A primary phosphorus-deficient skeletal phenotype in juvenile Atlantic salmon Salmo salar: the uncoupling of bone formation and mineralization.

To understand the effect of low dietary phosphorus (P) intake on the vertebral column of Atlantic salmon Salmo salar, a primary P deficiency was induced in post-smolts. The dietary P provision was reduced by 50% for a period of 10 weeks under controlled conditions. The animal's skeleton was subsequently analysed by radiology, histological examination, histochemical detection of minerals in bones and scales and chemical mineral analysis. This is the first account of how a primary P deficiency affects the skeleton in S. salar at the cellular and at the micro-anatomical level. Animals that received the P-deficient diet displayed known signs of P deficiency including reduced growth and soft, pliable opercula. Bone and scale mineral content decreased by c. 50%. On radiographs, vertebral bodies appear small, undersized and with enlarged intervertebral spaces. Contrary to the X-ray-based diagnosis, the histological examination revealed that vertebral bodies had a regular size and regular internal bone structures; intervertebral spaces were not enlarged. Bone matrix formation was continuous and uninterrupted, albeit without traces of mineralization. Likewise, scale growth continues with regular annuli formation, but new scale matrix remains without minerals. The 10 week long experiment generated a homogeneous osteomalacia of vertebral bodies without apparent induction of skeletal malformations. The experiment shows that bone formation and bone mineralization are, to a large degree, independent processes in the fish examined. Therefore, a deficit in mineralization must not be the only cause of the alterations of the vertebral bone structure observed in farmed S. salar. It is discussed how the observed uncoupling of bone formation and mineralization helps to better diagnose, understand and prevent P deficiency-related malformations in farmed S. salar.

Investigating the underlying mechanisms of temperature-related skin diseases in Atlantic salmon, Salmo salar L., as measured by quantitative histology, skin transcriptomics and composition.

Skin integrity is recognized as of vital consideration for both animal welfare and final product quality of farmed fish. This study examines the effects of three different rearing temperatures (4, 10 and 16 °C) on the skin of healthy Atlantic salmon post-smolts. Changes in skin condition were assessed by the means of skin composition analyses, quantitative histology assessments and transcriptome analysis. Level of protein, vitamin C and vitamin E was significantly higher at 16 °C compared with 4 °C. Quantitative histology measurements showed that the epidermal thickness decreased from low to high temperature, whereas the epidermal area comprising mucous cells increased. The difference was only significant between 4 and 16 °C. Both high and low temperature exhibited significant changes in the skin transcriptome. A number of immune-related transcripts responded at both temperatures. Contrary to well-described immunosuppressive effects of low water temperature on systemic immunity, a subtle increase in skin-mediated immunity was observed, suggesting a pre-activation of the mucosal system at 4 °C. Upregulation of a number of heat-shock proteins correlating with a decrease in epidermal thickness suggested a stress response in the skin at high temperature. The results demonstrate distinctive temperature-related effects on the skin of Atlantic salmon.

Proteomic analysis of epidermal mucus from sea lice-infected Atlantic salmon, Salmo salar L.

Health diets that contain immunostimulants and other functional ingredients can strengthen the immune response in Atlantic salmon, Salmo salar, and thereby reduce sea lice, Lepeophtheirus salmonis, infection levels. Such diets can be used to supplement other treatments and will potentially reduce the need for delousing and medication. A sea lice infection trial was conducted on fish with an average weight of 215 g. One control diet and four experimental diets containing functional ingredients were produced. The diets were fed to salmon for 4 weeks before infection with sea lice copepodids. When lice had developed to chalimus III/IV, 88 fish per diet were examined for lice loads. Mucus samples from fish fed the different diets were taken before and after lice infection. Mass spectrometry-based proteomics was used to characterize the protein composition in the epidermal mucus of Atlantic salmon and to identify quantitative alterations in protein expression. Multivariate analysis of the generated data sets was performed to identify protein biomarkers. Putative biomarkers associated with functional feed intake and with sea lice infection have been identified and can form the basis for strategic validation experiments with selected functional feeds.

Time-related changes of the intestinal morphology of Atlantic salmon, Salmo salar L., at two different soybean meal inclusion levels.

Abstract Soybean meal (SBM) induces enteritis in the distal intestine of Atlantic salmon. The present study assesses the effects of SBM concentrations on the kinetics of the enteritis process. Fish of 300 g, kept at 12 degrees C, were fed diets with different SBM inclusions: 0%, 10% and 20% SBM for 57 days. Samples of the distal intestine of five fish per treatment were taken for histological and electron microscopical analysis. A semi-quantitative scoring system was used to assess the degree of the morphological changes induced by SBM feeding in the distal intestinal epithelium. The first signs of enteritis appeared earlier in the salmon fed the 20SBM diet than in those fed the 10SBM diet. Thereafter, the condition increased steadily, displaying no signs of recovery. Furthermore, at the lower concentration, the process marking the onset of enteritis began more gradually than at the higher concentration and it displayed a tendency to stabilize after 13-20 days of continuous feeding. Electron microscopy indicated that the endocytosis process was hampered at day 3 of 20SBM and at 7 days of 10SBM. Furthermore, a strong reduction of microvilli was already evident after 7 days of 20SBM feeding, thus indicating a decreased uptake capacity of the distal enterocytes. In addition, transformation and migration of eosinophilic granulocytes was observed, which, in combination with the lysozyme C immunoreactivity supports their protective role during the inflammatory process in the distal gut of Atlantic salmon. It can be concluded that the severity of enteritis and its kinetics are concentration-dependent, showing no signs of recovery during feeding with diets containing SBM.

