Betaine increases net portal absorption of volatile fatty acids in Iberian pigs.

Betaine is an osmolyte with the potential to increase volatile fatty acids (VFAs) production and hence improve intestinal health.The present study investigated how betaine affects portal and arterial concentrations and net portal absorption (NPA) of VFA in growing Iberian pigs. Eight 30 kg BW Iberian growing barrows with indwelling catheters in portal vein, ileal vein and carotid artery were randomly assigned to a control diet or a diet supplemented with 0.5% betaine. Para-aminohippuric acid was infused into the ileal vein as a marker to determine portal blood flow using the dilution method. Blood samples were simultaneously taken from the carotid artery and portal vein at -60, 60, 120, 180, 240, 300 and 360 min after feeding 1 200 g of the diet. The NPA of VFA (acetate, propionate, butyrate, valerate, isobutyrate and caproate) was determined by multiplying the porto-arterial plasma concentration differences by portal plasma flow. Betaine increased NPA of acetate (1.44 fold; P < 0.001) and total VFA (0.55 fold; P < 0.001) while decreased NPA of propionate (-0.38 fold; P < 0.05) and valerate (-1.46 fold; P < 0.05) compared with control pigs. Estimated heat production potentially derived from NPA of VFA accounted for 0.20-0.27 of metabolizable energy for maintenance. Acetate and propionate accounted for most of the total VFA estimated heat production (0.83-0.89). Regarding bacterial communities, betaine apparently did not change the DNA abundance of fecal total bacteria, Lactobacillus, Bifidobacterium, Enterobacteriaceae, Bacteroides and the Clostridium clusters I, IV and XIV. In conclusion, betaine increased portal appearance and NPA of VFA, contributing to cover maintenance energy requirements.

Shifts in microbial populations in Rusitec fermenters as affected by the type of diet and impact of the method for estimating microbial growth (15N v. microbial DNA).

Rusitec fermenters are in vitro systems widely used to study ruminal fermentation, but little is known about the microbial populations establishing in them. This study was designed to assess the time evolution of microbial populations in fermenters fed medium- (MC; 50% alfalfa hay : concentrate) and high-concentrate diets (HC; 15 : 85 barley straw : concentrate). Samples from solid (SOL) and liquid (LIQ) content of fermenters were taken immediately before feeding on days 3, 8 and 14 of incubation for quantitative polymerase chain reaction and automated ribosomal intergenic spacer analysis analyses. In SOL, total bacterial DNA concentration and relative abundance of Ruminococcus flavefaciens remained unchanged over the incubation period, but protozoal DNA concentration and abundance of Fibrobacter succinogenes, Ruminococcus albus and fungi decreased and abundance of methanogenic archaea increased. In LIQ, total bacterial DNA concentration increased with time, whereas concentration of protozoal DNA and abundance of methanogens and fungi decreased. Diet×time interactions were observed for bacterial and protozoal DNA and relative abundance of F. succinogenes and R. albus in SOL, as well as for protozoal DNA in LIQ. Bacterial diversity in SOL increased with time, but no changes were observed in LIQ. The incubated diet influenced all microbial populations, with the exception of total bacteria and fungi abundance in LIQ. Bacterial diversity was higher in MC-fed than in HC-fed fermenters in SOL, but no differences were detected in LIQ. Values of pH, daily production of volatile fatty acids and CH4 and isobutyrate proportions remained stable over the incubation period, but other fermentation parameters varied with time. The relationships among microbial populations and fermentation parameters were in well agreement with those previously reported in in vivo studies. Using 15N as a microbial marker or quantifying total microbial DNA for estimating microbial protein synthesis offered similar results for diets comparison, but both methods presented contrasting results for microbial growth in SOL and LIQ phases. The study showed that fermentation parameters remained fairly stable over the commonly used sampling period (days 8 to 14), but shifts in microbial populations were detected. Moreover, microbial populations differed markedly from those in the inocula, which indicates the difficulty of directly transposing results on microbial populations developed in Rusitec fermenters to in vivo conditions.

