In silico Structural, Functional and Phylogenetic Analyses of cellulase from Ruminococcus albus.

BACKGROUND: Cellulose is the primary component of the plant cell wall and an important source of energy for the ruminant and microbial protein synthesis in the rumen. Cell wall content is digested by anaerobic fermentation activity mainly of bacteria belonging to species Fibrobacter succinogenes, Ruminicoccus albus, Ruminococcus flavefaciens, and Butyrivibrio fibrisolvens. Bacteria belonging to the species Ruminococcus albus contain cellulosomes that enable it to adhere to and digest cellulose, and its genome encodes cellulases and hemicellulases. This study aimed to perform an in silico comparative characterization and functional analysis of cellulase from Ruminococcus albus to explore physicochemical properties and to estimate primary, secondary, and tertiary structure using various bio-computational tools. The protein sequences of cellulases belonging to 6 different Ruminococcus albus strains were retrieved using UniProt. In in silico composition of amino acids, basic physicochemical characteristics were analyzed using ProtParam and Protscale. Multiple sequence alignment of retrieved sequences was performed using Clustal Omega and the phylogenetic tree was constructed using Mega X software. Bioinformatics tools are used to better understand and determine the 3D structure of cellulase. The predicted model was refined by ModRefiner. Structure alignment between the best-predicted model and the template is applied to evaluate the similarity between structures. RESULTS: In this study are demonstrated several physicochemical characteristics of the cellulase enzyme. The instability index values indicate that the proteins are highly stable. Proteins are dominated by random coils and alpha helixes. The aliphatic index was higher than 71 providing information that the proteins are highly thermostable. No transmembrane domain was found in the protein, and the enzyme is extracellular and moderately acidic. The best tertiary structure model of the enzyme was obtained by the use of Raptor X, which was refined by ModRefiner. Raptor X suggested the 6Q1I_A as one of the best homologous templates for the predicted 3D protein structure. Ramachandran plot analysis showed that 90.1% of amino acid residues are within the most favored regions. CONCLUSIONS: This study provides for the first time insights about the physicochemical properties, structure, and function of cellulase, from Ruminococcus albus, that will help for detection and identification of such enzyme in vivo or in silico.

Evaluation of the effects of dietary particle fractions on fermentation profile and concentration of microbiota in the rumen of dairy cows fed grass silage-based diets.

The study evaluated the effects of three different theoretical particle lengths (TPL) of grass silage on the distribution of particle fractions of the diet and the resulting effects on fermentation profile and concentrations of protozoa and mixed bacterial mass in the rumen of three lactating Holstein cows fed total mixed rations (45% grass silage, 5% grass hay and 50% concentrate) ad libitum. Decreasing TPL of grass silage (long, medium, short) reduced particles retained on the 19-mm sieve of the Penn State Particle Separator, while particle fractions from 8 mm to 19 mm and smaller than 8 mm were increased. Different TPL did not affect pH and the concentration of volatile fatty acids in the rumen. However, lowering the TPL from long to medium increased significantly the bicarbonate concentration, acetate proportion and protozoal number in the rumen, whereas the proportion of bacterial protein in ruminal digesta and its amino acid concentration were significantly increased by the short TPL. For the current feeding conditions, it can be concluded that increasing the fraction of particles between 8 and 19 mm and probably even the fraction below 8 mm by decreasing TPL of grass silage do not adversely affect rumen conditions and can be beneficial in terms of optimising concentration and activity of ruminal microbiota in high-yielding dairy cows.

Effect of ileal infusion of short-chain fatty acids on pancreatic prandial secretion and gastrointestinal hormones in pigs.

OBJECTIVES: Nutrients passing the ileum induce mechanisms regulating pancreatic secretion, but the effect of short-chain fatty acids (SCFAs) present in the ileum because of either intestinal fermentation or due to the cecoileal reflux is still unclear. This study investigated the effect of ileal SCFAs on pancreatic secretion and plasma levels of peptide YY, cholecystokinin, motilin, and neurotensin. METHODS: The pigs were fitted with pancreatic duct, ileal, and jugular vein catheters, and a duodenal T-shaped cannula. Saline, 85.0 or 170.0 mM acetate, 5.0 or 10.0 mM butyrate, 7.5 or 15.0 mM propionate were infused into the ileum during feeding. RESULTS: The ileal infusions of SCFAs did not affect the pancreatic juice outflow and the lipase output. The protein output was lower when 10.0 mM butyrate or 170.0 mM acetate were infused. The trypsin output decreased for most of the SCFA infusions. The alpha-amylase output decreased for the infusion of 10.0 mM butyrate and tended to decrease for 170.0 mM acetate. The infusions did not change gut hormone level. CONCLUSIONS: Ileal SCFAs might induce an inhibition of pancreatic enzyme secretion under prandial conditions. Ileal SCFAs do not inhibit pancreatic secretion by a hormonal pathway involving the release of peptide YY, motilin, neurotensin, or cholecystokinin.

Effects of betaine and condensed molasses solubles on nitrogen balance and nutrient digestibility in piglets fed diets deficient in methionine and low in compatible osmolytes.

