Stimulative effect of skipjack tuna soluble extract on pepsin-like protease in the stomach of rockfish (Sebastes schlegelii) using an in vitro perfusion method

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Abstract

The effect of intra-gastric infusion of skipjack tuna (Katsuwonus pelamis) muscle soluble extract and of carnosine, anserine and histidine, on pepsin-like protease activity was studied in the isolated, externally batch-cultured stomach of the rockfish (Sebastes schlegelii). Stomach was isolated from the fish, then intragastrically infused with the above solutions or with a balanced saline solution (control solution) as the stimulant at 0.1 mL/min for 6 h. Intra-gastric efflux was collected for measurement of pepsin-like protease activity. Skipjack tuna soluble extract, but not carnosine, anserine or histidine alone caused significant enhancement of pepsin-like protease activity during the infusion. Pepsin-like protease activity from skipjack tuna soluble extract responded immediately after infusion and was kept for 150 min after infusion. Stomach motility at the end of infusion was also observed. These results suggest that isolated stomach can receive mucosal side stimulants in vitro. It is likely that some effective components for stomach digestion in fish exist in skipjack tuna soluble extract.

Introduction

Studies of alternative protein sources of fish meal for aquaculture use have been conducted because the world-wide expansion of aquaculture has been increasing the demand for fish feeds (Tidwell and Allan, 2001) that depend on fish meal as the major dietary protein source. Among the various protein sources, soybean meal (SBM) is accepted for fish feeds (Watanabe, 2002). However, use of high levels of SBM in diets indicated reduced nutrient bioavailability and growth of major carnivorous species, such as European sea bass (Dicentrarchus labrax) (Tibaldi et al., 2006), Atlantic salmon (Salmo salar) (Øverland et al., 2009), red sea bream (Pagrus major) (Goto et al., 2001), Japanese flounder (Paralichthys olivaceus) (Deng et al., 2006) or yellowtail (Seriola quinqueradiata) (Watanabe et al., 1998). Thus, these findings suggested that there was a disruption in nutrient digestion and/or absorption (Tibaldi et al., 2006).

Digestion and absorption of nutrients depend on the activity of the digestive enzymes. The stomach is the primary digestive organ for dietary protein (except for stomach-less fish). Accordingly, the gastric protease pepsin plays an important role in the digestion of proteins in fish feeds. Compared with other vertebrate animals, fish are characterized by a high protein requirement from feeds (Steffen, 1989). However, in general, alternative plant proteins are less digestible than fish meal. In the above senses, it is suggested that enhancement of gastric pepsin secretion would enhance the gastric digestibility of plant proteins such as SBM. Thus, assessing of stimulants for pepsin secretion may represent a useful tool to help select alternative protein sources for fish feeds.

Gastric secretion of fish is affected by the chemical and physical properties of their stomach contents. It was reported that in vivo pepsin secretion increased after ingestion of chopped sprat by whiting (Merlangius merlangus) (Mazlan and Grove, 2004) and of formula diet by eel (Anguilla japonica) (Takii et al., 1985, Takii et al., 1986). However, there is a lack of information on the effect of dietary chemical components on pepsin secretion in fish stomach. In contrast, Igarashi et al., 2005, Aono et al., 1981 reported that some amino acids or peptides stimulated pepsin secretion in rat stomach in vivo. In any case, these in vivo techniques are not suitable for the screening method because much time is needed to confirm the effect of stimulants (Morales and Moyano, 2010). Thus, the present study was undertaken to develop a new in vitro screening method, which used the isolated, luminally infused and externally batch-cultured fish stomach. For this purpose, skipjack tuna (Katsuwonus pelamis) soluble extract (SJT-SE) was chosen as a stimulant and source of soluble amino acids, which include dipeptides, carnosine and anserine (Suyama and Yoshizawa, 1973), because the dipeptides enhance pepsin activity and pepsin secretion in rat stomach (Igarashi et al., 2005). If the peptides stimulate gastric secretion in the fish stomach and if the isolated stomach can receive dietary components during infusion, pepsin activity should be enhanced by intra-luminal infusion by the SJT-SE and peptide solution.

Accordingly, our aim in the present study was to clarify that the isolated stomach of fish could detect dietary components and differences between stimulants by enhancing the activity of pepsin-like protease to confirm whether the isolated stomach infusion method was able to be used as a gastric stimulant screening method. The experiments were also designed to evaluate the histological normality of the stomach mucosal layer after 6 h of infusion to confirm whether the above response was secretion or passive discharge by collapse due to an improper incubation condition. For these purposes, a carnivorous teleost, the rockfish (Sebastes schlegelii), was used. The species is cultured in Hokkaido, Japan.

Section snippets

Animals

Rockfish (S. schlegelii) of both sexes and approximately 150 g body mass were captured on the North coast of Uchiura Bay (Hokkaido, Japan) by hook and line, then stocked at our marine laboratory (UoRes, Toyoura, Hokkaido, Japan). Fish were transported to our inland laboratory within 3 h by car. Each fish was treated by a bath in fresh water at 17 °C for 2 min to eliminate external parasites before stocking at the laboratory. The experiment was performed during all seasons.

Rearing conditions

Fish were reared in 1000 L

Free amino acids in skipjack tuna soluble extract

Histidine, anserine, lysine, and taurine were easily detected in the SJT-SE infused into the stomach in this study (Table 1). Carnosine, anserine and histidine concentrations were 0.67, 5.9 and 20.4 mM respectively.

Pepsin-like protease activity and secretion

Pepsin-like protease activities from the efflux of the control group (BSS infusion) did not differ after 90 min incubation (p > 0.05). The skipjack tuna soluble extract group indicated considerable variation in pepsin-like protease activity (Fig. 2). Infusion of SJT-SE caused an increase

Discussion

Pepsin-like protease activities from isolated, luminally infused and externally batch-cultured rockfish stomach at the beginning of this infusion were high, then decreased and stabilized at low levels (approximately 75 μg tyrosine liberated/mL gastro-luminal efflux/30 min) after 90 min in control infusion (Fig. 2). Yellow colored efflux at the beginning of infusion also indicated the existence of acid in the efflux. Any activity and acid at the beginning should be from pre-existing proteases and

Acknowledgments

The authors appreciate Dr. Hidetoshi Matsuyama of Tokai University for support of this experiment. We also thank to Dr. Brian J. Harvey for his critical reading of the manuscript. This study was supported in part by grants from the Research for Promoting Technological Seeds of Japan Science and Technology Agency and the Research and Study Program of Tokai University Educational System General Research Organization.

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