Digestive enzymatic responses of chickens feed-restricted and refed as affected by age

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Pectinases From Sphenophorus levis Vaurie, 1978 (Coleoptera: Curculionidae): Putative Accessory Digestive Enzymes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparison of the digestive efficiency of extruded diets fed to ferrets (Mustela putorius furo), dogs (Canis familiaris) and cats (Felis catus)

The digestive tract of ferrets is anatomically simple, with no caecum, ileocolic valve or external differentiation between the transition of ileum and colon. The species has a short large intestine that provides minor contributions to the digestive process. Aiming to better understand the digestibility efficiency of ferrets, the present study compared the digestibility of extruded diets with different amounts of macronutrients fed to dogs, cats and ferrets. Three formulations for cat maintenance were used (values in % of DM basis): high carbohydrate (HC; nitrogen-free extract (NFE) = 54 %, protein = 31 % and fat = 8 %); moderate carbohydrate (MC; NFE = 37 %, protein = 41 % and fat = 10 %); and low carbohydrate (LC; NFE = 19 %, protein = 46 % and fat = 23 %). Apparent total tract macronutrient digestibility was determined by the method of total collection of faeces. Results were compared by ANOVA, considering the diet and species effects and their interactions. Means were compared by the Tukey's test (P < 0·05). Dogs and cats presented similar food intakes, but ferrets consumed almost two times more food (g/kg body weight). Species × diet interactions were verified for apparent total tract digestibility (ATTD; P < 0·05). Ferrets presented lower DM digestibility than dogs and cats for all three diets (P < 0·05), lower NFE digestibility than dogs for the three diets and lower NFE digestibility than cats for the HC and LC diets (P < 0·05). For crude protein (CP), ferrets presented lower ATTD than dogs and cats (P < 0·05), whereas for fat, dogs and ferrets presented similar ATTD, and higher values than those presented by cats (P < 0·05). Kibble diets had a lower DM, CP and NFE digestibility when fed to ferrets compared with dogs and cats. Fat digestibility was similar between dogs and ferrets and higher than that for cats.

Ultrastructure of the digestive tract in neotropical carnivorous catfish Hemisorubim platyrhynchos (Valenciennes, 1840) (Siluriformes, Pimelodidae)

The surface of the digestive tract of Hemisorubim platyrhynchos was analyzed by scanning electron microscopy. Morphometric studies by transmission electron microscopy were performed to analysis the intestinal microvilli. H. platyrhynchos is a Neotropical carnivorous freshwater catfish featuring a short digestive tract composed of a short esophagus, saccular stomach, and intestine with four regions: anterior, middle, posterior, and rectal. The esophageal surface is constituted by fingerprint-like microridges that anchor the mucosubstances secreted by goblet cells facilitating the passage of food. Goblet cells present the opening to the esophageal lumen, between the microridges. Club cells are in basal epithelium and they do not present the opening to the lumen. The gastric luminal surface shows polygon-shaped epithelial cells which secrete granules by exocytose to protect the gastric surface. The intestinal luminal surface reveals folds that are thicker in the anterior intestine than in the posterior intestine, increasing the absorptive surface area. The intestinal surface presents the microvilli of enterocytes and the opening of goblet cells. The morphometric analysis showed that the microvilli are longer in the anterior intestine, significantly decreasing towards the posterior intestine. The microvilli surface area significantly is greater in the anterior and middle intestine than in the posterior intestine. Numerous openings of goblet cells were observed in the posterior intestine acting in epithelial protection and lubrication. SCANNING 9999:1-8, 2015. © 2015 Wiley Periodicals, Inc.

The ultrastructural evaluation of digestive cells in the hepatopancreas of the Amazon River prawn, Macrobrachium amazonicum

The Amazon River prawn Macrobrachium amazonicum is endemic to tropical South America and is being intensively exploited by artisanal fisheries in Brazil. Limited information is available about the nutritional requirements of M. amazonicum, although the production of this species is now technically feasible. The digestive process in this species is still unclear and investigation into the digestive cells of its hepatopancreatic epithelium is required. Thus, the hepatopancreas from 15 specimens were fixed in Karnovsky Solution and processed for Transmission Electron Microscopy. Our results indicate that E cells are located at the distal portion of the hepatopancreatic tubule and are involved in mitotic activity. The cylindrical R cells are sparse and are only found in the proximal portion of the hepatopancreatic tubule. According to its ultrastructural characteristics, this cell is involved in pinocytosis. M cells are generally found near the R cells. The F cells are scattered throughout the length of the hepatopancreatic tubules, and B cells are observed mainly in the proximal and middle regions. F cells and B cells are likely related to, respectively, the synthesis of enzymes and the intracelular digestion. R- and M cells are probably related to material storage. Thus, these findings provide basic information on the cell types that perform protein digestion in M. amazonicum, and will be useful in further nutritional research. The identification and characterization of digestive cells is an important step towards understanding the digestive mechanisms.

