Manuel de la Higuera

Oxidative stress generated by dietary Zn-deficiency: studies in rainbow trout (Oncorhynchus mykiss).

To date, there is scarce information on the metabolic and biochemical repercussions of Zn-deficiency in fish. In this work, the effect of dietary Zn-deficiency on the diet utilization and the metabolism of activated oxygen species in rainbow trout (Oncorhynchus mykiss) has been studied. Fish were randomly separated in different lots according to their Zn-starvation and diet intake. In crude extracts of liver, gut and muscle, total and isoenzymatic superoxide dismutase and catalase activities were analysed. Lipid peroxidation was also determined in the same tissues. Western blotting was performed using antibodies against manganese- and copperzinc-containing superoxide dismutase. Lots fed on the Zn-deficient diet and with low intake showed significantly lower weight gain and feed-conversion efficiency indexes than control trouts. However, these parameters returned to control values when trouts were recovered by feeding them a control diet ad libitum. In control trouts, three independent copperzinc superoxide dismutase isozymes were detected in liver, whereas only one isozyme was present in the other lots. However, by Western blotting analysis the presence of a manganese superoxide dismutase was found in liver from all lots except in control trouts. Catalase activity and lipid peroxidation values were mainly detected in liver and gut, respectively, and both parameters increased in all lots with respect to the control group. Our results thus suggest that in rainbow trout an oxidative stress appears to occur as a consequence of Zn-deficiency.

Influence of nutritional composition of diet on sea bass, Dicentrarchus labrax L., reproductive performance and egg and larval quality

Abstract Two isocaloric formulated diets varying in protein and carbohydrate content (D1 = 51% (P)rotein, 13% (L)ipid, 10% (C)arbohydrate; D2 = 34% P, 14% L, 32% C) were fed to 2-year-old sea bass broodstock starting 6 months before spawning to determine their effects on reproductive performance and egg and larval survival. Fish were distributed in 8-m3 tanks, supplied with aerated running sea water (salinity 37.8‰, pH 8.3) and maintained under ambient photoperiod and temperature conditions. Before the experiment, fish were acclimated to the artificial diets by feeding D1 for 30 days. After this period, fish were fed D1 and D2 twice a day (1.5% of body weight per day). Fish fed D2, especially females, showed significantly diminished growth (weight, length and CF) and specific growth rates (P ≤ 0.05) over the first months. However, the dietary treatments did not have any effect on the histomorphology of ovarian development or gonad proximate composition. At spawning, D2 fish exhibited different reproductive performances compared to D1 fish. They did not have any peak in the production of eggs and showed a slightly extended spawning period. The mean number of eggs produced per female and the relative fecundity of fish given D2 diet were also appreciably lower than found for the D1 group. Although the egg proximate composition was the same regardless of dietary treatment, the eggs of the D2 fed fish had significantly lower buoyancy and hatchability (P ≤ 0.05) than those from D1 fish. In addition, more than 50% of hatched larvae from the D2 group showed body deformities. Taken together, these results indicate that the reproduction of sea bass and the survival of the progeny are greatly affected by the levels of energy-yielding nutrients in broodstock diets, and that a dietary protein level and a carbohydrate content of 32–33% have adverse consequences on sea bass reproduction. The sea bass dietary requirements to produce viable eggs seem also to be very specific since the hatching rates observed for both treatment groups were lower than those reported in other feeding trials in which natural diets were used.

Blood antioxidant defenses and hematological adjustments in crowded/uncrowded rainbow trout (Oncorhynchus mykiss) fed on diets with different levels of antioxidant vitamins and HUFA.

Rainbow trout maintained at crowding or noncrowding conditions were fed on five experimental diets that were formulated considering two levels of vitamin E (25.6 and 275.6 mg/kg diet), vitamin C (0 and 1000 mg/kg diet) and HUFA (12.5 and 30.5 g/kg diet): -E-HUFA, -E+HUFA, +E-HUFA, +E+HUFA, -C+E+HUFA. Hematological parameters, the activity of some antioxidant enzymes and lipid peroxidation from RBC were evaluated. The SOD isoenzyme pattern was analyzed by nondenaturing PAGE. Hematological response to crowding stress was manifested by increased hemoglobin and RBC count in most of the crowded groups. Antioxidant enzyme activity was clearly affected by dietary HUFA levels, with uncrowded fish fed on +HUFA diets showing a higher SOD activity compared to those fed on -HUFA diets. In uncrowded groups, only one CuZn-SOD isozyme was detected, whereas in the crowded fish a great variability was revealed with up to five isozymes. G6PDH activity was increased in uncrowded -E+HUFA fish compared to the remaining groups. Lipid peroxidation was significantly increased in -E+HUFA fish regardless of fish density. Data supported the negative correlation of lipid peroxidation and hematocrit or hemoglobin explained by decreased erythrocyte stability. Dietary imbalances in vitamin E and HUFA supplementation may promote oxidative stress which triggers hematological deterioration, which in turn would affect the whole hematological status and ultimately fish welfare.