Effect of disodium/calcium malate or supplementation on growth performance, carcass quality, ruminal fermentation products, and blood metabolites of heifers.

The aim of this study was to assess the effects of malate salts and culture on growth performance, carcass quality, ruminal fermentation products, and blood metabolites in heifers raised under southern Europe practical farm conditions. A total of 108 Charolaise cross heifers (214 ± 27.3 kg BW and 6.4 ± 1.1 mo of age) were housed in 18 pens of 6 animals each and used in a 114-d feedlot study. There was a totally randomized experimental design, and 6 pens were assigned to each of the following experimental diets: a control (no supplementation), the control plus 4 g of disodium/calcium malate mixture per kilogram of concentrate (2.12 g malate/kg), and the control plus 0.15 g of CBS 493.94 per kilogram of concentrate (1.5 × 10 cfu/kg). The control diet consisted of wheat-barley-based pelleted concentrate (32% starch, DM basis) and full-length barley straw. Concentrate and straw were fed separately ad libitum (5% orts) in an 88:12 ratio. On Days 0, 56, and 114, ruminal fluid and blood samples were obtained from each heifer between 2 and 2.5 h after the morning feeding by ruminocentesis and tail venipuncture, respectively. Body weight, concentrate ADFI, and G:F were recorded at 28, 56, 84, and 114 d. At slaughter, hot carcass weight and yield and carcass classification were determined in 2 representative heifers per pen (12 animals per dietary treatment). Supplementation with malate salts or did not affect concentrate ADFI ( = 0.98), ADG ( = 0.74), or G:F ( = 0.50) at any time during the experiment. At slaughter, there were no differences in carcass weight ( = 0.86), classification ( = 0.18), or carcass yield ( = 0.84) among experimental groups. Also, there were no differences treatments on ruminal pH ( = 0.24), ruminal fermentation products ( = 0.69, = 0.88, and = 0.93 for total VFA, NH-N, and lactate, respectively), and blood metabolites ( = 0.96, = 0.82, and = 0.15 for glucose, urea N, and lactate, respectively). In conclusion, under the feeding and management conditions of this study, diet supplementation with malate salts or did not have any significant effects on growth performance, carcass quality, ruminal fermentation products, and blood metabolites.

Treatment of tropical forages with exogenous fibrolytic enzymes: effects on chemical composition and in vitro rumen fermentation.

The effects of three treatments of fibrolytic enzymes (cellulase from Trichoderma longibrachiatum (CEL), xylanase from rumen micro-organisms (XYL) and a 1:1 mixture of CEL and XYL (MIX) on the in vitro fermentation of two samples of Pennisetum clandestinum (P1 and P2), two samples of Dichanthium aristatum (D1 and D2) and one sample of each Acacia decurrens and Acacia mangium (A1 and A2) were investigated. The first experiment compared the effects of two methods of applying the enzymes to forages, either at the time of incubation or 24 h before, on the in vitro gas production. In general, the 24 h pre-treatment resulted in higher values of gas production rate, and this application method was chosen for a second study investigating the effects of enzymes on chemical composition and in vitro fermentation of forages. The pre-treatment with CEL for 24 h reduced (p < 0.05) the content of neutral detergent fibre (NDF) of P1, P2, D1 and D2, and that of MIX reduced the NDF content of P1 and D1, but XYL had no effect on any forage. The CEL treatment increased (p < 0.05) total volatile fatty acid (VFA) production for all forages (ranging from 8.6% to 22.7%), but in general, no effects of MIX and XYL were observed. For both P. clandestinum samples, CEL treatment reduced (p < 0.05) the molar proportion of acetate and increased (p < 0.05) that of butyrate, but only subtle changes in VFA profile were observed for the rest of forages. Under the conditions of the present experiment, the treatment of tropical forages with CEL stimulated their in vitro ruminal fermentation, but XYL did not produce any positive effect. These results showed clearly that effectiveness of enzymes varied with the incubated forage and further study is warranted to investigate specific, optimal enzyme-substrate combinations.

