The expressions of trypsin and chymotrypsin in the pyloric caeca of Atlantic salmon (Salmo salar L.) were studied in three experiments. Two internal (trypsin phenotypes, life stages) and three common external factors (starvation, feeding, temperatures) influencing growth rates were varied. Growth was stimulated by increased temperature and higher feeding rate, and it was depressed during starvation. The interaction between trypsin phenotype and start-feeding temperature affected specific activity of trypsin, but not of chymotrypsin. Trypsin specific activity and the activity ratio of trypsin to chymotrypsin (T/C ratio) increased when growth was promoted. Chymotrypsin specific activity, on the other hand, increased when there was a reduction in growth rate whereas fish with higher growth had higher chymotrypsin specific activity resulting in lower T/C ratio value. During a rapid growth phase, trypsin specific activity did not correlate with chymotrypsin specific activity. On the other hand, a relationship between specific activities of trypsin and chymotrypsin could be observed when growth declined, such as during food deprivation. Trypsin is the sensitive key protease under conditions favouring growth and genetically and environmentally affected, while chymotrypsin plays a major role when growth is limited or depressed. Trypsin specific activity and the T/C ratio value are shown to be important factors in the digestion process affecting growth rate, and could be applicable as indicators for growth studies of fish in captive cultures and in the wild, especially when food consumption rate cannot be measured.

The responses of the digestive proteases trypsin and chymotrypsin and protein metabolism to differences in feed protein quality were investigated in Atlantic salmon (Salmo salar L.). Two sets of experimental feeds were produced. Each set of high and low quality feeds was provided to either 150 g or 2 kg salmon. Protein in the high quality feeds had significantly higher percentages of free (reactive) sulphydryl (SH) groups than the corresponding feeds based on low quality meals. After 90 days feeding, groups given high and low quality feeds did not differ in their specific growth rates (SGR) in either experiment. However, feed conversion efficiency (FCE) was significantly different between the high and low quality feed groups in 2 kg salmon, where the difference between the high and low feed protein qualities was larger, 10% versus 4% SH/[SH + (S-S)] in 150 g salmon. Higher FCE was preceded by significantly higher trypsin and chymotrypsin specific activities on day 60. SGR, in general, changed after the first month and was stable during the last 2 months in both experiments. Concurrently, both trypsin (T) and chymotrypsin (C) decreased with an increased activity ratio of trypsin to chymotrypsin (T/C ratio), and resulting in significantly lower T/C ratio on day 90 in salmon feeding on high quality feeds in both sizes of fish. Differences in FCE were associated with significant differences in levels of total free amino acids (TFAA) in the plasma and the white muscle, as well as in the ratio of essential to non-essential free amino acids (EAA/NEAA ratio), free hydroxyproline, and RNA in the white muscle. Interestingly, after 3 days starvation (day 93), 5-7 h postprandial EAA/NEAA ratio in the plasma was significantly lower in the high quality diet groups in both experiments. Trypsin specific activity inversely correlated with muscle TFAA levels in 2 kg salmon, concurrent with higher muscle levels of RNA, lower free hydroxyproline and higher FCE in fish fed higher quality diets. KEY WORDS

Atlantic krill meal and Antarctic krill meal were tested to replace fish meal in Atlantic salmon diets. Different techniques were applied to precisely evaluate dietary quality and quality of fish growth performance. Inverse relationship between trypsin specific activity (T) and ratio of trypsin to chymotrypsin (T/C ratio) as a result of increased chymotrypsin specific activity (C) in the pyloric ceca indicated fish growth status as a reduction in growth rate. These protease values in the feces could also indicate fish digestive efficiency, but could not predict fish growth status. There were relationships among feed conversion efficiency (FCE), in vitro digestibility, and pyloric cecal T/C ratio, with inverse levels of krill meal in the diets. Krill meal seemed to increase muscle protein concentration through increasing protein retention, as capacity for protein synthesis was reduced. Dietary quality tests by in vitro digestibility were corresponded with growth studies, and indicated a possibility of inclusion of krill meal at 50–60% replacements, and larger fish were more sensitive to dietary quality than smaller ones. At 80–100% replacements, in vitro digestibility and FCE reduced, and oocyte quality changed through increased trypsin‐like specific activity probably because of less or abnormal oocyte development. PRACTICAL APPLICATIONS The work illustrates differences in digestive efficiency and the quality of growth performance (protein growth efficiency and capacity for protein synthesis in muscle and oocytes) in fish feeding on diets with different levels of krill meal as fish meal replacer. The advantage of using various methods simultaneously for evaluating digestive efficiency and the quality of fish growth performance is to precisely provide reasonable information for some important biological differences between fish groups that could not be explained by using growth parameter alone, especially when the numbers of samples are not high. The methods are very practical for studying food quality, food utilization and growth quality of fish in different environmental conditions and with different behaviors in aquaculture as well as in natural ecosystem where food consumption rate cannot be measured. The in vitro digestibility method, using fish crude enzyme extracts and standardized by trypsin activity, could also compare the digestive ability between different species and provide information on food quality in fish growth trials that may not be necessary to be performed.

