BACKGROUND: Condensed tannin (CT) fractions of different molecular weights (MWs) may affect rumen microbial metabolism by altering bacterial diversity. In this study the effects of unfractionated CTs (F0) and five CT fractions (F1-F5) of different MWs (F1, 1265.8 Da; F2, 1028.6 Da; F3, 652.2 Da; F4, 562.2 Da; F5, 469.6 Da) from Leucaena leucocephala hybrid-Rendang (LLR) on the structure and diversity of the rumen bacterial community were investigated in vitro.
RESULTS: Real-time polymerase chain reaction assay showed that the total bacterial population was not significantly (P > 0.05) different among the dietary treatments. Inclusion of higher-MW CT fractions F1 and F2 significantly (P
Inclusion of phytase in animal feedstuff is a common practice to enhance nutrients availability. However, little is known about the effects of phytase supplementation on the microbial ecology of the gastrointestinal tract. In this study, freeze-dried Mitsuokella jalaludinii phytase (MJ) was evaluated in a feeding trial with broilers fed a low available phosphorus (aP) diet. A total of 180 male broiler chicks (day-old Cobb) were assigned into three dietary treatments: Control fed with 0.4% (w/w) of available phosphorus (aP); Group T1 fed low aP [0.2% (w/w)] supplemented with MJ; and T2 fed low aP and deactivated MJ. The source of readily available P, dicalcium phosphate (DCP), was removed from low aP diet, whereby additional limestone was provided to replace the amount of Ca normally found in DCP. For each treatment, 4 replicate pens were used, where each pen consisted of 15 animals. The animals' energy intake and caecal bacterial community were monitored weekly for up to 3 weeks. The apparent metabolizable energy (AME) and apparent digestibility of dry matter (ADDM) of broilers fed with different diets were determined. In addition, the caecal microbial diversities of broilers were assessed using high-throughput next-generation sequencing targeting the V3-V4 region of bacterial 16S rRNA. The results showed that broilers fed with T1 diet have better feed conversion ratio (FCR) when compared to the Control (p
AIMS: To evaluate the effects of condensed tannins (CTs) fractions of differing molecular weights (MWs) from a Leucaena leucocephala hybrid-Rendang on the rumen protozoal community in vitro.
METHODS AND RESULTS: The effects of unfractionated CTs (F0) and CT fractions of different MWs (F1 > F2 > F3 > F4 > F5) on protozoal population and community were evaluated in vitro using rumen microbes and ground guinea grass as the substrate. Higher-MW CT fractions F1 and F2 significantly (P
Molecular weights (MWs) and their chemical structures are the primary factors determining the influence of condensed tannins (CTs) on animal nutrition and methane (CH4 ) production in ruminants. In this study the MWs of five CT fractions from Leucaena leucocephala hybrid-Rendang (LLR) were determined and the CT fractions were investigated for their effects on CH4 production and rumen fermentation.
Condensed tannins (CTs) form insoluble complexes with proteins and are able to protect them from degradation, which could lead to rumen bypass proteins. Depending on their degrees of polymerization (DP) and molecular weights, CT fractions vary in their capability to bind proteins. In this study, purified condensed tannins (CTs) from a Leucaena leucocephala hybrid were fractionated into five different molecular weight fractions. The structures of the CT fractions were investigated using 13C-NMR. The DP of the CT fractions were determined using a modified vanillin assay and their molecular weights were determined using Q-TOF LC-MS. The protein-binding affinities of the respective CT fractions were determined using a protein precipitation assay. The DP of the five CT fractions (fractions F1-F5) measured by the vanillin assay in acetic acid ranged from 4.86 to 1.56. The 13C-NMR results showed that the CT fractions possessed monomer unit structural heterogeneity. The number-average molecular weights (Mn) of the different fractions were 1265.8, 1028.6, 652.2, 562.2, and 469.6 for fractions F1, F2, F3, F4, and F5, respectively. The b values representing the CT quantities needed to bind half of the maximum precipitable bovine serum albumin increased with decreasing molecular weight--from fraction F1 to fraction F5 with values of 0.216, 0.295, 0.359, 0.425, and 0.460, respectively. This indicated that higher molecular weight fractions of CTs from L. leucocephala have higher protein-binding affinities than those with lower molecular weights.
