Åshild Krogdahl

Effects of dietary soyabean meal, inulin and oxytetracycline on intestinal microbiota and epithelial cell stress, apoptosis and proliferation in the teleost Atlantic salmon (Salmo salar L.).

Soyabean meal (SBM)-induced enteritis in the distal intestine of the teleost Atlantic salmon (Salmo salar L.) and other salmonids may be considered a model for diet-related mucosal disorders in other animals and man. The role of the intestinal microbiota in its pathogenesis was explored. Compared to diets containing fishmeal (FM) as the sole protein source, responses to extracted SBM or the prebiotic inulin, with or without oxytetracycline (OTC) inclusion, were studied following a 3-week feeding trial. Intestinal microbiota, organosomatic indices and histology, as well as immunohistochemical detection of proliferating cell nuclear antigen (PCNA), heat shock protein 70 (HSP70) and caspase-3-positive cells in the distal intestine, were studied. Distal intestine somatic indices (DISI) were higher in inulin and lower in SBM compared to FM-fed fish. The low DISI caused by SBM corresponded with histological changes, neither of which was affected by OTC, despite a significant decrease in adherent bacteria count. Image analysis of PCNA-stained sections showed a significant increase in the proliferative compartment length in SBM-fed fish, accompanied by apparent increases in reactivity to HSP70 and caspase-3 along the mucosal folds, indicating induction of cellular repair and apoptosis, respectively. Fish fed the SBM diet had higher total number as well as a more diverse population composition of adherent bacteria in the distal intestine. Thus SBM-induced enteritis is accompanied by induction of distal intestinal epithelial cell protective responses and changes in microbiota. Putative involvement of bacteria in the inflammatory response merits further investigation.

Soybean proteinase inhibitors affect intestinal trypsin activities and amino acid digestibilities in rainbow trout (Oncorhynchus mykiss)

Abstract Rainbow trout ( Oncorhynchus mykiss ), 265 g kept in fresh water, 1 m 2 surface area tanks, 10 kg per tank were fed diets based on high quality fish meal added 0.00, 0.37, 0.74, 1.11 and 1.48% soybean proteinase inhibitors, and chromic oxide as internal marker. After 10 days, 10 fish were dissected to obtain total contents of intestinal segments, and faeces were stripped from the remainder. The inhibitors decreased protein digestibility from 93 to 70%. Cysteine accumulated increasingly in the small intestine with increasing dietary inhibitor level. A linear relationship between intestinal proteolytic activities and protein digestibility was seen. Lipid digestibility was not significantly affected.

Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) digest and metabolize nutrients differently. Effects of water salinity and dietary starch level

Abstract Atlantic salmon (average weight: 199 g, group size: 55) and rainbow trout (average weight: 565 g, group size: 25) of the same age, kept in freshwater (FW) or full-strength seawater (SW), were fed diets containing 7% (LCH) or 23% (HCH) precooked maize starch, replacing dietary protein. The fish were fed ad libitum in triplicate groups. Feed intake was monitored. The species appeared quite similar regarding thermal growth coefficient (TGC); 3.09% and 3.14% for the salmon and the trout, respectively, and retention of digestible protein; 45.9% and 43.9%, and energy; 45.7% and 46.9%, respectively. Rainbow trout showed higher digestibilities than Atlantic salmon, most pronounced with the HCH diet. Digestibilities were negatively affected by starch level in Atlantic salmon, less severe in rainbow trout. Hepatosomatic index was higher in rainbow trout than Atlantic salmon when fed the HCH diet. Liver lipid, liver glycogen, plasma glucose and plasma insulin were higher in rainbow trout than in Atlantic salmon at both starch levels. The difference was greatest with the HCH diet. In Atlantic salmon, the HCH diet elevated only liver glycogen. Specific activities of maltase and iso-maltase were substantially higher in the rainbow trout than in the Atlantic salmon. In seawater, Atlantic salmon showed higher TGC than in FW. Rainbow trout seemed unaffected by salinity. Salinity did not affect body composition significantly, except for ash, which was higher in SW than in FW in both species. Digestibility of protein, starch, freeze dry matter and energy was higher in FW than in SW. For starch digestibility, this was true for Atlantic salmon only. Retention of protein, but not energy, was higher in SW than FW. No significant effects of water salinity were observed with regards to condition factor, dress-out percentage, HSI, liver lipid, liver glycogen, plasma glucose or plasma insulin. The high starch diet reduced TGC in both species with no significant effect on FCR or body composition. Negative effects of starch level were observed on digestibility of macronutrients and energy in Atlantic salmon, but not in rainbow trout. Retention of digestible energy was not significantly affected by starch level, indicating that starch energy can replace protein energy. The results clearly showed stimulating effects of dietary starch on mucosal maltase, sucrase and lactase activities in both Atlantic salmon and rainbow trout, contrary to results of previous research. The response in leucine aminopeptidase showed adaptation according to protein level.