Biochemical structure of protein (reactive SH content, content ratio of SH/SÐS and concentration of D-Asp as % of total (D L)-Asp) indicating digestibility of dietary protein was changed under different processing conditions. Based on ®sh crude enzyme extract, in vitro digestibility of different ®sh materials processed under different conditions correlated positively with reactive SH content and content ratio of SH/SÐS and negatively with D-Asp concentration. In vitro digestion of different experimental feeds, based on Atlantic salmon crude enzyme extracts, was studied in association with growth trials in order to investigate its value as a criterion for industrial strategy in predicting feed quality. Crude enzymes were extracted from the pyloric caeca before feeding. Signi®cant differences in in vitro digestibility between the experimental feeds were observed whereby there would be differences in feed conversion ef®ciency within 3 months of feeding. There were associations between the in vitro digestibility and other parameters for dietary quality, such as mink digestibility and the biochemical structure parameters of the dietary protein due to different processing conditions. Crude enzyme extracts from rainbow trout and European seabass were also used for in vitro digestibility study of different experimental feeds by standardising trypsin activity to that of Atlantic salmon crude enzyme extract. The results indicated that different ®sh species have different digestion ability to the same feed types, and the effective time for feed utilisation and growth is dependent on ®sh sensitivity and the extent of difference in digestibility between the feeds consumed as observed in the Atlantic salmon trials. For the species investigated, sensitivity ranking of the enzymes to feed quality under the condition studied was Atlantic salmon > rainbow trout > European seabass. The results indicated that in vitro digestibility study of experimental feeds using pyloric caecal crude enzyme extract from a speci®c species at an age of interest could be a practical, quick and reliable method for testing feed quality in growth trials. By standardising the crude enzyme extract with regards to trypsin activity, the in vitro digestibility values could be comparable not only within the same species but also between different species.

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Havforskningsinstituttets institusjonelle arkiv Brage IMR - Institutional repository of the Institute of Marine Research b r a g e i m rDette er forfatters siste versjon av den fagfellevurderte artikkelen, vanligvis omtalt som postprint. I Brage IMR er denne artikkelen ikke publisert med forlagets layout fordi forlaget ikke tillater dette. Du finner lenke til forlagets versjon i Brage-posten. Det anbefales at referanser til artikkelen hentes fra forlagets side. Ved lenking til artikkelen skal det lenkes til post i Brage IMR, ikke direkte til pdf-fil.This is the author's last version of the article after peer review and is not the publisher's version, usually referred to as postprint. You will find a link to the publisher's version in Brage IMR. It is recommended that you obtain the references from the publisher's site.Linking to the article should be to the Brage-record, not probiotic-supplemented and carbohydrases-supplemented diets) were studied on digestive 25 enzyme specific activities and growth performance quality of juvenile Siamese fighting fish 26 (Betta splendens Regan, 1910) during 2 weeks of critical and intensive rearing period. The 27 modified procedures did not change biochemical compositions and gross energy of diets, but 28 generally resulted in relatively higher in vitro digestibilities of protein and carbohydrate and 29 fish survival rate, albeit insignificant. Only gamma irradiation significantly increased in vitro 30 protein digestibility of the diet, and microwave irradiation increased starch gelatinization and 31 water solubility (P < 0.05). Fish fed microwave-treated diet showed highest values in all 32 studied growth indicators and digestive enzyme specific activities (except lipase), with 33 significantly higher amylase specific activity and activity ratio of amylase to trypsin (A/T 34 ratio). Correlation analysis indicated significant relationships (P < 0.05) among the levels of 35 total protease, amylase and trypsin, and between SGR and A/T ratio. Muscle and body 36 compositions of juveniles fed on microwave-or gamma-irradiated diets were similar to the 37 control, while the juveniles fed on probiotic-or carbohydrases-supplemented diets showed 38 lower protein depositions (P < 0.05). Similar levels of RNA, RNA/Protein ratio, and 39Protein/Lipid ratio in body and muscle in all dietary groups fed ad libitum suggested that the 40 improved growth performance in juvenile Siamese fighting fish fed on microwave-irradiated 41 diet may not be only due to improved physicochemical properties of the diet but also 42 improved fish consumption rate. 43 44

Hyriopsis (Hyriopsis) bialatus has been cultured during the mussel life cycle from glochidia to the adult stage with a low total survival of 6% up to 130-day-old juveniles. The main digestive enzymes (amylase and proteinases) were not detectable in one-day-old juveniles, and increased during development. The stomach, including digestive glands, was the major digestive organ for both carbohydrate and protein. The optimum conditions for amylase activity were 40°C and pH 7; for acidic proteinases they were 60°C and pH 5. Two main alkaline proteinases were found in the intestine, with optimum conditions of 30°C and pH 8 and 60°C and pH 8. To improve mussel survival by finding suitable phytoplankton species and age as food for juveniles and adults, an in-vitro digestibility test was performed on ten algal species three and seven days old using amylase and proteinases in crude enzyme extracts from different mussel life stages. Among the phytoplankton selected, the three most efficiently digested by juveniles were sevenday-old Chlorella sp.2, seven-day-old Chlorococcum sp. and seven-day-old Kirchneriella incurvata, in the ratio 1:1:3 for 30-day-old juveniles and 3:1:1 for 130-day-old juveniles. For the adult mussel, three-day-old Chlorella sp.2, seven-day-old Coccomyxa sp., and seven-day-old Monoraphidium sp., in the ratio 3:1:1, were the most digestible phytoplankton. Levels of in-vitro digestibility were related to the quality (not the concentrations) of carbohydrate and protein in the phytoplankton mixtures, and protein digestibility seemed to be the key factor determining food quality for the mussel.