Digestive protease activities, growth and feed utilisation in Atlantic salmon (Salmo salar L.)
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The specific activities of trypsin and chymotrypsin in the pyloric caeca were investigated in individually labeled Atlantic salmon (Salmo salar L.). Photoperiod (natural or 24 h) and feed protein quality (two levels of digestibility) were used as growth affecting factors in two grow-out experiments. Parameters indicative of protein growth and metabolism, i.e. plasma and white muscle free amino acid (FAA) concentrations, white muscle RNA concentrations, and white muscle protein synthesis capacity (RNA/protein ratio) were also measured. The feasibility of using free hydroxyproline (Hyp) concentration in the white muscle as an indicator of the rate of tissue protein breakdown (turnover) was assessed. Further, an in vitro digestibility assay was developed to evaluate the effects of industrial processing conditions on feed protein digestibility. Digestion using crude pyloric caecal extracts was standardised by trypsin activity and compared with growth experiments to predict the effects of feed protein quality on specific growth rate (SGR) and feed conversion efficiency (FCE). Finally, dorsal aorta cannulation of fish was assessed as a tool for evaluating feed protein quality (containing no free or supplemented amino acids) through repeated measurements of plasma FAA concentrations after feeding. Trypsin (T) and chymotrypsin (C) showed high covariation in all experiments, regardless of whether growth was affected indirectly (through photoperiod manipulation) or directly (through feed protein quality). Groups exhibiting different feed conversion efficiencies (FCE) had different activity ratios of trypsin to chymotrypsin (T/C ratio). The T/C ratio seemed to be more sensitive than growth measurements to slight differences in feed protein quality and might have an application as an indicator of growth performance. In salmon reared under different photoperiods, SGR correlated with trypsin activity and T/C ratio on individual basis. These correlations could possibly be explained by a predominant influence of feed intake on growth under these conditions. In contrast to trypsin activity, chymotrypsin activity was uncorrelated to SGR variation. Plasma essential (EAA) and total (TFAA) free amino acid concentrations did not show consistent relationships with other biochemical parameters and growth rate. White muscle EAA, however, decreased with SGR, while white muscle TFAA and Hyp concentrations showed an increasing trend. Of all measured parameters, Hyp level in white muscle showed the highest correlation with growth rate. An observed inverse relation between SGR and white muscle RNA concentrations indicated a lower relative protein synthetic activity at higher growth rates and could indicate a shift to a higher importance of lipid deposition at high feed intakes. This was consistent with an increased protein turnover, indicated by elevated white muscle free Hyp levels, suggesting a lower efficiency of protein retention at high growth rates and that a higher fraction of ingested amino acids were used as energy substrates. Fish meal raw material, drying temperature and duration of drying affected feed protein digestibility in vitro. This reduction in digestibility was concomitant with a higher incidence of disulphide bond formation in the feed proteins, demonstrating a negative effect of disulphide bond formation on feed protein quality. Digestibility of the experimental diets measured in vitro correlated with SGR and FCE after three months of feeding, but differences in SGR between feed quality groups did not reach statistical significance in either 150 g or 2 kg salmon. However, 2 kg salmon fed restricted rations showed significantly higher FCE in the ‘high’ protein quality feed groups. Differences in FCE at the end of the experiment seemed to be preceded by differences in trypsin and chymotrypsin specific activities one month earlier. Trypsin activity was unaffected by feed protein quality, possibly only reflecting the similar feed intake in the experimental groups. This resulted in a relationship between chymotrypsin activity and feed in vitro digestibility when standardised by trypsin activity. Fish groups given feeds of ‘high’ protein quality had relatively higher RNA concentrations in the white muscle than groups given ‘low’ quality feeds, indicating a positive effect of feed protein digestibility on muscle ribosome concentration, and possibly protein