Carbohydrates affect protein-turnover rates, growth, and nucleic acid content in the white muscle of rainbow trout (Oncorhynchus mykiss)

Abstract We have investigated the effect of dietary carbohydrate on different parameters of protein-turnover rate, nature of growth, and nucleic acid content in the muscle of rainbow trout in order to better understand the molecular nature of these growth parameters in the absence of this dietary component. For this, we used a methodology based on the incorporation rate of tritium labelled phenylalanine in muscle protein. Juvenile rainbow trout of an initial body weight of 110 g were fed near to satiety with a control or a non-carbohydrate diet during 7 weeks. The absence of dietary carbohydrate significantly depressed fish growth, as well as daily body weight gain, as a consequence of muscular hypotrophy (the cell size diminished by almost 50%) and not by a reduction of number of cells (hypoplasia). This nutritional situation also significantly slowed (by almost 11%) muscle-protein accumulation rate ( K G ) as a result of a significant increase (eight-fold) in muscle-protein degradation rate ( K D ), without changing the other protein-turnover rates, protein synthesis rate ( K S ), protein synthesis capacity ( C S ), protein synthesis efficiency ( K RNA ), protein synthesis rate per cell unit ( K DNA ), or protein retention efficiency (PRE). These results, together with the nucleic acid content, clearly indicate that the absence of carbohydrate significantly exacerbates the muscular-protein degradation without affecting protein synthesis. In conclusion, carbohydrates are needed to prevent amino acids released during protein degradation from being used to synthesize carbohydrates and/or to be used for energy and not for growth.

Impact of starvation-refeeding on kinetics and protein expression of trout liver NADPH-production systems

Herein we report on the kinetic and protein expression of glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase, and malic enzyme (ME) in the liver of the trout (Oncorhynchus mykiss) during a long-term starvation-refeeding cycle. Starvation significantly depressed the activity of these enzymes by almost 60%, without changing the Michaelis constant. The time response to this nutritional stimulus increased with fish weight. The sharp decline in G6PDH and ME activities was due to a specific protein-repression phenomenon, as demonstrated by molecular and immunohistochemical analyses. Also, the dimeric banding pattern of liver G6PDH shifted from the fully reduced and partially oxidized forms, predominant in control, to a fully oxidized form, more sensitive to proteolytic inactivation. Refeeding caused opposite effects in both protein concentration and enzyme activities of about twice the control values in the first stages, later reaching the normal enzyme activity levels. Additionally, the partially oxidized form of G6PDH increased. The kinetics of these enzymes were examined in relation to the various metabolic roles of NADPH. These results clearly indicate that trout liver undergoes protein repression-induction processes under these two contrasting nutritional conditions.

Growth, protein-turnover rates and nucleic-acid concentrations in the white muscle of rainbow trout during development.

We have studied the growth rate, nucleic-acid concentration, protein-accumulation rate (K(G)), and several other parameters relating to protein turnover, such as the protein-synthesis (K(S)), and protein-degradation rates (K(D)), protein-synthesis capacity (C(S)), protein-synthesis efficiency (K(RNA)), protein-synthesis rate per DNA unit (K(DNA)) and protein-retention efficiency (PRE), in the white muscle of rainbow trout during development. Both growth rate and relative food intake decreased significantly with age and weight, as did the food-efficiency ratio (FER) and protein-efficiency ratio (PER). Although absolute RNA and DNA contents increased with age, their relative concentrations decreased. The RNA/DNA ratio increased sharply from 14 to 28 weeks but afterwards decreased towards initial values. Hypertrophy increased rapidly to the 28-week stage but henceforth increased much more slowly. Hyperplasia, on the other hand, continued to increase linearly, resulting in a significant four- to fivefold predominance in this type of growth at the end of the 96-week experimental period. K(G) decreased significantly with age, as did K(S), and C(S), whereas at the 14-week stage, K(D) was significantly lower than at other ages. K(RNA) increased until 28 weeks. K(DNA) increased significantly in juvenile fish compared to both fingerlings and adults, where it showed similar lower values. PRE remained high at all ages.

