Introduction
The replacement of fishmeal in diets for salmonids has often resulted in manifold effects on digestive enzyme activities, in increased immune response and in significantly different growth. The ability to digest different types of feed is primarily based on the individual digestive potential of the fish. This includes feed intake and the breakdown of feed particles and nutrients via enzymes. The first step in protein digestion is the breakdown of large proteins in the stomach by pepsin. Pepsin is an endopeptidase and its main cleavage sites are between aromatic amino acids, such as phenylalanine and tyrosine, which occur in different abundances in different protein resources. Plant meals used as feedstuff additionally contain high amounts of long-chain carbohydrates which are difficult to digest for carnivorous fish. One of the main enzymes involved in the process of carbohydrate breakdown is amylase. α-amylase, specialized on the cleavage of starch, is highly conserved between organisms and for instance in Sea bass amylolytic activity increased in direct relation to dietary carbohydrate level (Péres et al., 1996). Thus this study with two independent feeding experiments has been conducted in order to evaluate pepsin and amylase activities in relation to different plant protein inclusion levels in first feeding Rainbow trout (Oncorhynchus mykiss; RT) and Brown trout (Salmo trutta; BT).
Materials & Methods
Eyed Rainbow trout eggs (Oncorhynchus mykiss, Kamloops strain) were purchased from Forellenzucht Trostadt GbR (Reurieth-Trostadt, Germany), originating from Troutlodge Inc. (Sumner, Washington USA). Eyed Brown trout eggs (Salmo trutta) were reproduced from wild Sea trout (Salmo trutta trutta) in Schleswig-Holstein at Fischbrutanstalt Altmühlendorf (Germany). After 28 days post hatch fish were fed ad libitum with three isonitrogenous and amino acid balanced diets. For Rainbow trout the diets contained 0%, 50% and 97% plant derived proteins (A, B and C) and for Brown trout 0%, 50% and 89% (X, Y and Z; diets were produced in cooperation with Skretting ARC, Norway). The remaining percentage derived from animal protein sources. After two months of rearing in recirculating hatchery troughs, fish were transferred to a recirculating aquarium system and fed with 3.8% (RT) and 2.2% (BT) for two and a half months. Fish were reared with weekly assessment of growth parameters. Whole body samples (first feeding fry) and whole digestive tract samples (juveniles) were taken to determine individual amylase and pepsin activities photometrically (Molecular Probes, Inc.).
Results
Average amylase activities for both species five days prior to hatching were statistically equal to activities on hatching day; nevertheless amylase activities of Rainbow trout eggs were significantly higher than Brown trout eggs in terms of mU per mg whole egg homogenate. The average activity for the period from hatching until first feeding is significantly higher (p=0.03) in Rainbow trout (0.76 mUmg-1 whole body homogenate) than in Brown trout (0.38 mUmg-1). Pepsin activities have also been assessed prior to first feeding. In comparison to amylase however, pepsin activities do not differ significantly (p=0.77) if averaged over time (4.49 mUmg-1 for Rainbow trout and 4.10 mUmg-1 for Brown trout). With the event of first feeding on, Amylase activities are influenced differently by diet type for Rainbow trout and Brown trout. Rainbow trout do not differ in their amylase activities when fed the diets types A, B or C one day after first feeding. However at day 31 post hatch the experimental diets have a significant influence on amylase activity. This difference between experimental groups is also present in the whole digestive tract samples of day 75 and at the end of the experiment. On both sampling days amylase is significantly more active in fish of group A than of group C (p<0.0001). Evaluating the influence of diet types X, Y and Z on amylase activity in Brown trout shows no significant effect at any point in time. Comparable to Rainbow trout however, amylase activities are increasing with time until the end of the experiment. After first feeding pepsin activities are strongly increasing in fry of both species. After the first 10 days of feeding, pepsin activities in Rainbow trout are two times higher compared to the yolk sac stage. For Brown trout, activities are more than 10 times higher. The influence of the different diets is not significant for Rainbow trout at first feeding, however Rainbow trout fed diet C shows a significant increase (p<0.001) in pepsin activities until the end of the trial. The extreme increase of pepsin activities for Brown trout after first feeding resulted in similar activities than those for Rainbow trout at the end of the experiment. The diets had only a small influence on pepsin activities of brown trout, like a reduced average activity in diet group Z. At the end of the trial, activities are still similar between groups - without an influence by experimental diets.
Discussion
The assessment of growth parameters in this study showed that a balanced mixture of animal and plant based proteins in early fry and fingerling feed generates a growth superior to that of diets completely based on one or the other resource. Higher amylase levels in Rainbow trout than in Brown trout could be an effect of an already occurring adaptation to plant proteins during the last decades of intensive breeding. A direct comparison of Brown trout and Rainbow trout displays that Brown trout might have more the character of a carnivorous fish in terms of digestive enzyme activity. Typical carnivorous fish have high protease and low carbohydrate levels (Ugolev and Kuz'mina, 1994). The extreme increase of pepsin activity within the first 20 days after first feeding in Brown trout is about 10 to 20 times higher compared to Rainbow trout activities. This might thus be the result of the strict carnivorous feeding habit of wild Sea trout. During the first days of feeding in both species activities of amylase and pepsin were alternating likewise the study conducted by Darias et al. (2006), which showed clear alternating expression patterns of amylase in red porgy (Pagrus pagrus) during the first weeks after hatching and it has already been discussed that digestive enzymes in early trout fry are controlled via intrinsic expression mechanisms (Polakof et al., 2012). Of interest in the present study is therefore the high variability of amylase activities in Rainbow trout and the quite homogeneous amylase and pepsin activities in Brown trout.
References
Péres, A., Cahu, C.L., Zambonino Infante, J., Le Gall, M.M., Quazuguel, P. 1996. Amylase and trypsin responses to intake of dietary carbohydrate and protein depend on the developmental stage in sea bass (Dicentrarchus labrax) larvae. Fish Physiology and Biochemistry 15: 237-242.
Darias, M.J., Murray, H.M., Gallant, J.W., Astola, A., Douglas, S.E., Yúfera, M., Martínez-Rodríguez, G. 2006. Characterization of a partial α-amylase clone from red porgy (Pagrus pagrus): Expression during larval development. Comparative Biochemistry and Physiology Part B 143 (2): 209-218.
Polakof, S., Panserat, S., Soengas, J.L., Moon, T.W. 2012. Glucose metabolism in fish: a review. Comparative Biochemistry and Physiology Part B 182 (8): 1015-1045.
Ugolev, A.M., Kuz'mina, V.V. (1994): Fish enterocyte hydrolases. Nutrition adaptations. Comparative Biochemistry and Physiology Part A 107 (1), pp. 187-193.