synthetic activity. At the same time, white muscle Hyp concentrations were significantly lower in the ‘high’ quality dietary groups, indicating a lower protein turnover rate and potentially higher protein retention efficiency in these fish. However, this was not detectable as a difference in fillet protein content after three months of feeding. White muscle ratios of essential to nonessential free amino acids (EAA/NEAA ratio) were higher in groups with higher FCE, whereas plasma values showed no specific pattern, except after starvation and refeeding, where EAA/NEAA ratios were higher with higher FCE. No correlations were found between digestive protease activity and other parameters on an individual basis. A possible explanation for these findings could be that growth rate in this case was limited by feed protein digestibility and unrelated to feed intake. High and low quality feeds were selected from the protein quality study in order to investigate amino acid uptake following feeding. Feed intake was positively correlated with the sum of EAA in plasma 6 h post-feeding. Variation in plasma FAA profiles was to a large extent explained by individual differences in feed intake, but individual differences in metabolism of specific amino acids were indicated. However, feeds of different protein qualities could still be distinguished through significant differences in plasma EAA profiles after statistical correction for these factors. The results indicate that the relationship between trypsin and chymotrypsin activity (T/C ratio) may have an application as an indicator of differences in growth performance between groups of fish, both when growth is affected by external factors and diet quality. The different mechanisms through which growth differences were affected under the two specific experimental setups suggest that the T/C ratio could have a broader application also outside the limitations of the current studies. In particular, the method may be useful for determining the nutritional and growth status of fish in the wild where food consumption cannot be measured. However, this remains to be validated. The relationship between white muscle Hyp concentrations and protein turnover deserves further investigation, as this parameter showed strong correlation with growth rates. Further studies of the hormonal and genetic mechanisms regulating trypsin and chymotrypsin activity, and how they are affected by dietary and exogenous factors need to supplement future studies in this field. These studies demonstrated the suitability and efficiency of small-scale assays for the evaluation of feed protein quality. Further development of such methods is recommended as alternatives or supplements to regular time-consuming growth experiments.
This dissertation consists of the following papers, referred to in the text by their Roman numerals.
Paper 1: Paper 1: Reprinted with kind permission of Springer Science and Business Media: J. Sunde, G.L. Taranger and K. Rungruangsak-Torrissen, Digestive protease activities and free amino acids in white muscle as indicators for feed conversion efficiency and growth rate in Atlantic salmon (Salmo salar L.), Fish Physiology and Biochemistry 2001, 25 (4). Published version available at: http://dx.doi.org/10.1023/A:1023233024001Part 2: Paper 2: Sunde, J., Eiane, S.A., Rustad, A., Jensen, H.B., Opstvedt, J., Nygård, E., Venturini, G. & Rungruangsak-Torrissen, K. (2004) Effect of fish feed processing conditions on digestive protease activities, free amino acid pools, feed conversion efficiency and growth in Atlantic salmon (Salmo salar L.). Aquaculture Nutrition 10 (4), 261-277. Published version available at: http://dx.doi.org/10.1111/j.1365-2095.2004.00300.xPaper 3: Reprinted from In vitro digestibility based on fish crude enzyme extract for prediction of feed quality in growth trials. J. Sci. Food Agric., 82, 644-654, Rungruangsak-Torrissen, K, Rustad, A., Sunde, J., Eiane, S.A., Jensen, H.B., Opstvedt, J., Samuelsen, T.A., Mundheim, H., Luzzana, U. & Venturini, G., Copyright 2002 Society of Chemical Industry. Reproduced with permission. Permission is granted by John Wiley & Sons Ltd on behalf of the SCI.Paper 4: Sunde, J., Kiessling, A., Higgs, D., Opstvedt, J., Venturini, G. & Rungruangsak-Torrissen, K. (2003) Evaluation of feed protein quality by measuring plasma free amino acids in Atlantic salmon (Salmo salar L.) after dorsal aorta cannulation. Aquaculture Nutrition 9 (6): 351-360. Published version available at: http://dx.doi.org/10.1046/j.1365-2095.2003.00263.x