with media replacement resulted in higher E₂ production than in static culture. The results indicate that a 6 h incubation period is sufficient to produce significant increases in E₂ production in response to hCG, 17P and T, and that incubations longer than 12 h result in losses E₂ from the incubation media. These findings have implications for the validity of using static cultures to examine the effects of hormone treatment on the activity of steroid converting enzymes in vitro.     

Ameliorating effect of β-carotene on antioxidant response and hematological parameters of mercuric chloride toxicity in Nile tilapia (Oreochromis niloticus)

The impact of different levels of dietary β-carotene to alleviate the effect of mercuric chloride toxicity in Nile tilapia was assessed. Semi-purified diets containing 0, 40, and 100 mg β-carotene kg^?1 dry diet were fed for 21 days, which were subjected to sublethal concentration of mercuric chloride (0.05 ppm). Hematological and biochemical parameters, lipid profile, and antioxidant response were examined. All hematological parameters of tilapia fish starting from second week of toxicity were significantly decreased. A significant increasing trend in liver enzymes (ALT and AST) were observed parallel to the time of toxicity and peroxide radicals (MDA) appearing significantly increased in toxicated group without carotene supplement, although carotene supplementation return all parameters within the control levels. Mercury accumulated significantly in fish liver and white muscles in toxicated group while it showed a significant reduction in dietary β-carotene-treated group. Overall, it can be used as immunostimulant and alleviate the suppression effect resulted from immune depressive stressful condition in farmed Nile tilapia.     

Effect of diet and β-naphthoflavone on hepatic and renal glutathione S-transferase isoenzymes in carp (Cyprinus carpio)

Glutathione S-transferase (GST) isoenzymes were isolated from liver and kidney of carp (Cyprinus carpio) by glutathione affinity chromatography and chromatofocusing. Ten hepatic and eight renal catalytically active isoenzymes were identified. GST subunits from purified isoenzymes were further separated by high-performance liquid chromatography (HPLC) and were used as standards for the experimental sample analysis. Experimental samples came from carp that were fed fish meal (standard diet) or soybean bas ed diets for one year, injected or not with -naphthoflavone (BNF; i.p. injection of 50 mg kg-1). HPLC did not allow us to identify precisely the GST isoenzyme pattern in experimental carp. However, GSTs could be pooled in three categories: homodimeric, heterodimeric and unidentified GST subunits. On this basis, the effect of diet and BNF on the GST isoenzyme pattern was investigated. The homodimer/heterodimer ratio was decreased in liver of carp fed a standard diet and in kidney of both dietary groups. BNF increased the total specific GST activity in liver and kidney. However, the GST isoenzyme pattern was not modified in carp fed the standard diet while tissue specific modifications occured in carp fed the soybean diet. BNF decreased the homodimer/heterodimer ratio in liver and increased it in kidney. Abbreviations: BNF - -naphthoflavone; CDNB - 1-chloro-2,4-dinitrobenzene; GSH - reduced glutathione; GST - glutathione S-transferase; HPLC - high-performance liquid chromatography; SDS-PAGE - SDS-polyacrylamide gel electrophoresis.     

Effect of long-term administration of dietary β-1,3-glucan on growth, physiological, and immune responses in Litopenaeus vannamei (Boone, 1931)

β-1,3-Glucan at different dietary doses was administered to enhance the growth, immunity, and survival against nitrite stress in Pacific white shrimp, Litopenaeus vannamei. Four different diets supplemented with 0, 250, 500, or 1,000 mg of β-1,3-glucan kg⁻¹ diets were fed to L. vannamei. Growth performance (weight gain and survival rate), physiological conditions (blood total protein, glucose, lactate, triacylglycerols, cholesterol levels) and immunological responses (superoxide dismutase, catalase, lysozyme, acid phosphatase, and alkaline phosphatase activities) of shrimp were recorded after 84-day feeding and 120 h after exposed to nitrite-N. After 84-day feeding, 250 mg kg⁻¹ β-1,3-glucan diet resulted in better weight gain (P < 0.05). Before the nitrite stress, blood lactate, triacylglycerols, and cholesterol level in shrimp fed with 250 mg kg⁻¹ β-1,3-glucan diet were significantly higher than those observed in shrimp fed with other diets (P < 0.05). Higher activities of catalase, lysozyme, and alkaline phosphatase were observed in shrimp fed with 500 or 1,000 mg kg⁻¹ β-1,3-glucan diet as compared to those obtained in shrimp fed with other diets (P < 0.05). After 120-h nitrite stress, blood protein, lactate, superoxide dismutase, catalase, and alkaline phosphatase activities in shrimp fed with 500 or 1,000 mg kg⁻¹ β-1,3-glucan were significantly higher than those observed in shrimp fed with other diets (P < 0.05). Glucose and triacylglycerol levels of shrimp fed with 500 or 1,000 mg kg⁻¹ β-1,3-glucan were significantly lower than those observed in other diets (P < 0.05). In shrimp fed with 500 and 1,000 mg kg⁻¹ β-1,3-glucan and 120-h after nitrite stress, the mortality was significantly lower than that observed in shrimp of control. Together, in this 84-day feeding trial, 250 mg kg⁻¹ β-1,3-glucan improved growth, whereas 500 mg kg⁻¹ β-1,3-glucan preferentially improved nitrite resistance, probably through accelerating energy metabolism and activating immune system.     