Effects of soybean meal and salinity on intestinal transport of nutrients in Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss).

Groups of fresh- and seawater-adapted Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss) were fed diets with (SBM diet) or without (control diet) extracted soybean meal (30% of protein substituted with SBM) for 3 weeks. Average fish size per group ranged from 597 to 1763 g. One tank or net pen per species, dietary group and water salinity was used. In vitro nutrient transport (D-glucose, the L-amino acids aspartate, lysine, methionine, phenylalanine and proline, and the dipeptide glycyl-sarcosine) was measured using intact tissue (everted sleeve method) from the different postgastric intestinal regions. The dimensions of the different intestinal regions were also measured for each treatment group. Results indicate that SBM causes decreased carrier-mediated transport and increased permeability of distal intestinal epithelium for the nutrients, and the capacity of this region to absorb nutrient was diminished. Salinity may also affect the relative contribution of carrier-mediated and independent uptake to total nutrient absorption.

A glucokinase-like-enzyme in the liver of Atlantic salmon (Salmo salar).

An enzyme with properties similar to rat liver glucokinase (Hexokinase IV or D) is present in salmon liver in addition to low-Km hexokinase(s). The specific activity of this enzyme increases about 1.6 fold, comparing activities after feeding diets with 25% and 0% digestive energy from starch. The enzyme has a low affinity for glucose, S0.5 = 25.2-26.8 mM (95% confidence interval) and a low activity with fructose, approximately 8% of the activity with glucose. Its molecular mass was estimated to 50.7 +/- 0.6 kDa (SEM. n = 3) by gel filtration, and it displays positive cooperativity with respect to glucose. The Hill constant = 1.73-1.81 (95% confidence interval). The enzyme is competitively inhibited by N-acetyl glucosamine, K(i) approximately 0.28 mM.

2 - Feeding, digestion and absorption of nutrients

Publisher Summary The main function of the alimentary tract of any animal is the acquisition of food with subsequent assimilation of vital nutrients. The natural diet of fishes varies tremendously between fish species and their natural habitats. This chapter summarizes the current state of knowledge, or lack thereof, highlighting the similarities and differences of fish digestive physiology relative to that of the more well studied mammalian systems. Important references, especially review articles, are discusses in the chapter to aid in finding more detailed information as needed. Feeding strategies and development of digestive processes in fish larvae and juveniles, as well as physiological responses to changes in ingredient composition of formulated feeds for cultured fish species have been reviewed.

Effects of methionine, cysteine and medium chain triglycerides on nutrient digestibility, absorption of amino acids along the intestinal tract and nutrient retention in Atlantic salmon (Salmo salar L.) under pair-feeding regime.

Abstract Atlantic salmon, kept in seawater (8–11°C), initial weight 180 g, were pair-fed four high energy, low protein diets for 65 days in triplicate. The diets were supplemented isonitrogenously with amino acids: methionine (6.2 g kg −1 ), cysteine (5.0 g kg −1 ) or alanine (3.7 g kg −1 ) (control). In the fourth diet, also supplemented with alanine, medium chain triglycerides (MCT) (100 g kg −1 ) replaced fish oil. Feed intake was estimated and feeding level adjusted based on the difference between delivered and wasted feed. Apparent digestibilities and pancreatic enzyme activities along the intestinal tract were determined from analyses of chyme from different regions and of faeces, using yttrium oxide as the marker. Plasma amino acid concentrations were determined in blood from the caudal vein. Whole fish taken at start and termination of the experiment were used to estimate nitrogen and energy retention. Feed intake in fish fed the MCT-diet determined the feeding level of fish on the other diets. MCT increased digestibilities of protein and starch and increased nitrogen retention from 52% to 66%, while energy retention was significantly reduced. MCT caused accumulation of cysteine in the proximal regions of the post-gastric intestinal tract and increased pancreatic proteolytic activity of the chyme. Methionine supplementation increased fat digestibility. Cysteine increased digestibilities of protein, fat, starch, as well as nitrogen retention. Hence, sulphur amino acids seemed to be the first limiting amino acids and methionine less effective than cysteine in relieving the limitation. Plasma levels of taurine did not, however, indicate any limitation in the capability to convert methionine into cysteine.

