Establishment of a comprehensive reference transcriptome for vertebral bone tissue to study the impacts of nutritional phosphorus deficiency in rainbow trout (Oncorhynchus mykiss, Walbaum).

Reducing dietary phosphorus (P) is a common approach to reduce effluent P outputs. The potential resulting P-deficiency is known to negatively impact fish bone condition and might result in vertebral deformities. To date, no large-scale study involving deep sequencing of the bone transcriptome has been conducted in salmonids and vertebral molecular changes remain poorly described. This study aims to provide the first comprehensive vertebral transcriptome for rainbow trout (Oncorhynchus mykiss) to allow functional and quantitative expression studies. Fish weighing 60.8±1.6g, were fed for 27weeks using two practical diets having 0.29% (deficient) and 0.45% (sufficient) available phosphorus (P), respectively. Deep sequencing was conducted using HiSeq2000 Illumina 100 paired-end technology from pooled P-deficient and P-sufficient fish and individuals displaying vertebral deformities. Over 140 million trimmed paired-end reads were assembled de novo with Trinity and resulted in 679,869 transcripts with a mean length of 542.5bp. From these sequences, 340,747 matched with referenced ESTs from rainbow trout. Furthermore, 141,909 and 117,564 sequences were functionally annotated against Nr and Uniprot databases, respectively. Interestingly, we observed putative homologue sequences for most of the key components involved in bone formation and turnover in mammals.

Factors affecting protein release from alginate-chitosan coacervate microcapsules during production and gastric/intestinal simulation.

A series of experiments was performed to evaluate the influence of a number of physico-chemical factors on the diffusion of a model protein, bovine serum albumin (BSA), from dried chitosan-coated alginate microcapsules. Diffusion of BSA was quantified during the microcapsule manufacture processes (gelation, washing, rinsing) and during incubation in conditions simulating the pH encountered during the gastric (0.1 N HCl; pH 1.5) and intestinal (200 mM Tris-HCl; pH 7.5) phases of digestion. Factors tested included alginate and chitosan concentration, calcium chloride (CaCl2) concentration in the gelation medium, loading rate, chitosan molecular mass and pH of the gelation medium. Microcapsule size and gelation time were altered in order to determine their effects on protein retention. Alginate and chitosan concentration significantly influenced BSA retention during microcapsule manufacture and acid incubation, as did calcium chloride concentration in the gelation medium (P<0.05). BSA retention during manufacture was not significantly altered by protein loading rate or pH of the encapsulation medium, however, protein retention during acid incubation decreased significantly with increasing protein loading rate and encapsulation medium pH (P<0.05). Microcapsules that were washed with acetone following manufacture demonstrated significantly increased protein retention during acid incubation (P<0.05). In microcapsules that had been acetone-dried to a point whereby their mass was reduced to 10% of that immediately following encapsulation, protein retention was over 80% following 24-h acid incubation vs. only 20% protein retention from non acetone-dried microcapsules. The presence of calcium in the neutral buffer medium significantly reduced BSA diffusion in a concentration-dependent manner (P<0.05).

Evaluation of protein release from chitosan-alginate microcapsules produced using external or internal gelation.

A series of experiments was undertaken to evaluate the diffusion of a model protein, i.e. bovine serum albumin (BSA), from chitosan-alginate microcapsules produced using either internal or external gelation. Diffusion of BSA was quantified during the microcapsule manufacture processes (gelation, washing, rinsing) and during incubation in conditions simulating the pH encountered during the gastric and intestinal phases of digestion. Encapsulation of an acid phosphmonoesterase permitted in situ protein localization, providing evidence to explain results obtained with BSA. There was significantly greater protein loss from internally versus externally-gelled chitosan-alginate microcapsules during the manufacture process (37.6% versus 4.7%, respectively). Similar trends were observed during 24 h incubation in 0.1 N hydrochloric acid. Increasing alginate concentration from 2-4% (w:v) did not significantly reduce losses from internally-gelled microcapsules. Addition of 0.25 M NaCl to the gelling medium significantly increased protein diffusing during microcapsule manufacture and acid incubation from externally gelled microcapsules. In situ protein localization revealed a higher level of protein near the surface of the microcapsules of externally gelled microcapsules versus internal gelation. The above data indicate that externally-gelled microcapsules are inhomogeneous with a higher concentration of alginate near the microcapsule surface, thus reducing the porosity of the resulting microcapsules. These results suggest that the porous nature of internally-gelled chitosan-alginate microcapsules may result in low encapsulation efficiency, depending on the nature of the product being encapsulated.