The objective of this study was to isolate, identify, and characterize some lactic acid bacterial strains from human milk, infant feces, and fermented grapes and dates, as potential probiotics with antimicrobial activity against some human pathogenic strains. One hundred and forty bacterial strains were isolated and, after initial identification and a preliminary screening for acid and bile tolerance, nine of the best isolates were selected and further identified using 16 S rRNA gene sequences. The nine selected isolates were then characterized in vitro for their probiotic characteristics and their antimicrobial activities against some human pathogens. Results showed that all nine isolates belonged to the genus Lactobacillus. They were able to tolerate pH 3 for 3 h, 0.3% bile salts for 4 h, and 1.9 mg/mL pancreatic enzymes for 3 h. They exhibited good ability to attach to intestinal epithelial cells and were not resistant to the tested antibiotics. They also showed good antimicrobial activities against the tested pathogenic strains of humans, and most of them exhibited stronger antimicrobial activity than the reference strain L. casei Shirota. Thus, the nine Lactobacillus strains could be considered as potential antimicrobial probiotic strains against human pathogens and should be further studied for their human health benefits.
In this study, a Salmonella Typhimurium lytic bacteriophage, Φ st1, which was isolated from chicken faecal material, was evaluated as a candidate for biocontrol of Salmonella in chickens. The morphology of Φ st1 showed strong resemblance to members of the Siphoviridae family. Φ st1 was observed to be a DNA phage with an estimated genome size of 121 kbp. It was found to be able to infect S. Typhimurium and S. Hadar, with a stronger lytic activity against the former. Subsequent characterisation of Φ st1 against S. Typhimurium showed that Φ st1 has a latent period of 40 min with an average burst size of 22 particles per infective centre. Approximately 86.1% of the phage adsorbed to the host cells within the initial 5 min of infection. At the optimum multiplicity of infection (MOI) (0.1), the highest reduction rate of S. Typhimurium (6.6 log₁₀ CFU/ml) and increment in phage titre (3.8 log₁₀ PFU/ml) was observed. Φ st1 produced adsorption rates of 88.4-92.2% at pH7-9 and demonstrated the highest bacteria reduction (6.6 log₁₀ CFU/ml) at pH9. Φ st1 also showed an insignificant different (P>0.05) reduction rate of host cells at 37 °C (6.4 log₁₀ CFU/ml) and 42 °C (6.0 log₁₀ CFU/ml). The in vivo study using Φ st1 showed that intracloacal inoculation of ~10¹² PFU/ml of the phage in the chickens challenged with ~10¹⁰ CFU/ml of S. Typhimurium was able to reduce (P<0.05) the S. Typhimurium more rapidly than the untreated group. The Salmonella count reduced to 2.9 log₁₀ CFU/ml within 6h of post-challenge and S. Typhimurium was not detected at and after 24h of post-challenge. Reduction of Salmonella count in visceral organs was also observed at 6h post-challenge. Approximately 1.6 log₁₀ FU/ml Φ st1 was found to persist in the caecal wall of the chicks at 72 h of post-challenge. The present study indicated that Φ st1 may serve as a potential biocontrol agent to reduce the Salmonella count in caecal content of chickens.
The effect of Leucaena leucocephala hybrid-Bahru (LLB), which contains a high concentration of condensed tannins, on cellulolytic rumen fungal population in goats was investigated using real-time PCR. The fungal population in goats fed LLB was inhibited during the first 10 days of feeding, but after 15 days of feeding, there was a tremendous increase of fungal population (157.0 μg/ml), which was about fourfold more than that in control goats (39.7 μg/ml). However, after this period, the fungal population decreased continuously, and at 30 days of feeding, the fungal population (50.6 μg/ml) was not significantly different from that in control goats (55.4 μg/ml).