CYP1A1 immunolocalization in the olfactory organ of rainbow trout and its possible induction by β-naphthoflavone: analysis in adults and embryos around hatching

Cytochrome P4501A1 (CYP1A1) isoform, which is known as being of major toxicological significance, has been well-studied in the mammalian olfactory mucosa. Only few studies have dealt with this biotransformation system in the fish olfactory organ which is particularly vulnerable to waterborne xenobiotics since sensory neurons are in direct contact with the aquatic environment. The present immunocytochemical study describes the cellular and subcellular distributions of CYP1A1 in the olfactory organ of rainbow trout in both adults and embryos around hatching. The enzyme inducibility in response to a 4-day exposure to waterborne -naphthoflavone (0.1 mg l^–1), a model inducer of CYP1A1, was also examined. In untreated adult fish, CYP1A1 was almost exclusively expressed in the nonsensory epithelium which covers the edges and the tip of the lamellae. Both goblet and ciliated nonsensory cells appeared immunoreactive. In -naphthoflavone-treated fish, in addition to a strong labeling in the nonsensory epithelium, ciliated nonsensory cells in the olfactory epithelium appeared well-labeled. Four days before hatching, only a few cells were weakly stained in the placodal epithelium of some embryos. By 7 days post-hatching, the enzyme expression was increased in the olfactory pit and it was restricted to ciliated nonsensory cells. No evident CYP1A1 induction was detected in either embryos or alevins. Results suggest the presence of a two-line CYP1A1 biotransformation system in the adult fish olfactory organ: a basal level of enzyme expression insured by the nonsensory epithelium and an additional line in which the sensory epithelium is activated in response to CYP1A1 inducers. This system might take place during development in parallel with the onset of the nonsensory epithelium.     

Identification of haematological markers in shrimp health assessment from the immune profile of Fenneropenaeus indicus on β-1,3-glucan administration and white spot syndrome virus challenge

Haematological alterations in Fenneropenaeus indicus fed marine yeast glucan-incorporated diet and challenged with white spot syndrome virus were analysed. Adult F. indicus 16.45 ± 2.12 g (mean ± SD) were reared in 25 ‰ sea water and allowed to acclimate for a period of 7 days. Diet was prepared incorporating glucan (0.2 %) extracted from marine yeast, Debaryomyces hansenii S169. The shrimps were reared on experimental diet for 28 days and then challenged with white spot syndrome virus through oral administration. Random sampling was done (n = 6 shrimps) on 1st, 15th and 28th day of the experiment and on post-challenge day 2 (PCD 2) and PCD 7 for haematological analysis. The immune parameters viz. plasma protein, total haemocyte count, phenoloxidase activity, superoxide anion production, alkaline phosphatase and acid phosphatase activities were estimated in the haemolymph. Superoxide dismutase, catalase, glutathione peroxidase, hydroperoxide, conjugated dienes and malondialdehyde concentrations in the haemolymph of shrimps were also analysed. Analysis of variance showed that there are significant differences (p < 0.05) in the immune and antioxidant parameters in different treatment groups of F. indicus. The shrimps fed glucan-incorporated diet showed higher survival rate with comparatively lower accumulation of lipid peroxidation products. Correlation coefficients showed that all haematological variables except hydroperoxides and conjugated dienes exhibited positive correlation with the survival rate. The immune variables and antioxidant parameters exhibited a greater degree of correlation with each other. When multiple regression of survival rate on all immune parameters was considered, the amount of variability explained was 93 % (R Square = 0.932). When significant regression coefficients among the immune parameters were taken into account, it was found that total haemocyte count (p < 0.001), phenoloxidase activity (p < 0.05), alkaline phosphatase activity (p < 0.05) and plasma protein concentration (p < 0.05) together are explaining 84 % (R ² = 0.842) of the variability, indicating that these four parameters are mainly supporting the survival. The amount of variability explained by the antioxidant parameters was 94 % (R ² = 0.938). When significant regression coefficients among the antioxidant parameters were taken into account, it was found that superoxide dismutase activity (p < 0.01) and catalase activity (p < 0.05) together are explaining 87.4 % (R ² = 0.874) of the variability, indicating that these two antioxidants act as the major immune effectors supporting the survival in shrimps.     