Early response of gene expression in the distal intestine of Atlantic salmon (Salmo salar L.) during the development of soybean meal induced enteritis.

Plant products in general and soybeans in particular can challenge the function and health of the intestinal tract. Salmonids develop an intestinal inflammation when fed diets containing soybean meal (SBM) and certain other legume ingredients. In the present study a 44K oligonucleotide salmonid microarray, qPCR and histology were used to investigate early response mechanisms in the distal intestine of Atlantic salmon (Salmo salar L.) during the first week of oral exposure to a diet containing 20% extracted SBM. The distal intestine transcriptome was profiled on days 1, 2, 3, 5 and 7 and compared to a control group fed fishmeal as the sole protein source. Histological evaluation of the distal intestine revealed the first signs of inflammation on day 5. The most prominent gene expression changes were seen on days 3 and 5. Up-regulation in immune-related genes was observed during the first 5 days, including GTPase IMAP family members, NF-kB-related genes and regulators of T cell and B cell function. Many functional genes involved in lipid metabolism, proteolysis, transport, metabolism and detoxification were initially up-regulated on days 1-3, possibly as an attempt by the tissue to compensate for the initiating immune response. Cell repair and extracellular matrix remodeling genes were up-regulated (heparanase, collagenase) on days 3 and 5. Down regulation of genes related to endocytosis, exocytosis, detoxification, transporters and metabolic processes from day 5 indicated initiation of dysfunction of digestive and metabolic functions that may occur as a result of inflammation or as a response to the introduction of soybean meal in the diet. This is the first study conducting transcriptomic profiling to characterize early responses during the development of SBMIE. Switching Atlantic salmon from a fishmeal to a 20% SBM diet resulted in rapid changes to the intestinal transcriptome, indicating an immune reaction with subsequent impaired epithelial barrier function and other vital intestinal functions.

Transcriptional regulation of IL-17A and other inflammatory markers during the development of soybean meal-induced enteropathy in the distal intestine of Atlantic salmon (Salmo salar L.).

Progression of soybean meal (SBM)-induced enteropathy in Atlantic salmon (Salmo salar L.) distal intestine (DI) was studied to investigate pathophysiological mechanisms and immune responses. Seawater-adapted salmon were fed an extracted SBM-containing diet (200 g kg(-1)) from day 1-21 and compared with fish fed a fishmeal-based diet (day 0). Histological evaluation of the DI revealed signs of inflammation from day 5, which progressively increased in severity and affected more fish with increasing SBM exposure time. The expression profiles of 16 genes were analyzed by quantitative PCR. The pro-inflammatory cytokines interleukin 17A (IL-17A), IL-1β, interferon α (IFNα) and IFNγ, as well as IL-17A receptor, T-cell receptor γ (TCRγ), cluster of differentiation 4α (CD4α), CD8β, transforming growth factor β (TGFβ), trypsin, protease-activated receptor 2 (PAR2) and myeloid differentiation primary response gene 88 (MyD88) were significantly up-regulated during early and/or late inflammation stages, whereas interferon-γ-inducible lysosomal thiol reductase (GILT) was downregulated. Up-regulation of TCRγ from day seven suggests proliferation of intraepithelial γδ T cells. IL-17A, up-regulated by 218-fold during early inflammation, indicates involvement of T helper 17 cells in the pathogenesis of the SBM-induced inflammatory response.

Pancreatic proteinases from man, trout, rat, pig, cow, chicken, mink and fox. Enzyme activities and inhibition by soybean and lima bean proteinase inhibitors

1. The specific activities of the trypsins and chymotrypsins, measured with synthetic substrates, varied within one order of magnitude. 2. The trout trypsin and chymotrypsin were 5-15 times as efficient in hydrolyzing casein as the remaining animals. 3. The inhibition of total caseinolytic activity in extracts of pancreatic tissue by SBTI and inhibitors in crude extracts of raw soybeans varied ten-fold. 4. The animals may be ranked as follows according to the sensitivity of the caseinolytic activity to SBTI; trout greater than fox, chicken, greater than pig greater than rat, cow greater than mink greater than man.