Bacteriophage EC1-UPM is an N4-like bacteriophage which specifically infects Escherichia coli O78:K80, an avian pathogenic strain that causes colibacillosis in poultry. The complete genome sequence of bacteriophage EC1-UPM was analysed and compared with other closely related N4-like phage groups to assess their genetic similarities and differences.
Lovastatin, a natural byproduct of some fungi, is able to inhibit HMG-CoA (3-hydroxy-3 methyl glutaryl CoA) reductase. This is a key enzyme involved in isoprenoid synthesis and essential for cell membrane formation in methanogenic Archaea. In this paper, experiments were designed to test the hypothesis that lovastatin secreted by Aspergillus terreus in fermented rice straw extracts (FRSE) can inhibit growth and CH4 production in Methanobrevibacter smithii (a test methanogen). By HPLC analysis, 75% of the total lovastatin in FRSE was in the active hydroxyacid form, and in vitro studies confirmed that this had a stronger effect in reducing both growth and CH4 production in M. smithii compared to commercial lovastatin. Transmission electron micrographs revealed distorted morphological divisions of lovastatin- and FRSE-treated M. smithii cells, supporting its role in blocking normal cell membrane synthesis. Real-time PCR confirmed that both commercial lovastatin and FRSE increased (P < 0.01) the expression of HMG-CoA reductase gene (hmg). In addition, expressions of other gene transcripts in M. smithii. with a key involvement in methanogenesis were also affected. Experimental confirmation that CH4 production is inhibited by lovastatin in A. terreus-fermented rice straw paves the way for its evaluation as a feed additive for mitigating CH4 production in ruminants.
Four repetitive element sequence-based polymerase chain reaction (rep-PCR) methods, namely repetitive extragenic palindromic PCR (REP-PCR), enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), polytrinucleotide (GTG)₅ -PCR and BOX-PCR, were evaluated for the molecular differentiation of 12 probiotic Lactobacillus strains previously isolated from the gastrointestinal tract of chickens and used as a multistrain probiotic. This study represents the first analysis of the comparative efficacy of these four rep-PCR methods and their combination (composite rep-PCR) in the molecular typing of Lactobacillus strains based on a discriminatory index (D).
Depending on their source, concentration, chemical structure, and molecular weight, condensed tannins (CTs) form insoluble complexes with protein, which could lead to ruminal bypass protein, benefiting animal production. In this study, CTs from Leuceana leucocephala hybrid were fractionated into five fractions by a size exclusion chromatography procedure. The molecular weights of the CT fractions were determined using Q-TOF LC-MS, and the protein-binding affinities of the respective CT fractions were determined using a protein precipitation assay with bovine serum albumin (BSA) as the standard protein. The calculated number-average molecular weights (M(n)) were 1348.6, 857.1, 730.1, 726.0, and 497.1, and b values (the b value represents the CT quantity that is needed to bind half of the maximum precipitable BSA) of the different molecular weight fractions were 0.381, 0.510, 0.580, 0.636, and 0.780 for fractions 1, 2, 3, 4, and 5, respectively. The results indicated that, in general, CTs of higher molecular weight fractions have stronger protein-binding affinity than those of lower molecular weights. However, the number of hydroxyl units within the structure of CT polymers also affects the protein-binding affinity.
Molecular diversity of rumen archaeal populations from bovine rumen fluid incubated with or without condensed tannins was investigated using 16S rRNA gene libraries. The predominant order of rumen archaea in the 16S rRNA gene libraries of the control and condensed tannins treatment was found to belong to a novel group of rumen archaea that is distantly related to the order Thermoplasmatales, with 59.5% (15 phylotypes) and 81.43% (21 phylotypes) of the total clones from the control and treatment clone libraries, respectively. The 16S rRNA gene library of the control was found to have higher proportions of methanogens from the orders Methanomicrobiales (32%) and Methanobacteriales (8.5%) as compared to those found in the condensed tannins treatment clone library in both orders (16.88% and 1.68% respectively). The phylotype distributed in the order Methanosarcinales was only found in the control clone library. The study indicated that condensed tannins could alter the diversity of bovine rumen methanogens.