Catalytic activity and immunochemical quantification of hepatic cytochrome P-450 in β-naphthoflavone and isosafrol treated rainbow trout (Oncorhynchus mykiss)

In addition to catalytical assays, immunochemical techniques have recently been employed to measure induction of the cytochrome P-450 (P450) monooxygenase system in fish with polyaromatic hydrocarbons (PAH). In the present study, polyclonal antibodies were raised against rainbow trout P450IA1. Levels of rainbow trout P450IA1 determined using protein blotting- and ELISA procedures were compared with levels of 7-ethoxyresorufin-O-deethylase (7-EROD) activity in liver microsomes from rainbow trout. These comparisons showed that values of P450A1 were positively correlated (r=0.99 and r=0.97) with 7-EROD activities. In addition, the effects of isosafrol (ISF) or -naphthoflavone (NF) treatments on P450 levels in rainbow trout liver were investigated using immunochemical and catalytical methods. ISF treatment induced 7-EROD activity as well as 7-methoxycoumarin-O-demethylase-, 7-ethoxycoumarin-O-deethylase-, 7-propoxy-coumarin-O-depropylase and 7-butoxycoumarin-O-debutylase activities, although to a lesser extent, compared with the NF treatment. In contrast, immunochemical quantification of rainbow trout P450IA1 protein revealed a slightly different pattern. ISF appeared to be a weak inducer of P450IA1 in rainbow trout compared with NF. In addition, the degree of inhibition of 7-alkoxycoumarin-O-dealkylase activities in ISF microsomes differed from that measured in control- and NF microsomes. The discrepancies between catalytic and immunochemical estimates of rainbow trout P450IA1 in ISF treated fish in addition to differencs between specific inhibitory pattern by specific polyclonal antibodies raised against rainbow trout P450IA1, indicate that important differences exists between the responses induced by NF- and ISF treatments in the rainbow trout liver.Part of this work was presented at the 6th International Conference on Biochemistry and Biophysics of Cytochrome P-450, Vienna, Austria, July 3–8, 1988.     

Partial or total replacement of fish meal byplant protein affects gonadal development and plasma17 β-estradiol levels in female Nile tilapia

This study was conducted to evaluate theeffect of plant proteinbased diets on gonadaldevelopment and plasma 17 -estradiol (E2) levelin female Nile tilapia Oreochromis niloticus.Fish with a mean body weight of 6.7 (0.1) g were fedfour different diets with the same digestible protein(DP) and digestible energy (DE) containing gradedlevels of a mixture of plant ingredients as partial ortotal replacement of fish meal protein for 20 weeks.The control diet (D0) was based on fish meal, twodiets containing 33% (D33) and 66% (D66) of plantprotein, and one diet containing only plant protein(D100). Fish were sampled at 12 and 20 weeks. Nosignificant differences were found in different stagesof oocyte development and plasma E2 levels betweentilapia fed diets D0 and D100 at 12 weeks. Eight weekslater tilapia fed diet D0 showed a higher (P < 0.05)level of E2 than the D100 group. This difference andthe reduced proportion of vitellogenic and matureoocytes demonstrated that diets containing only plantprotein are less efficient in terms of tilapia growthand consequently ovarian development.     

Histological changes in insulin-immunoreactive pancreatic β-cells,and suppression of insulin secretion and somatotrope activity in brook trout (Salvelinus fontinalis) maintained on reduced food intake or exposed to acidic environment

Immature brook trout (Salvelinus fontinalis) were randomly divided into a pH control, a pH and food control and an acid-stressed group. Fish in the first two groups were held at neutral pH and those in the last group were maintained at pH 4.2 for up to two months. The food supply to the pH and food control group was restricted to simulate the reduction in food intake demonstrated for acid-stressed trout. Plasma insulin levels were significantly decreased from 5–20 ng/ml to 1–2 ng/ml and plasma cortisol levels were significantly increased from 5–10 ng/ml to as high as 70 ng/ml in the acid-stressed brook trout. Concomitantly, a significant decrease of 21–39% in the proportion (volume density) of insulin immunoreactive -cells was observed within the principal pancreatic islets. Somatic growth was stunted and ultrastructural morphometry revealed the suppression of somatotrope secretory activity in the acid-stressed fish. Restriction of food supply induced a smaller but still significant decrease in circulating levels of insulin which was however not accompanied by a reduction in insulin immunoreactive -cells. The rise in plasma cortisol levels was not significant, and the plasma levels of glucose and protein were unaffected. Nevertheless, somatotrope secretory activity was suppressed and somatic growth was stunted. This study demonstrates for the first time the complexity of the endocrine response to acid stress and that some of the response to acid stress can be attributed to the lowering of food intake.