RESUMEN
This study aimed to evaluate in vitro fermentation characteristics, microbial protein synthesis and microbial community composition when replacing maize silage (MS) with red clover silage (RCS) in total mixed rations (TMR) of dairy cows. Treatments included TMR containing forage (MS and RCS) and concentrates (0.75:0.25) with targeted proportions (dry matter (DM) basis) of RCS in TMR of 0.15 (RCS15 ), 0.30 (RCS30 ), 0.45 (RCS45 ), and 0.60 (RCS60 ), in substitution of MS. Samples of the TMR were incubated using the in vitro Ankom RF technique with a mixture of rumen fluid and buffer solution (1:2 v/v) for 8 and 24 hr. Gas production and total short-chain fatty acids concentration did not differ between diets, whereas ammonia-nitrogen concentration increased with increasing level of RCS. Acetate proportion was not affected by RCS level, but propionate showed a linear increase with increasing level of RCS at the expenses of butyrate. Branched fatty acids proportions linearly declined, reflecting a reduced deamination of true protein. Gene copy numbers of protozoa linearly decreased with increasing RCS levels, while total numbers of bacteria and methanogens were not affected by diet. The amylolytic bacteria Ruminobacter amylophillus and Prevotella bryantii showed evidence to increase with higher RCS levels after 8 hr and 24 hr, respectively, whereas no effects of diet where observed for the fibrolytic bacteria Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes. Concentrations of purine bases, and total N production in liquid-associated microbes declined with increasing RCS levels, suggesting a negative impact of this feed on microbial growth. The findings of this study suggest that in general, microbial protein synthesis might be impaired by the substitution of MS by RCS, therefore caution should be taken when formulating diets for dairy cows using high levels of RCS as ingredient.
Asunto(s)
Bacterias/metabolismo , Rumen/fisiología , Ensilaje/análisis , Trifolium/química , Zea mays/química , Animales , Digestión , Fermentación , Biosíntesis de Proteínas/fisiologíaRESUMEN
Compared to grass silage (GS)-, corn silage (CS)-based diets appear to increase the efficiency of microbial protein synthesis (EMPS) in the rumen. Opposite results for the EMPS obtained in vitro have raised the question of whether an inadequate supply of amino N for microbes might explain the low EMPS. We examined the effects of supplementation with different N sources in CS on the EMPS and microbial populations in vitro. GS and CS were used as substrates for in vitro incubation. CS was non-supplemented or supplemented with urea, mixed amino acids (AA), peptone, or protein to adjust the N content to that of GS. Degradation of organic matter (OM) and crude protein (CP) revealed a positive effect of all N supplements, except protein. Additionally, N supplementation increased fiber degradation of CS. Peptone primarily stimulated hemicellulolytic activity and urea stimulated cellulolytic activity. The EMPS of CS was improved by all N supplements, with peptone and urea exhibiting the highest increase (57% and 54%, respectively), followed by AA mix (40%) and protein (11%) compared to that of CS alone (111â¯g microbial CP kg-1 fermented OM). However, the level of EMPS detected with GS (200â¯g microbial CP kg-1 fermented OM) was not achieved. Protozoal 18S rRNA gene copy numbers were negatively correlated with the EMPS, whereas no correlation was found between total bacteria and the EMPS. A stimulating effect of urea, AA mix, and peptone was detected for Ruminococcus albus and Prevotella bryantii, whereas Fibrobacter succinogenes was inhibited by N supplementation. This indicated that neither the amount of available N nor the N source was the only limiting factor in the low EMPS values of CS in vitro. Information is also provided on the stimulating effects of different N sources on several microbial species in mixed rumen culture.