Lipidic and proteic absorption in digestive tract of tropical fat snook (Centropomus parallelus, POEY 1860)

The tropical fat snook Centropomus parallelus is a species of recognized ecological importance and with a high economical potential for fisheries and aquaculture. The investigations of digestive tube morphology in association with their feeding abilities are fundamental to improve techniques for aquaculture feeding procedures. Sudan black staining and Perl`s method were used to evaluate their absorption capacity of fat and protein respectively. The Sudan black stain was performed 12 h after the ingestion of lipids. The lipids are intensely absorbed in the ceca epithelium and less intensely in the intestine and rectum. The Perl`s method was performed 12 h after the ingestion of ferritin. The proteins are absorbed only in the rectum. This is the first description of fat and protein absorption ability by the digestive tube of fat snook. These data enhance the possibility of the addition of macromolecules in rations that can show a diversity of physiological effects. The histological implications of each segment of the digestive tube in association with fish biology are further discussed. (C) 2010 Elsevier B.V. All rights reserved.

Digestive peptidases and proteinases in the midgut gland of the pink shrimp Farfantepenaeus paulensis (Crustacea, Decapoda, Penaeidae)

Proteases from the midgut gland of the Farfantepenaeus paulensis juveniles were assessed. Enzyme activity was determined using protease substrates and inhibitors. The effect of pH, temperature and calcium on proteolytic activity was assayed. Caseinolytic activity was analysed in substrate-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin, chymotrypsin and leucine aminopeptidase activity was detected. Proteolytic activity was strongly inhibited by the specific trypsin inhibitors. Tosyl-phenylalanine chloromethyl ketone inhibited 59.3% of chymotrypsin activity. The greatest trypsin-like activity occurred at pH 8.0 and 45 degrees C. Chymotrypsin-like activity reached maximal values at alkaline pH (7.2-9.0) and 55 degrees C. CaCl(2) did not increase trypsin-like activity, but rather inhibited it at concentrations of 30 (20%), 50 (30%) and 100 mM (50%). The substrate-SDS-PAGE zymogram revealed eight proteinase bands. Two possibly thermal-resistant (85 degrees C, 30 min) chymotrypsin isoforms were found, which were inhibited by phenyl-methyl-sulphonyl-fluoride. Aminopeptidase activity of enzyme extracts (Arg, Leu, Lys, Phe and Val) and the recommended concentrations of these essential amino acids in penaeid shrimp diets were positively correlated (P < 0.05). Beause protein digestion involves the combined action of different enzymes, adequate knowledge of shrimp digestion and enzyme characteristics is required for the assessment of the digestive potential of different feed sources and development of in vitro digestibility protocols.

The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut

Cathepsin L-like proteinases (CAL) are major digestive proteinases in the beetle Tenebrio molitor. Procathepsin Ls 2 (pCAL2) and 3 (pCAL3) were expressed as recombinant proteins in Escherichia coil, purified and activated under acidic conditions. Immunoblot analyses of different T. molitor larval tissues demonstrated that a polyclonal antibody to pCAL3 recognized pCAL3 and cathepsin L 3 (CAD) only in the anterior two-thirds of midgut tissue and midgut luminal contents of T. molitor larvae. Furthermore, immunocytolocalization data indicated that pCAL3 occurs in secretory vesicles and microvilli in anterior midgut Therefore CAL3, like cathepsin L 2 (CAL2), is a digestive enzyme secreted by T. molitor anterior midgut CAD hydrolyses Z-FR-MCA and Z-RR-MCA (typical cathepsin substrates), whereas CAL2 hydrolyses only Z-FR-MCA. Active site mutants (pCAL2C25S and pCAL3C265) were constructed by replacing the catalytic cysteine with serine to prevent autocatalytic processing. Recombinant pCAL2 and pCAL3 mutants (pCAL2C25S and pCAL3C26S) were prepared, crystallized and their 3D structures determined at 1.85 and 2.1 angstrom, respectively. While the overall structure of these enzymes is similar to other members of the papain superfamily, structural differences in the S2 subsite explain their substrate specificities. The data also supported models for CAL trafficking to lysosomes and to secretory vesicles to be discharged into midgut contents. (C) 2012 Elsevier Ltd. All rights reserved.