Effect of dietary fish oil substitution with linseed oil on the performance, tissue fatty acid profile, metabolism, and oxidative stability of Atlantic salmon.

The objective of this experiment was to test the effect of total or partial substitution of dietary fish oil (FO) by linseed oil (LO) in Atlantic salmon feeding on performance, liver and muscle fatty acid composition, selected lipogenic and lipolytic enzyme activities, and flesh oxidative stability. For 12 wk, fish (220 +/- 12 g of initial BW) were fed five experimental diets in which the FO was serially replaced by 25, 50, 75, and 100% LO. Total FO replacement by LO did not (P = 0.20) affect fish final weight, biometric indices, or i.m. fat contents. Liver and muscle neutral lipid (NL) composition responded to dietary treatments in different ways. Whereas the sum of n-3 PUFA in muscle followed a linear and quadratic pattern with increasing levels of LO, a linear (P = 0.005) effect was observed in the liver NL fraction. Total n-3 and n-6 PUFA contents in the polar lipid fraction (PL) were unaffected (P = 0.356) by dietary input of LO in muscle. Activity of liver glucose-6-P-dehydrogenase (G6PD) was greater with increasing levels of LO (P = 0.004). A time effect (P < 0.001) was observed in the concentration of lipid peroxidation products, expressed as thiobarbituric acid reactive substances, in fish flesh stored under refrigeration for 9 d; however, the progressive inclusion of LO in the feed did not affect (P = 0.125) flesh oxidation stability. In summary, LO can totally replace FO in Atlantic salmon feed without affecting growth performance and muscle susceptibility to lipid oxidation. Fatty acid metabolism in the liver was affected by LO, promoting G6PD activity and eicosatetraenoic acid accumulation; however, a 100% LO replacement decreased (P < 0.001) concentrations of eicosapentaenoic and docosahexaenoic acids in salmon muscle.

Influence of temperature and high dietary linoleic acid content on esterification, elongation, and desaturation of PUFA in Atlantic salmon hepatocytes.

The esterification, desaturation, and elongation of [1-14C]18:3n-3, [1-14C]18:2n-6, and [1-14C]20:5n-3 at 5 and at 12 degrees C were studied using cultivated hepatocytes from Atlantic salmon. The salmon were fed diets, in which 0, 50, or 100% of the supplementary fish oil had been replaced by soybean oil, for 950 day-degrees at 5 and 12 degrees C. The endogenous percentage of 18:2n-6 in hepatocyte lipids was 2% in cells from fish fed a diet with 100% of the supplemental lipid from fish oil, and it was slightly less than 25% in cells from fish fed the diet with 100% of the supplemental lipid from soybean oil. Furthermore, the percentages of 20:3n-6 and 20:4n-6 were significantly higher in hepatocytes from fish fed on soybean oil than they were in those of fish fed on fish oil. The percentages of 20:5n-3 and 22:6n-3, on the other hand, were lower. The endogenous levels of n-6 FA were not significantly correlated with the total amounts of radiolabeled FA esterified in hepatocyte lipids. The main radiolabeled products formed from 18:2n-6 were 20:2n-6 and 20:3n-6. The level of the important eicosanoid precursor 20:4n-6 was twice as high in hepatocyte phospholipids from fish fed the 100% soybean oil diet as it was in hepatocytes from fish fed the diet with 100% of supplemental lipid from fish oil. The main products formed from 18:3n-3 were 20:4n-3, 20:5n-3, and 22:6n-3. High levels of dietary 18:2n-6 do allow, or even seem to increase, the production of 22:6n-3 from 18:3n-3 in hepatocytes. The main products formed from 20:5n-3 were 22:5n-3 and 22:6n-3. The production of 22:6n-3 from 20:5n-3 was higher at 5 degrees C than at 12 degrees C. The percentage of 24:5n-3 was higher at 5 degrees C than it was at 12 degrees C, as was the ratio of 24:5 to 22:5. These results suggest that the elongation rate of 22:5n-3 to 24:5n-3 is higher at the lower temperature.

Location of superoxide production sites in turbot neutrophils and gilthead seabream acidophilic granulocytes during phagocytosis of glucan particles.

Here we show, by spectrophotometry and enzyme cytochemical methods, that turbot neutrophils and gilthead seabream acidophils responded in a similar way when incubated with PMA or with particulate glucans. Cells stimulated with PMA released high amounts of superoxide both intra- and extracellularly. However, O2- was mainly released intracellularly when cells were incubated with particulate glucans. Small glucan particles were quickly phagocytosed and O2- was initially produced in intracellular vesicles and tubular structures that later fused with the phagosome or with the cell membrane. Large glucan filaments that were not phagocytosed also induced cell stimulation and O2- was also produced in intracellular vesicles which then appeared to fuse with the cell membrane. We conclude that, in stimulated turbot neutrophils and gilthead seabream acidophils, superoxide production is carried out initially in intracellular compartments that are very similar to those described in mammalian neutrophils.

Meningioangiomatosis in the brain stem of a dog.

Meningioangiomatosis (MA) is a malformation of the vasculature of the central nervous system, characterized by the proliferation of meningothelial cells around blood vessels. MA has been reported, albeit rarely, in main, cattle and dogs. This paper records a case of MA affecting the brain stem in a 9-year-old male dog, and provides histochemical and immunohistochemical evidence for a perivascular fibroblast-like cell origin of the proliferating cells.