Asunto(s)
Alimentación Animal , Biota , Suplementos Dietéticos , Compuestos de Nitrógeno/metabolismo , Rumen/microbiología , Ensilaje , Animales , Bovinos , FermentaciónRESUMEN
Here, we examined diurnal changes in the ruminal microbial community and fermentation characteristics of dairy cows fed total mixed rations containing either corn silage (CS) or grass silage (GS) as forage. The rations, which consisted of 52% concentrate and 48% GS or CS, were offered for ad libitum intake over 20 days to three ruminal-fistulated lactating Jersey cows during three consecutive feeding periods. Feed intake, ruminal pH, concentrations of short chain fatty acids and ammonia in rumen liquid, as well as abundance change in the microbial populations in liquid and solid fractions, were monitored in 4-h intervals on days 18 and 20. The abundance of total bacteria and Fibrobacter succinogenes increased in solids in cows fed CS instead of GS, and that of protozoa increased in both solid and liquid fractions. Feeding GS favored numbers of F. succinogenes and Selenomonas ruminantium in the liquid fraction as well as the numbers of Ruminobacter amylophilus, Prevotella bryantii and ruminococci in both fractions. Minor effects of silage were detected on populations of methanogens. Despite quantitative changes in the composition of the microbial community, fermentation characteristics were less affected by forage source. These results suggest a functional adaptability of the ruminal microbiota to total mixed rations containing either GS or CS as the source of forage. Diurnal changes in microbial populations were primarily affected by feed intake and differed between species and fractions, with fewer temporal fluctuations evident in the solid than in the liquid fraction. Interactions between forage source and sampling time were of minor importance to most of the microbial species examined. Thus, diurnal changes of microbial populations and fermentative activity were less affected by the two silages.
Asunto(s)
Fenómenos Fisiológicos Nutricionales de los Animales , Ritmo Circadiano/fisiología , Microbioma Gastrointestinal/fisiología , Rumen/microbiología , Ensilaje , Amoníaco/metabolismo , Alimentación Animal/análisis , Animales , Bovinos , Ácidos Grasos/metabolismo , Femenino , Fermentación , Fibrobacter/metabolismo , Fístula Gástrica , Concentración de Iones de Hidrógeno , Lactancia/fisiología , Poaceae/química , Poaceae/metabolismo , Prevotella/metabolismo , Ruminococcus/metabolismo , Selenomonas/metabolismo , Ensilaje/análisis , Zea mays/química , Zea mays/metabolismoRESUMEN
The chicken gastrointestinal tract (GIT) harbours a complex microbial community, involved in several physiological processes such as host immunomodulation and feed digestion. For the first time, the present study analysed dietary effects on the protein inventory of the microbiome in crop and ceca of broilers. We performed quantitative label-free metaproteomics by using 1-D-gel electrophoresis coupled with LC-MS/MS to identify the structural and functional changes triggered by diets supplied with varying amount of mineral phosphorous (P) and microbial phytase (MP). Phylogenetic assessment based on label-free quantification (LFQ) values of the proteins identified Lactobacillaceae as the major family in the crop section regardless of the diet, whereas proteins belonging to the family Veillonellaceae increased with the P supplementation. Within the ceca section, proteins of Bacteroidaceae were more abundant in the P-supplied diets, whereas proteins of Eubacteriaceae decreased with the P-addition. Proteins of the Ruminococcaceae increased with the amount of MP while proteins of Lactobacillaceae were more abundant in the MP-lacking diets. Classification of the identified proteins indicated a thriving microbial community in the case of P and MP supplementation, and stressed microbial community when no P and MP were supplied. Data are available via ProteomeXchange with identifier PXD003805.
Asunto(s)
Pollos/microbiología , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/microbiología , Microbiota/efectos de los fármacos , Fósforo/farmacología , Alimentación Animal/análisis , Animales , Proteínas Bacterianas/metabolismo , Ciego/microbiología , Pollos/metabolismo , Dieta , Tracto Gastrointestinal/metabolismo , Fósforo/análisis , Proteínas de Plantas/química , ProteómicaRESUMEN
This study examined ruminal microbial community composition alterations during initial adaption to and following incubation in a rumen simulation system (Rusitec) using grass or corn silage as substrates. Samples were collected from fermenter liquids at 0, 2, 4, 12, 24, and 48 h and from feed residues at 0, 24, and 48 h after initiation of incubation (period 1) and on day 13 (period 2). Microbial DNA was extracted and real-time qPCR was used to quantify differences in the abundance of protozoa, methanogens, total bacteria, Fibrobacter succinogenes, Ruminococcus albus, Ruminobacter amylophilus, Prevotella bryantii, Selenomonas ruminantium, and Clostridium aminophilum. We found that forage source and sampling time significantly influenced the ruminal microbial community. The gene copy numbers of most microbial species (except C. aminophilum) decreased in period 1; however, adaption continued through period 2 for several species. The addition of fresh substrate in period 2 led to increasing copy numbers of all microbial species during the first 2-4 h in the fermenter liquid except protozoa, which showed a postprandial decrease. Corn silage enhanced the growth of R. amylophilus and F. succinogenes, and grass silage enhanced R. albus, P. bryantii, and C. aminophilum. No effect of forage source was detected on total bacteria, protozoa, S. ruminantium, or methanogens or on total gas production, although grass silage enhanced methane production. This study showed that the Rusitec provides a stable system after an adaption phase that should last longer than 48 h, and that the forage source influenced several microbial species.