Properties and secretory mechanism of Musca domestica digestive chymotrypsin and its relation with Drosophila melanogaster homologs

Musca domestica larvae present two different digestive chymotryptic activities found in the posterior midgut (PMG): one major soluble activity in the lumen and another minor present in cell membrane fractions. Both soluble and membrane-bound chymotryptic activities have different half lives of thermal inactivation (46 degrees C) in the presence and absence of 10 mM Triton X-100, indicating that they are two different molecular species. Purified soluble chymotryptic activity has pH optimum 7.4 and a molecular mass of 28 kDa in SDS-PAGE. It does not cleave short substrates, such as Suc-F-MCA, preferring longer substrates, such as Suc-AAPF-MCA, with a primary specificity (kcat/Km) for Phe rather than Tyr and Leu residues. In-gel activity revealed a unique band against S-AAPF-MCA with the same migration as purified chymotrypsin. One chymotrypsinogen-like sequence (MdChy1) was sequenced, cloned and recombinantly expressed in Escherichia coli (DE3) Star. MdChy1 is expressed in the proximal posterior midgut (PMG1), as seen by RT-PCR. Expression analysis of other chymotrypsin genes revealed genes expressed at the anterior midgut (AMG) and PMG. Western blot of M. domestica midgut tissues using anti-MdChy1 antiserum showed a single band in samples from AMG and PMG, co-migrating with recombinant and purified enzymes. Immunogold labeling corresponding to Mdchy1 was found in small vesicles (thus indicating exocytosis) and in the lumen of AMG and PMG, corroborating the existence of two similar groups of chymotrypsins. Transcriptomes of M. domestica AMG and whole midgut prepared by pyrosequencing disclosed 41 unique sequences of chymotrypsin-like enzymes (19 probably functional), from which MdChy1 is highly expressed. Phylogenetic reconstruction of Drosophila melanogaster and M. domestica chymotrypsin-like sequences revealed that the chymotrypsin genes expanded before the evolutionary separation of Musca and Drosophila. (C) 2012 Elsevier Ltd. All rights reserved.

Gross morphology and topography of the digestive apparatus in rheas (Rhea americana americana)

Rodrigues M.N., Oliveira G.B., Silva R.S.S, Tivane C.T., Albuquerque J.F.G., Miglino M.A. & Oliveira M.F. 2012. [Gross morphology and topography of the digestive apparatus in rheas (Rhea americana americana).] Macroscopia e topografia do aparelho digestorio de emas (Rhea americana americana). Pesquisa Veterinaria Brasileira 32(7):681-686. Departamento de Cirurgia, Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao Paulo, Cidade Universitaria, Av. Prof. Dr. Orlando Marques de Paiva 87, Sao Paulo, SP 05508270, Brazil. E-mail: marcio_medvet@hotmail.com Rheas are birds belonging to the ratites group and, among ostriches and emus, are the largest birds currently alive. In this work we studied the macroscopic aspects of rheas' digestive tract in order to provide important information to a better understanding of these birds' eating habits as well their anatomy. Twenty young animals aging between two and six months from the Centre for Wild Animals Multiplication (Cemas, scientific breeding license form Ibama no.1478912) were used. After dissection it was observed that their tongue was small and presented a rhomboid form, being disposed on the oral cavity floor, and inserted in its base by a frenulum. The esophagus was a rectilinear tube with elastic aspect and longitudinal elastic fibers, without dilation, which gives it an absence of crop. The proventriculus presented a fusiform form and the gastric ventricle showed and slightly oval form when filled, and was internally coated with a thick gastric cuticle. The small intestine was composed of three distinct regions: duodenum, jejunum and ileum. The duodenum had a light gray color and showed a "U" curved shaped. The jejunum was dark green, long and composed of several short loops arranged above each other. The ileum had a gray color and was connected with the jejunum. In ventral line to the rectum and cloaca, the ileum extended cranially, dorsally to the ascending duodenum. The large intestine was composed of two caeca, one right and one left, and colon-rectum and ileum were continuous with the cloaca. The structures of the rhea digestive tract resemble those described in the literature regarding to its shape and topography, even though rhea's caeca are well developed and relatively long.

