Addition of ß-glucans in diets for tropical gar (Atractosteus tropicus) larvae: effects on growth, digestive enzymes and gene expression of intestinal epithelial integrity and immune system.

The effect of ß-glucans 1,3/1,6 from Saccharomyces cerevisiae yeast at different inclusion percentages (0.0, 0.2, 0.4, 0.6, and 0.8%) in the diet for tropical gar (Atractosteus tropicus) larvae was evaluated on growth, digestive enzyme activity and, relative expression of the immune system genes. The bioassay started on the third day after hatching (DAH) and lasted 21 days, using a total of 1500 larvae of 0.055 ± 0.008 g and, a total length of 2.46 ± 0.26 cm. Larviculture was carried out in a recirculation system with 15 tanks of 70 L using a density of 100 organisms per experimental unit. No significant differences in larval growth were observed by the inclusion of ß-glucans (p > 0.05). Digestive enzymes showed changes in lipase and trypsin activities, presenting higher values in fish fed 0.6% and 0.8% ß-glucans diets compared to the other treatments (p < 0.05). Leucine-aminopeptidase, chymotrypsin, acid phosphatase, and alkaline phosphatase activity showed higher activities in larvae fed with a 0.4% ß-glucan diet compared to the control group. The relative expression of intestinal membrane integrity (mucin 2) muc-2, (occludins) occ, (nucleotide-binding oligomerization domain) nod-2, and immune system lys (lysosome) genes showed over-expression in larvae fed the 0.4% ß-glucan diet to the rest of the treatments (p < 0.05). The inclusion of ß-glucans at 0.4-0.6% in diets for A. tropicus larvae could improve larviculture, as effects on the increase in the activity of several digestive enzymes and the expression of genes of the immune system.

Digestive proteases and in vitro protein digestibility of feed ingredients for the Central American river turtle, Dermatemys mawii.

Functional characteristics of digestive proteases and in vitro digestibility of several protein sources were studied in hatchlings of Central American river turtles, Dermatemys mawii. Acidic and alkaline proteases from the stomach and intestines were used, and optimums for acidic proteases were registered at 55°C and pH 2, while alkaline proteases were found at 55°C and pH 9. Ten protein ingredients, both vegetable and animal sources, were explored by the pH-STAT method, only for alkaline proteases. The degree of hydrolysis was at its highest for squid meal and lowest for blood meal, while the total free amino acids level was at its highest for squid meal, and lowest for wheat gluten meal. Our results indicate that D. mawii has a broad capacity to digest both animal and vegetable sources, and suggests some ingredients more suitable to design artificial diets for this species.

Changes on digestive enzymes during initial ontogeny in the three-spot cichlid Cichlasoma trimaculatum.

A study was performed in order to understand the development of digestive enzymes during initial ontogeny of Cichlasoma trimaculatum, for which the activity of acidic and alkaline proteases, lipases, amylases and phosphatases was determined by means of biochemical and electrophoretic analysis. Our results showed that the activity of alkaline proteases, trypsin and chymotrypsin is present from day 6 after hatching (dah) during exogenous feeding with Artemia nauplii. The activities of carboxypeptidase A and leucine aminopeptidase are present from the first days, increasing at 6 dah and reaching their maximum activity at 9 dah while acid protease activity started at 9 dah. Furthermore, the lipase activity is detected on 6 dah and keeps increasing and decreasing on 17 dah. Amylase activity is detected on 3 dah, presenting fluctuations until 45 dah, where it reaches its maximum activity. Acid and alkaline phosphatases are detected from 3 dah and reach a maximum activity between 13 and 19 dah. The SDS-PAGE electrophoresis revealed six types of bands in the alkaline proteases, with molecular weight between 113.4 and 20.4 kDa. First three bands appear on 6 dah, but it is until 11 dah when all isoforms appear. Based on these results, it is considered that this species completes its digestive enzymatic machinery from day 9 after hatching, therefore is recommended to perform the transition from live feed to inert feed at 15 dah.

Partial characterization of digestive proteases in tropical gar Atractosteus tropicus juveniles.

Tropical gar (Atractosteus tropicus) is an economically and socially important freshwater species from Southeastern Mexico, with a high aquaculture potential. With this in mind, the purpose of this study was to characterize the digestive proteases of tropical gar juveniles through biochemical and electrophoretic analyses. Twenty specimens with an average weight of 73.6 ± 12.7 g were used to obtain stomach and intestinal tissue from which multienzymatic extracts were prepared. The general activities of the acid and alkaline proteases were evaluated, as well as the specific activities of trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A. The effect of the pH and temperature on the proteases was also analyzed, together with the composition of the multienzymatic extracts using protease inhibitors and electrophoretic tests. Results showed that A. tropicus have a functional stomach in which protein hydrolysis starts with pepsin and which contains endo- and exopeptidases (trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A) and proteases that are resistant to high temperatures (45 and 55 °C for alkaline and acid proteases, respectively) and pH values. Using zymogram technique, we found two acid protease isoforms (0.35 and 0.71 rf) and five alkaline protease isoforms (83.7, 43.7, 27.5, 24.0 and 19.4 kDa), which decrease or disappear with the different inhibitors. Thus, this species is considered to be a carnivore capable of adapting to its environment by consuming different types of proteins from preys and also could adapt rapidly to consume a compound diet with different animal protein sources.

Partial characterisation of digestive proteases of the Mayan cichlid Cichlasoma urophthalmus.

The characterisation of digestive proteases in native freshwater fish such as the Mayan cichlid Cichlasoma urophthalmus provides scientific elements that may be used to design balanced feed that matches with the digestive capacity of the fish. The purpose of this study was to characterise the digestive proteases, including the effect of the pH and the temperature on enzyme activity and stability, as well as the effect of inhibitors using multienzymatic extracts of the stomach and intestine of C. urophthalmus juveniles. Results showed that the optimum activities of the acid and alkaline proteases occurred at pH values of 3 and 9, respectively, whereas their optimum temperatures were 55 and 65 °C, respectively. The acid proteases were most stable at pH values of 2­3 and at temperatures of 35­45 °C, whereas the alkaline proteases were most stable at pH values of 6­9 and at 25­55 °C. The inhibition assays recorded a residual activity of 4% with pepstatin A for the acid proteases. The inhibition of the alkaline proteases was greater than 80% with TPCK, TLCK, EDTA and ovalbumin, and of 60 and 43.8% with PMSF and SBT1, respectively. The results obtained in this study make it possible to state that C. urophthalmus has a sufficiently complete digestive enzyme machinery to degrade food items characteristic of an omnivorous fish species, although specimens showed a tendency to carnivory.