Asunto(s)
Adaptación Fisiológica , Fermentación , Rumen/microbiología , Adaptación Fisiológica/genética , Alimentación Animal/análisis , Animales , Bovinos , Industria Lechera , Femenino , Dosificación de Gen , Poaceae/química , Zea mays/químicaRESUMEN
BACKGROUND: Diet influences the porcine intestinal microbial ecosystem. Barrows were fitted with ileal T-cannulas to compare short-term effects of eight different wheat or barley genotypes and period-to-period effects on seven bacterial groups in ileal digesta and faeces by qPCR. RESULTS: Within genotypes of wheat and barley, there was no difference (P > 0.05) in contents of analysed NSP, yet cereal types differed (P < 0.001) except for soluble arabinoxylans. Genotypes showed no effect on bacterial gene copy numbers. In ileal digesta of barley- compared to wheat-fed pigs, log10 copy numbers were lower (P < 0.05) for total eubacteria (9.6-9.8), Bacteroides-Prevotella-Porphyromonas (6.5-6.8), Clostridium cluster IV (6.7-6.9), and Roseburia spp. (6.6-7.2), while higher copy numbers were found for Lactobacillus spp. (9.4-8.8). Enterobacteriaceae (7.0-7.8) and Bifidobacterium spp. (7.0-7.7) were lower (P < 0.001) in faeces of barley compared to wheat-fed pigs. Ileal eubacteria, Clostridium cluster IV and Roseburia spp. linearly increased from period 1 to 8 for both cereals (P < 0.05). CONCLUSION: Wheat and barley differently influence microbial composition particularly in the small intestine, with barley increasing the Lactobacillus spp.:Enterobacteriaceae ratio, underlining its potential to beneficially manipulate the intestinal microbial ecosystem.
Asunto(s)
Bacterias/metabolismo , Hordeum/genética , Porcinos/fisiología , Triticum/genética , Alimentación Animal/análisis , Animales , Bacterias/genética , Dieta/veterinaria , Heces/microbiología , Contenido Digestivo , Genotipo , Íleon/microbiología , MasculinoRESUMEN
Molecular fingerprinting and sequencing based techniques have been widely used to characterize microbial communities. Terminal restriction fragment length polymorphism (T-RFLP) and 454-pyrosequencing were used to determine the microorganisms present in the different sections of the chicken gastrointestinal tract (GIT) (crop, jejunum, ileum and caeca). Broilers fed with diets differing in phosphorous (P) and calcium (Ca) as well as in phytase levels were used to study the microbiota of the upper and lower part of the GIT. A database with terminal restriction fragments (T-RF) of the most important organism present in the different gastrointestinal sections was constructed. The analysis revealed a distinct microbial assemblage on each section. Regardless of the diet, crop, jejunum and ileum were mainly colonized by Lactobacillaceae, and caeca were the most diverse site. The correlation between Lactobacillus crispatus and L. reuteri was positive in the crop, but negative in the jejunum. In crop samples, higher P and Ca levels led to a shift in the abundance of L. reuteri and L. crispatus to L. salivarius and L. taiwanensis whereas in the ileum supplementation of phytase favored L. salivarius and L. taiwanensis but resulted in decreased abundance of L. crispatus. Both methods were correlating significantly, being T-RFLP a reliable fingerprinting method to rapidly analyze large numbers of samples in a cost-effective and rapid manner. Results are easy to interpret with no need of deep bioinformatics knowledge and can be integrated with taxonomic information.