The effects of supplementation of decapod zoea to Artemia basal diet on the digestive gland histology and the fatty acid composition in common octopus (Octopus vulgaris) paralarvae.

[EN]The present study aimed to evaluate the effect of the supplementation of different crab zoeas to enriched Artemia basal diet for O. vulgaris paralarvae during the first month of life. Paralarvae were fed using: enriched Artemia nauplii alone and Artemia co-fed with either first zoea stages of Grapsus adscensionis or Plagusia depressa. The experiment was carried out over a period of 28 days, in 0.12 m3 tanks with a flow-through rearing system. Growth in dry weightas well as mantle length and width were assessed weekly. Additionally, prey and paralarvae fatty acid composition and digestive gland (DG) histology were evaluated.

Study of the components of quality in SO2-free wines obtained by innovative vinification protocols. Evaluation of the pre-fermentative addition of lysozyme and oenological tannins.

The research performed during the PhD candidature was intended to evaluate the quality of white wines, as a function of the reduction in SO2 use during the first steps of the winemaking process. In order to investigate the mechanism and intensity of interactions occurring between lysozyme and the principal macro-components of musts and wines, a series of experiments on model wine solutions were undertaken, focusing attention on the polyphenols, SO2, oenological tannins, pectines, ethanol, and sugar components. In the second part of this research program, a series of conventional sulphite added vinifications were compared to vinifications in which sulphur dioxide was replaced by lysozyme and consequently define potential winemaking protocols suitable for the production of SO2-free wines. To reach the final goal, the technological performance of two selected yeast strains with a low aptitude to produce SO2 during fermentation were also evaluated. The data obtained suggested that the addition of lysozyme and oenological tannins during the alcoholic fermentation could represent a promising alternative to the use of sulphur dioxide and a reliable starting point for the production of SO2-free wines. The different vinification protocols studied influenced the composition of the volatile profile in wines at the end of the alcoholic fermentation, especially with regards to alcohols and ethyl esters also a consequence of the yeast’s response to the presence or absence of sulphites during fermentation, contributing in different ways to the sensory profiles of wines. In fact, the aminoacids analysis showed that lysozyme can affect the consumption of nitrogen as a function of the yeast strain used in fermentation. During the bottle storage, the evolution of volatile compounds is affected by the presence of SO2 and oenological tannins, confirming their positive role in scaveging oxygen and maintaining the amounts of esters over certain levels, avoiding a decline in the wine’s quality. Even though a natural decrease was found on phenolic profiles due to oxidation effects caused by the presence of oxygen dissolved in the medium during the storage period, the presence of SO2 together with tannins contrasted the decay of phenolic content at the end of the fermentation. Tannins also showed a central role in preserving the polyphenolic profile of wines during the storage period, confirming their antioxidant property, acting as reductants. Our study focused on the fundamental chemistry relevant to the oxidative phenolic spoilage of white wines has demonstrated the suitability of glutathione to inhibit the production of yellow xanthylium cation pigments generated from flavanols and glyoxylic acid at the concentration that it typically exists in wine. The ability of glutathione to bind glyoxylic acid rather than acetaldehyde may enable glutathione to be used as a ‘switch’ for glyoxylic acid-induced polymerisation mechanisms, as opposed to the equivalent acetaldehyde polymerisation, in processes such as microoxidation. Further research is required to assess the ability of glutathione to prevent xanthylium cation production during the in-situ production of glyoxylic acid and in the presence of sulphur dioxide.

Digestive processes in ruminal drinkers characterized by means of the acetaminophen absorption test

The aim of the present study was to measure transit patterns of nutrients and the absorptive ability in ruminal drinkers (RDs) compared with healthy unweaned calves. The acetaminophen (paracetamol) absorption test was used to characterize the oroduodenal transit rate. Clinical examination and the analysis of various blood parameters provided supplementary information on digestive processes. Three unweaned bucket-fed calves (one RD and two healthy controls) each from seven Swiss dairy farms were included in the study. Measurements (tests 1 and 2) were performed twice at an interval of 10 days. Between tests, the feeding technique of the RDs and one control calf per farm was changed to feeding with a nipple instead of by bucket (without nipple). Acetaminophen appearance in the blood was delayed and reduced in RDs compared with the controls. Acid-base metabolism and several haematological and metabolic parameters differed markedly between RDs and healthy controls. The characteristics of the oroduodenal transit rate, absorptive abilities and clinical status in RDs were nearly normalised within 10 days of reconditioning.