Josefina Blasco

Fasting and refeeding in carp, Cyprinus carpio L. : the mobilization of reserves and plasma metabolite and hormone variations

SummaryCarp, Cyprinus carpio, were subjected to a short term of fasting (2 months) and 12 days of refeeding. The early changes produced in plasma metabolites and hormones (insulin and glucagon) and their respective energy contribution in liver and muscle during fasting and refeeding was studied. Two phases of fasting were differentiated. The first phase (until day 8 of fasting) was characterized by a reduction in the hepatosomatic index mainly due to glycogen mobilization. A transitory increase in plasma glucose and lactate suggested an initial increase in energy demand. No changes were produced in the percentage of glycogen and protein in muscle, but musculosomatic index and the total body muscle protein decreased. Although the most depleted tissue in this phase was the liver, the loss of energy content of total muscle was higher. Stabilization of liver glycogen content, plasma glucose and lactate levels, decreased muscle protein levels and a reduction in the rate of body weight loss characterized the second phase (from day 8 of fasting). Protein content in whole muscle decreased by 22%, similar to the first phase. The energy expenditure of both liver and muscle was lower in this phase. Plasma insulin levels decreased two-fold and plasma glucagon three-fold in the first phase and remained low in the second phase of fasting. Twelve days of refeeding produced a greater increase in daily growth rate than in the control group and a recovery of plasma insulin, glucagon and glucose levels. Liver completely recovered. In contrast, musculosomatic index, protein and lipid content indicated that muscle did not completely recover from the 2 months of fasting, although and overshoot of muscle glycogen was observed.

Plasma glucose kinetics and tissue uptake in brown trout in vivo: effect of an intravascular glucose load

The object of the present study was to elucidate whether a glucose load modifies glucose uptake by tissues in brown trout in vivo. By the use of 2-[1,2-3H]-deoxyglucose, plasma glucose disappearance rate and tissue glucose uptake were measured after an intraaortic glucose load of 500 mg·kg-1 (glucose load group) and under normoglycemic conditions (control). We also attempted to determine whether fasting modifies the glucose load disposal (fasted glucose load group). The procedure used to calculate 2-deoxyglucose uptake by tissues was evaluated, and the levels of 2-deoxyglucose uptake were compared with those of 2-deoxyglucose phosphorylation. Uptake and phosphorylation rates were similar in all tissues, except in brain and heart. In all the groups glucose uptake rates were highest in spleen, kidney, brain and gills, and lowest in red muscle, heart and white muscle. However, white muscle was the main site of glucose uptake on a whole tissue basis. The glucose load led to strong, long-lasting hyperglycemia, in spite of the increases observed in plasma insulin levels and in glucose uptake rate by the whole body (control: 4.9 μmol·min-1·kg-1; glucose load group: 6.5 μmol·min-1·kg-1). This higher rate was due to the higher glucose uptake only in white and red muscles (four- and threefold, respectively). Fasting halved the uptake of glucose by both red and white muscles in the load condition. In consequence the use of exogenous glucose decreased with fasting (fasted glucose load group: 5.1 μmol·min-1·kg-1), causing still longer hyperglycemia.

Plasma glucagon levels in different species of fish

Plasma glucagon levels in 11 different teleosts and in dogfish were evaluated using an adapted classical mammalian radioimmunoassay (RIA). The values obtained are considered relative; sensitivity was inferior to the original method, but the validation experiment results are acceptable. The mean value of the coefficient of the variation for interassay was 6% and for intraassay 4%, recovery was 98%, and a good linearity in the dilution test was found. Some species (Sparus aurata and cyprinids) presented high plasma glucagon concentrations (0.50-1.60 ng/ml) versus the lower levels (0.08-0.40 ng/ml) found in salmonids and others, and the lowest in dogfish (0.02-0.20 ng/ml). A positive correlation (P less than 0.02) between insulin and glucagon plasma levels was found in different species. The low levels obtained in all parameters analyzed in the selachian were remarkable. This adapted RIA could serve as a useful tool in amplifying knowledge on the endocrine pancreatic response in fish in different biological situations.