Asunto(s)
Pollos/microbiología , Dieta , Microbioma Gastrointestinal , Fósforo Dietético , Alimentación Animal , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Calcio/administración & dosificación , Clonación Molecular , Digestión , Femenino , Tracto Gastrointestinal/microbiología , Perfilación de la Expresión Génica , Concentración de Iones de Hidrógeno , Lactobacillaceae/clasificación , Datos de Secuencia Molecular , Análisis Multivariante , Fósforo Dietético/administración & dosificación , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Componente Principal , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
Phytate breakdown in the digestive tract of broilers is affected by supplements of mineral phosphorus (P) and phytase with unknown interactions between the 2 factors. It was the objective to study phytate hydrolysis and the presence of inositol phosphate isomers (InsPs) as affected by supplements of mineral P and phytase in the small intestine of broilers. Fifteen-day old broilers were assigned to 48 pens of 20 broilers each (n = 8 pens/treatment). Two low-P corn-soybean meal-based diets without (BD-; 4.4 g P/kg dry matter) or with monocalcium phosphate (MCP; BD+; 5.2 g P/kg dry matter) were supplied without or with added phytase at 500 or 12,500 FTU/kg. On d 24, digesta from the duodenum/jejunum and lower ileum was pooled per segment on a by-pen basis, freeze-dried, and analyzed for P, InsPs, and the marker TiO2. Another 180 broilers (n = 6 pens/treatment, 10 birds each) were fed the 3 BD+ diets from d 1 to 21 to assess the influence of supplemented phytase on tibia mineralization and strength. Significant interactions between MCP and phytase supplements on myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate) (InsP6) hydrolysis (duodenum/jejunum: P ≤ 0.001; ileum: P = 0.004) and level of specific lower InsPs were detected. Supplementation with 12,500 FTU/kg phytase resulted in 92% InsP6 hydrolysis and strong degradation of InsP5. This treatment resulted in higher P net absorption, affirmed by higher BW gain, tibia strength, and mineralization compared to treatments without or with 500 FTU/kg phytase (P ≤ 0.05). MCP supplementation reduced the degradation of InsP6 and specific lower InsPs in birds fed diets without or with 500 FTU/kg of phytase (P ≤ 0.05), but did not reduce InsP6 hydrolysis or degradation of InsP5 at the high phytase dose. Effects of added MCP on phytase efficacy depend on the dose of supplemented phytase. Differences in the concentrations of lower InsPs indicated that the initial step of InsP6 hydrolysis is not the only catabolic step that is influenced by MCP or phytase levels.
Asunto(s)
6-Fitasa/farmacología , Fosfatos de Calcio/farmacología , Pollos/metabolismo , Intestino Delgado/metabolismo , Fósforo/farmacología , Ácido Fítico/metabolismo , 6-Fitasa/administración & dosificación , Alimentación Animal/análisis , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Calcificación Fisiológica/efectos de los fármacos , Dieta/veterinaria , Suplementos Dietéticos , Interacciones Farmacológicas , Femenino , Hidrólisis , Fosfatos de Inositol/metabolismo , Masculino , Fósforo/administración & dosificación , Tibia/fisiologíaRESUMEN
Changes of the rumen microbial community structure, as it can be established with a rumen simulation technique (RUSITEC) were studied using PCR and single-strand conformation polymorphism (SSCP) of small subunit rDNA genes (SSU rDNA). Four total mixed rations were incubated and two ammonia levels in the artificial saliva were applied. Three replicated vessels were used for each treatment. Mixed microbial fractions were isolated by stepwise centrifugation from the liquid fraction (reference microbes, RM) and from the solids of the feed residues (solid-associated microbes, SAM). PCR-primers targeting archaea, fibrobacter, clostridia, and bacteria, respectively, were applied to represent the individual taxonomic groups by SSCP profiles. These SSCP profiles were converted into a binary matrix and distances among treatments were visualised by non-metric multidimensional scaling. Between replicates belonging to one treatment only small differences were found, indicating a high reproducibility of the RUSITEC and the chosen SSCP method. The ammonia concentration seems to be affecting the SSCP profiles. Great differences occurred between RM and SAM, especially for profiles targeting bacteria and clostridia. Differences in the profiles of RM were also found between mixed rations that contained the same feedstuffs in different ratios and between rations with similar nutrient content but based on different feedstuffs. In conclusion, the PCR-SSCP-based technique in conjunction with non-metric multidimensional scaling was sufficiently sensitive to detect and compare changes in composition of rumen microbial community structure in vitro as affected by diet and other environmental factors.