The efficacy of bacteriophage EC1, a lytic bacteriophage, against Escherichia coli O78:K80, which causes colibacillosis in poultry, was determined in the present study. A total of 480 one-day-old birds were randomly assigned to 4 treatments groups, each with 4 pens of 30 birds. Birds from the control groups (groups I and II) received PBS (pH 7.4) or 10(10) pfu of bacteriophage EC1, respectively. Group III consisted of birds challenged with 10(8) cfu of E. coli O78:K80 and treated with 10(10) pfu of bacteriophage EC1 at 2 h postinfection, whereas birds from group IV were challenged with 10(8) cfu of E. coli O78:K80 only. All the materials were introduced into the birds by intratracheal inoculation. Based on the results of the present study, the infection was found to be less severe in the treated E. coli-challenged group. Mean total viable cell counts of E. coli identified on eosin methylene blue agar (designated EMB + E. coli) in the lungs were significantly lower in treated, E. coli-challenged birds than in untreated, E. coli-challenged birds on d 1 and 2 postinfection. The EMB + E. coli isolation frequency was also lower in treated birds; no E. coli was detectable in blood samples on any sampling day, and E. coli were isolated only in the liver, heart, and spleen of treated chickens at a ratio of 2/6, 1/6, and 3/6, respectively, at d 1 postinfection. The BW of birds from the E. coli-challenged group treated with bacteriophage EC1 were not significantly different from those of birds from both control groups but were 15.4% higher than those of the untreated, E. coli-challenged group on d 21 postinfection. The total mortality rate of birds during the 3-wk experimental period decreased from 83.3% in the untreated, E. coli-challenged birds (group IV) to 13.3% in birds treated with bacteriophage EC1 (group III). These results suggest that bacteriophage EC1 is effective in vivo and could be used to treat colibacillosis in chickens.
Molecular diversity of protists from bovine rumen fluid incubated with condensed tannins of Leucaena leucocephala hybrid-Rendang at 20 mg/500 mg dry matter (treatment) or without condensed tannins (control) was investigated using 18S rRNA gene library. Clones from the control library were distributed within nine genera, but clones from the condensed tannin treatment clone library were related to only six genera. Diversity estimators such as abundance-based coverage estimation and Chao1 showed significant differences between the two libraries, although no differences were found based on Shannon-Weaver index and Libshuff.
A 16-wk feeding experiment was conducted to investigate the effects of a prebiotic, isomaltooligosaccharide (IMO), a probiotic, PrimaLac®, and their combination as a synbiotic on the chemical compositions of egg yolks and the egg quality of laying hens. One hundred and sixty 16-wk-old Hisex Brown pullets were randomly assigned to 4 dietary treatments: (i) basal diet (control), (ii) basal diet + 1% IMO (PRE), (iii) basal diet + 0.1% PrimaLac® (PRO), and (iv) basal diet + 1% IMO + 0.1% PrimaLac® (SYN). PRE, PRO, or SYN supplementation not only significantly (P < 0.05) decreased the egg yolk cholesterol (24- and 28-wk-old) and total saturated fatty acids (SFA; 28-, 32-, and 36-wk-old), but also significantly (P < 0.05) increased total unsaturated fatty acids (UFA; 28-, 32-, and 36-wk-old), total omega 6 and polyunsaturated fatty acids (PUFA), including linoleic and alpha-linolenic acid levels in the eggs (28-wk-old). However, the total lipids, carotenoids, and tocopherols in the egg yolks were similar among all dietary treatments in the 24-, 28-, 32-, and 36-wk-old hens. Egg quality (Haugh unit, relative weights of the albumen and yolk, specific gravity, shell thickness, and yolk color) was not affected by PRE, PRO, or SYN supplementation. The results indicate that supplementations with IMO and PrimaLac® alone or in combination as a synbiotic might be useful for improving the cholesterol content and modifying the fatty acid compositions of egg yolk without affecting the quality of eggs from laying hens between 24 and 36 wk of age.