Proteomics in the liver of gilthead sea bream (Sparus aurata) to elucidate the cellular response induced by the intake of maslinic acid

Maslinic acid (MA) is a pentacyclic triterpene used as a feed additive to stimulate growth, protein‐turnover rates, and hyperplasia in fish. To further our understanding of cellular mechanisms underlying the action of MA, we have used 2‐DE coupled with MS to identify proteins differentially expressed in the livers of juvenile gilthead sea bream (Sparus aurata) grown under fish‐farm conditions and fed with a 100 mg/kg MA‐enriched diet (MA100). After the comparison of the protein profiles from MA100 fed fish and from control, 49 protein spots were found to be altered in abundance (≥2‐fold). Analysis by MALDI‐TOF/TOF allowed the unambiguous identification of 29 spots, corresponding to 19 different proteins. These proteins were: phosphoglucomutase, phosphoglucose isomerase, S‐adenosyl methionine‐dependent methyltransferase class I, aldehyde dehydrogenase, catalase, 6‐phosphogluconate dehydrogenase, fumarylacetoacetate hydrolase, 4‐hydroxyphenylpyruvic dioxygenase, methylmalonate‐semialdehyde dehydrogenase, lysozyme, urate oxidase, elongation factor 2, 60 kDa heat‐shock protein, 58 kDa glucose‐regulated protein, cytokeratin E7, type‐II keratin, intermediate filament proteins, 17‐β‐hydroxysteroid dehydrogenase type 4, and kinase suppressor of Ras1. Western blot analysis of kinase suppressor of Ras1, glucose 6‐phosphate dehydrogenase, elongation factor 2, 60 kDa heat‐shock protein, and catalase supported the proteome evidence. Based on the changes found in the protein‐expression levels of these proteins, we proposed a cellular‐signalling pathway to explain the hepatic‐cell response to the intake of a diet containing MA.

Selective changes in the protein-turnover rates and nature of growth induced in trout liver by long-term starvation followed by re-feeding

We report upon the effects of a cycle of long-term starvation followed by re-feeding on the liver-protein turnover rates and nature of protein growth in the rainbow trout (Oncorhynchus mykiss). We determined the protein-turnover rate and its relationship with the nucleic-acid concentrations in the livers of juvenile trout starved for 70 days and then re-fed for 9 days. During starvation the total hepatic-protein and RNA contents decreased significantly and the absolute protein-synthesis rate (AS) also fell, whilst the fractional protein-synthesis rate (KS) remained unchanged and the fractional protein-degradation rate (KD) increased significantly. Total DNA content, an indicator of hyperplasia, and the protein:DNA ratio, an indicator of hypertrophy, both fell considerably. After re-feeding for 9 days the protein-accumulation rates (KG, AG) rose sharply, as did KS, AS, KD, protein-synthesis efficiency (KRNA) and the protein-synthesis rate/DNA unit (KDNA). The total hepatic protein and RNA contents increased but still remained below the control values. The protein:DNA and RNA:DNA ratios increased significantly compared to starved fish. These changes demonstrate the high response capacity of the protein-turnover rates in trout liver upon re-feeding after long-term starvation. Upon re-feeding hypertrophic growth increased considerably whilst hyperplasia remained at starvation levels.

Dietary protein effects on growth and fractional protein synthesis and degradation rates in liver and white muscle of rainbow trout (Oncorhynchus mykiss)

Abstract We have studied the effects of a decrease in dietary protein on the growth and proteinturnover parameters [fractional protein synthesis ( K S ), degradation ( K D ) and accumulation ( K G ) rates, synthesis capacity ( C S ), synthesis efficiency ( K RNA and K DNA ), and protein retention efficiency (PRE)]. The administration of a low-protein diet caused a significant decrease in the availability of protein precursors due to a decline in feed efficiency and thus a concomitant decrease in whole-body, white-muscle and liver growth capacity throughout the experimental period. The reduction in dietary protein caused a significant decrease in the K G value in the liver as a consequence of a decrease in protein retention efficiency (PRE). In white muscle, however, a considerable reduction in the K G value was due to a decrease in K S , C S , K DNA and K RNA rather than a decrease in PRE.

The influence of dietary protein on the kinetics of NADPH production systems in various tissues of rainbow trout (Oncorhynchus mykiss)

Abstract The effects of a decrease in dietary protein level on the kinetic behaviour of the four NADPH production systems [glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), malic enzyme (ME), and NADP-isocitrate dehydrogenase (NADP-IDH)] were investigated in four different trout tissues. Typical hyperbolic saturation curves were always obtained for the activity of these enzymes. A low-protein diet caused a significant decrease in the maximum velocity values ( V max ) of all the hepatic enzyme systems studied. The decrease in hexose monophosphate dehydrogenases (G6PDH and 6PGDH) was about 35 and 50%, respectively, whereas for ME and NADP-IDH it was about 35 and 25%. No significant changes were found in the Michaelis constant. These kinetic characteristics are compatible with an increase in the catalytic efficiency of these enzymes without there being any changes in their activity ratio values. The kinetic parameters for these enzymes in the kidney, spleen, and gill tissue did not undergo any significant change.