Fate of plasma glucose in tissues of brown trout in vivo: effects of fasting and glucose loading

We report the fate of glucose, both as a source of energy and as a temporary store, in the tissues of brown trout (Salmo trutta) in control, fasted and glucose-loaded fish. Tissue glucose utilization (3H-2-deoxyglucose phosphorylation) and storage (conversion of 14C-glucose into glycogen, protein, and lipid) were measured in immature brown trout, and the oxidation rate was calculated as glucose utilization minus storage and 14C-ionic metabolites remaining in the tissue. The glucose utilization rate is tissue-specific, the highest values being found in spleen, kidney, hindgut, brain, and gill. All these tissues also showed a highly active glycolytic pathway. The lowest utilization indices were observed in white and red muscles, skin, stomach and caeca, which also presented the largest proportion of glucose converted into stores (mainly protein and glycogen). Fasting reduced the glucose disappearance rate by 24%, although there were no significant variations in glucose utilization indices or distribution profile. After a glucose load, plasma glucose and insulin levels rose and the rates of glucose utilization, storage, and oxidation also increased in all tissues (from 1.5- to 4-fold). The relative importance of each tissue in glucose disposal was similar to that in normoglycaemia. In liver, only glucose storage was measured reliably; the conversion of glucose to glycogen was higher than in other tissues, and rose markedly (35-fold) in glucose-loaded fish. In most tissues glucose flux into lipids, glycogen and protein increased. The distribution of glucose may not be a merely substrate-mediated process because fasting in glucose-loaded fish caused lower tissue glucose utilization, particularly in gut, red muscle and gills. Conversion of glucose to tissue stores was reduced, lipids being the most affected.

Low-temperature challenges to gilthead sea bream culture: review of cold-induced alterations and ‘Winter Syndrome’

Although gilthead sea bream have been cultured successfully for the last two decades they are particularly sensitive to low temperature. Especially in the northern Mediterranean area, cold affects fish health and decreases fish-farm production, and may even cause mortality through what is known as ‘Winter Disease’ or ‘Winter Syndrome’. This paper reviews the diagnosis and physiological effects of this disease, focusing on recent studies of cold-induced alterations in gilthead sea bream physiology. ‘Winter Syndrome’ is characterised by multi-organ dysfunction entailing hyposensitivity, erratic swimming, pale and friable livers, necrotic muscles, atrophy of the exocrine pancreas, and distended digestive tract. Its complex aetiology involves several factors such as thermal stress, metabolic depression, immune suppression, and occasional opportunistic pathogens. Low temperatures may be the initial cause of all these factors, except pathogen action. Indoor studies have demonstrated that a drop in temperature causes cold-induced fasting, thermal stress and metabolic depression. These immediate effects are related to an ionic imbalance caused by malfunctions of the gills and digestive system. They are also related to a fatty liver, which appeared steatotic and affected hepatic metabolism and blood composition. The result is a lower immune capacity and fish that are more susceptible to infection. There is no significant thermal compensation under cold conditions and in this situation any additional stress factors can cause fish to suffer metabolic collapse. This study reviews the physiological and zootechnical origins of the disease and, where possible, recommends ways of improving culture conditions during pre-cold, cold and recovery periods.

Tracing metabolic routes of dietary carbohydrate and protein in rainbow trout ( Oncorhynchus mykiss ) using stable isotopes ([ 13 C]starch and [ 15 N]protein): effects of gelatinisation of starches and sustained swimming

Here we examined the use of stable isotopes, [¹³C]starch and [¹⁵N]protein, as dietary tracers to study carbohydrate assimilation and distribution and protein utilisation, respectively, by rainbow trout (Oncorhynchus mykiss). The capacity of glucose uptake and use by tissues was studied, first, by varying the digestibility of carbohydrate-rich diets (30 % carbohydrate), using raw starch and gelatinised starch (GS) and, second, by observing the effects of two regimens of activity (voluntary swimming, control; sustained swimming at 1·3 body lengths/s, exercise) on the GS diet. Isotopic ratio enrichment (¹³C and ¹⁵N) of the various tissue components (protein, lipid and glycogen) was measured in the liver, muscles, viscera and the rest of the fish at 11 and 24 h after a forced meal. A level of 30 % of digestible carbohydrates in the food exceeded the capacity of rainbow trout to use this nutrient, causing long-lasting hyperglycaemia that raises glucose uptake by tissues, and the synthesis of glycogen and lipid in liver. Total 13C recovered 24 h post-feeding in the GS group was lower than at 11 h, indicating a proportional increase in glucose oxidation, although the deposition of lipids in white muscle (WM) increased. Prolonged hyperglycaemia was prevented by exercise, since sustained swimming enhances the use of dietary carbohydrates, mainly through conversion to lipids in liver and oxidation in muscles, especially in red muscle (RM). Higher recoveries of total 15N for exercised fish at 24 h, mainly into the protein fraction of both RM and WM, provide evidence that sustained swimming improves protein deposition, resulting in an enhancement of the protein-sparing effect.