Voluntary food intake, digestibility and water turnover were determined in adult Malaysian lesser mouse-deer (Tragulus javanicus) given unlimited access to lundai foliage (Sapium baccatum). Daily dry matter (DM) intake was 42.4 g/kg metabolic live mass (M0.73) or 3.7% M. Digestible energy intake was 853 kJ/day (571 kJ metabolisable energy per M0.73), calculated to be used with 79% efficiency. Apparent digestibility (%) of organic matter was 83.8, crude fibre 63.7, acid detergent fibre 60.5, neutral detergent fibre 72.1 and crude protein 65.0. Urinary excretion of the purine derivative, allantoin, was 0.05 mg/g digestible DM intake suggesting rumen microbial yield efficiency may be lower than in other ruminant species. Total water intake was 182 ml/M0.82. The body-water content of the fed mouse-deer, from tritiated water dilution, was 77% M, consistent with a very lean carcass. Turnover of body water was 17% per day. The mouse-deer produced relatively dry, well-defined faecal pellets.
Chicken gut microbiota has paramount roles in host performance, health and immunity. Understanding the topological difference in gut microbial community composition is crucial to provide knowledge on the functions of each members of microbiota to the physiological maintenance of the host. The gut microbiota profiling of the chicken was commonly performed previously using culture-dependent and early culture-independent methods which had limited coverage and accuracy. Advances in technology based on next-generation sequencing (NGS), offers unparalleled coverage and depth in determining microbial gut dynamics. Thus, the aim of this study was to investigate the ileal and caecal microbiota development as chicken aged, which is important for future effective gut modulation.
A total of 46 Lactobacillus isolates obtained from chicken intestine were assessed on their ability to adhere to the chicken ileal epithelial cell (IEC) in vitro. Twelve out of the 46 isolates showed moderate to good ability to adhere to the IEC. Temperature (between 4 degrees C and 42 degrees C) did not affect attachment. Incubation (contact) time of 30 min was found to be insufficient for the attachment of bacteria to the IEC, but contact time beyond 1 h did not increase this ability. The pH values (4-7) of the suspending buffer did not have any significant effect on the attachment of bacteria to the IEC, but at pH 8 it was reduced significantly (P < 0.05).
A rotatable central composite design (CCD) was used to study the effect of cryoprotectants (skim milk, sucrose and lactose) on the survival rate of a probiotic Lactobacillus strain, L. reuteri C10, for poultry, during freeze-drying and storage. Using response surface methodology, a quadratic polynomial equation was obtained for response value by multiple regression analyses: Y = 8.59546-0.01038 X(1)-0.09382 X(2)-0.07771 X(3)-0.054861 X(1)(2)-0.04603 X(3)(2)-0.10938 X(1)X(2). Based on the model predicted, sucrose exerted the strongest effect on the survival rate. At various combinations of cryoprotectants, the viability loss of the cells after freeze-drying was reduced from 1.65 log colony forming units (CFU)/mL to 0.26-0.66 log CFU/mL. The estimated optimum combination for enhancing the survival rate of L. reuteri C10 was 19.5% skim milk, 1% sucrose and 9% lactose. Verification experiments confirmed the validity of the predicted model. The storage life of freeze-dried L. reuteri C10 was markedly improved when cryoprotectants were used. At optimum combination of the cryoprotectants, the survival rates of freeze-dried L. reuteri C10 stored at 4°C and 30°C for 6 months were 96.4% and 73.8%, respectively. Total viability loss of cells which were not protected by cryoprotectants occurred after 12 and 8 weeks of storage at 4°C and 30°C, respectively.