A balance experiment was carried out to investigate the effects of betaine monohydrate (BET) or betaine derived from condensed molasses solubles (CMS) as a substitute for methionine and choline on nitrogen (N) balance and total tract nutrient digestibility in weaned piglets. The experiment included four treatments with 32 barrows with an average initial body weight (BW) of 13.5 kg. The supplementation of DL-methionine and choline (positive control = PC) to the basal diet, which was deficient in methionine and low in compatible osmolytes in the form of betaine or its precursor choline (negative control = NC) resulted in a significant increase in N retention of 0.8 g/d. The substitution of DL-methionine and choline with BET or CMS did not affect N retention compared to the PC and the NC treatment either. Feeding the PC diet increased the digestibilities of organic matter, NDF, ADF, NFE, crude ash, Ca, P, methionine, tryptophan and cystine by 1.9%, 7.3%, 9.7%, 1.1%, 6.3%, 13.9%, 7.7%, 15.9%, 4.3% and 2.8%, respectively, and tended (p < 0.20) to increase the digestibilities of most other amino acids by 1.6-3.4%. Digestibility of CP, EE (HCl), Mg and Na was 3.1% (p=0.09), 5.1% (p=0.09), 5.1% (p= 0.06) and 3.3% (p= 0.17) higher, respectively, when compared to the NC treatment. BET and CMS supplementation increased most nutrient digestibilities in the same magnitude as for the PC treatment. In summary, the supplementation of betaine, originating from different sources, to a diet with low contents of compatible osmolytes increased in particular the fermentation of fibre and enhanced mineral absorption. The supplementation of the NC with DL-methionine was more efficient in improving N retention than the replacement of DL-methionine by betaine originating from BET or CMS.

Effects of fibre concentration of diets consisting of hay and slowly degradable concentrate on ruminal fermentation and digesta particle size in mid-lactation dairy cows.

Four multiparous ruminally cannulated Holstein cows (mean bodyweight [BW] 615 kg) in mid-lactation (103 days in milk and 32 kg milk x d(-1) at start of the experiment) were used in an one-factorial experiment to evaluate the effects of fibre level (19, 24, 28, 32 and 39% physically effective NDF [peNDF] in dry matter [DM]) in diets consisting of hay and slowly degradable concentrate on rumen fermentation patterns and digesta particle size, under a constant intake level (146 g DM x kg(-0.75). The different fibre concentrations in the diet were achieved by adjusting the hay to concentrate ratio. The above-mentioned levels of peNDF corresponded to 70, 60, 50, 40 and 25% concentrate in diet DM, respectively, and followed the lactation curve of the cows. The ruminal pH was positively and linearly correlated to the percentage of fibre (peNDF, NDF or CF) in ration DM with R2 of 0.76-0.88 (p < 0.001) for solid digesta (particle-associated rumen fluid, PARL), and R2 of 0.26-0.29 (p < 0.05) for fluid digesta (free rumen liquid, FRL). The lowest fibre level in the diet (19% peNDF) or the highest level of concentrate (70% on DM basis) caused pH values lower than 6.0 at almost all sampling times only in PARL but not in FRL, and significantly increased the proportion of large particles in rumen digesta, which in turn was reflected by a depression of fibre digestibility. A level of 24% peNDF or 60% concentrate in the diet maintained the ruminal pH higher than 6.0 and 5.8 in FRL and PARL, respectively. Therefore, the inclusion of more than 60% slowly degradable concentrate in dairy cows diets fed approximately 18 kg DM x d(-1) is discouraged. Based on the response of ruminal solid digesta to dietary fibre, it can be concluded that the recommendations of feeding a structural value > or =1 per kg DM (De Brabander et al. 1999) underestimated, and 400 g CF per 100 kg BW (Hoffmann 1990) overestimated the evaluation of structural effectiveness of the present diet.

Effects of physically effective fibre concentration of diets consisting of hay and slowly degradable concentrate on chewing activity in mid lactation dairy cows under constant intake level.

Four ruminally cannulated Holstein cows (BW 615 kg) in mid lactation were used to evaluate the effects of fibre level (39, 32, 28, 24 and 19% physically effective NDF [peNDF] in DM) in diets consisting of hay and slowly degradable concentrate on chewing activity, under a constant intake level (18 kg DM x d(-1)). The different dietary fibre concentrations were achieved by adjusting the hay to concentrate ratio. The above-mentioned levels of peNDF corresponded to 25, 40, 50, 60 and 70% concentrate respectively. The diets with decreasing share of concentrate were offered in sequence according to the progressive lactation of the cows. A decrease of the peNDF from 39-28% reduced total chewing and rumination time, total number of chews and number of boluses per day as well as chewing time spent per unit of DM. No significant change in chewing behaviour occurred between peNDF level of 28 and 24%. This peNDF level (i.e., 24%) might be considered as a tolerable fibre level. A further reduction of peNDF to 19% led to a significant reduction of chewing activity. This level can be considered as critical for chewing activity and presumably for the rumen health. The chewing parameters correlated linearly to fibre and non-fibre carbohydrates with R2 of 23-51%.

Serum IgA-promoting effects induced by feed loads containing isolated deoxynivalenol (DON) in growing piglets.

Deoxynivalenol (DON), a Fusarium toxin belonging to the trichothecene group, has been reported to produce a variety of adverse health effects in farm animals, such as inhibition of protein synthesis, reduction of feed intake, and alteration of the immune system. In pigs, the effects of increasing levels of chemically pure DON in a semisynthetic diet on performance, health, and serum immunglobulin A (IgA) levels were examined. A diet, without grain components and trichothecene free (8 main trichothecenes), with doses of 0, 300, 600, and 1200 microg pure DON/kg was fed to 34 female pigs for a period of 8 wk after weaning under standardized conditions. Body weight gain and biochemical and hematological values in the blood and serum, including concentrations of IgA, blood glucose, cortisol, and insulinlike growth factor 1 (IGF-1), were determined. Increasing levels of DON in the feed induced a significant depression of glucose levels. Cortisol and IGF-1 levels were not significantly affected but differed between groups at the end of the experiment. A significant increase of IgA concentration in the serum even at a dosage level of 600 microg DON/kg feed was observed. This is the first report demonstrating in vivo that limited dosages of DON are able to stimulate IgA levels in the serum of growing piglets.