Oxygen consumption and feeding rates of gilthead sea bream (Sparus aurata) 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.

Variations in tissue reserves, plasma metabolites and pancreatic hormones during fasting in immature carp (Cyprinus carpio)

Abstract 1. 1. Immature carp were subjected to 2-month fasting periods. Mobilization of reserves in liver and muscle, and the energy contribution of each reserve were studied. Changes in plasma glucose, amino acids, insulin and glucagon levels were determined throughout the experiment. 2. 2. No changes were observed in plasma glucose, insulin or glucagon at 19 days of fasting, but plasma amino acids increased. At 50 days of fasting, both plasma glucagon and amino acids increased, liver glycogen decreased and muscle proteolysis began. 3. 3. Between 50 and 67 days of fasting, plasma glucose and insulin decreased significantly, while glucagon and amino acids continued to increase. Strong muscular proteolysis was observed while liver glycogen stabilized. 4. 4. The contribution of each reserve in liver and muscle to energy production throughout fasting is considered.

Effects of fasting and feeding on plasma amino acid levels in brown trout

Plasma amino acid (AA) levels were analysed at various intervals during fasting and after feeding in juvenile brown trout, Salmo trutta. After 3 days of food deprivation, total plasma AAs decreased significantly (–26% vs. control) due to a fall in the most abundant essential AAs (EAA): branched-chain amino acid (BCAA) and Thr, and there was also a decrease in non-essential AA (NEAA) levels, especially Ala and Asn. After 15 days of fasting, BCAA increased, possibly related to muscle protein mobilization. After 50 days of food deprivation, a significant increase in Gln levels (40% vs. 15 days of fasting) and a decrease in BCAA (–25% vs. 15 days of fasting) was noted. Feeding a commercial trout pellet caused a clear and progressive increase in plasma AA levels. Both EAA and NEAA concentrations peaked at 11 h post-feeding (4068 ± 160 and 4261 ± 239 µM NEAA, respectively) with a greater increase in EAA (+62%) than NEAA (40%) with respect to control values. These data show that changes in the plasma AA pool clearly reflect the nutritional state of fish and help us to understand the complex AA metabolism.

New Insights into Fish Swimming: A Proteomic and Isotopic Approach in Gilthead Sea Bream

Moderate exercise enhances fish growth, although underlying physiological mechanisms are not fully known. Here we performed a proteomic and metabolic study in white (WM) and red (RM) muscle of gilthead sea bream juveniles swimming at 1.5 body lengths per second. Continuous swimming for four weeks enhanced fish growth without increasing food intake. Exercise affected muscle energy stores by decreasing lipid and glycogen contents in WM and RM, respectively. Protein synthesis capacity (RNA/protein), energy use (estimated by lipid-δ(13)C and glycogen-δ(13)C), and enzymatic aerobic capacity increased in WM, while protein turnover (expressed by δ(15)N-fractionation) did not change. RM showed no changes in any of these parameters. 2D-PAGE analysis showed that almost 15% of sarcoplasmic protein spots from WM and RM differed in response to exercise, most being over-expressed in WM and under-expressed in RM. Protein identification by MALDI-TOF/TOF-MS and LC-MS/MS revealed exercise-induced enhancement of several pathways in WM (carbohydrate catabolism, protein synthesis, muscle contraction, and detoxification) and under-expression of others in RM (energy production, muscle contraction, and homeostatic processes). The mechanism underpinning the phenotypic response to exercise sheds light on the adaptive processes of fish muscles, being the sustained-moderate swimming induced in gilthead sea bream achieved mainly by WM, thus reducing the work load of RM and improving swimming performance and food conversion efficiency.