Technical note: Comparison of automated ribosomal intergenic spacer analysis and denaturing gradient gel electrophoresis to assess bacterial diversity in the rumen of sheep.

The aim of this study was to compare automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) techniques to assess bacterial diversity in the rumen of sheep. Sheep were fed 2 diets with 70% of either alfalfa hay or grass hay, and the solid (SOL) and liquid (LIQ) phases of the rumen were sampled immediately before feeding (0 h) and at 4 and 8 h postfeeding. Both techniques detected similar differences between forages, with alfalfa hay promoting greater (P < 0.05) bacterial diversity than grass hay. In contrast, whereas ARISA analysis showed a decrease (P < 0.05) of bacterial diversity in SOL at 4 h postfeeding compared with 0 and 8 h samplings, no variations (P > 0.05) over the postfeeding period were detected by DGGE. The ARISA technique showed lower (P < 0.05) bacterial diversity in SOL than in LIQ samples at 4 h postfeeding, but no differences (P > 0.05) in bacterial diversity between both rumen phases were detected by DGGE. Under the conditions of this study, the DGGE was not sensitive enough to detect some changes in ruminal bacterial communities, and therefore ARISA was considered more accurate for assessing bacterial diversity of ruminal samples. The results highlight the influence of the fingerprinting technique used to draw conclusions on factors affecting ruminal bacterial diversity.

Short- to medium-term effects of consumption of quebracho tannins on saliva production and composition in sheep and goats.

Eight Merino sheep (49.4 ± 4.23 kg BW) and 8 Alpine goats (53.2 ± 2.51 kg BW) were used to study the effect of ingestion of quebracho tannins on salivation. Four sheep and 4 goats were individually fed a daily allotment of 20 g DM of alfalfa hay/kg BW (Control). Another 4 sheep and 4 goats were also given 20 g DM of alfalfa hay/kg BW supplemented with 50 g of quebracho/kg DM (Tannin) for a period of 64 d. The saliva secretion from the left parotid gland was collected by insertion of a polyvinyl chloride catheter into the parotid duct and the amount of parotid saliva produced recorded over three 48-h periods on d 1 and 2 (P1), d 31 and 32 (P2), and d 61 and 62 (P3) after the tannin feeding was initiated. The total amount of saliva produced was estimated from rumen water kinetics determined on d 4, d 34, and d 64 of the experiment. Experimental design was completely randomized, with repeated measures on each experimental unit, performing separate analysis for sheep and goats. Parotid saliva production was not affected by the sampling period in either animal species receiving the Control diet. Corresponding values for sheep were 2.04, 2.12, and 2.27 L/d (P = 0.89) and for goats 1.65, 1.79, and 1.86 L/d (P = 0.95). Sheep fed the Tannin diet produced 55, 73, and 107% of the amount of saliva recorded in sheep fed the Control diet on P1, P2, or P3, respectively. Corresponding values in goats were 88, 130, and 134% on P1, P2, or P3, respectively. Estimated total saliva production was not affected (P = 0.50 for sheep and P = 0.97 for goats) by the ingestion of quebracho. There was no difference (P > 0.10) in osmotic pressure, P, Mg, Ca, urea, and protein concentrations in parotid saliva. There were, however, differences in Na and K concentrations in response to the ingestion of quebracho tannins, with Na concentrations increasing (P = 0.05) and K concentrations decreasing (P = 0.04) in sheep saliva and pH increasing (P = 0.05) in goat saliva. In conclusion, the inclusion of quebracho at 50 g/kg DM for 64 d does not appear to alter saliva production in sheep and goats.

Postprandial changes of fiber-degrading microbes in the rumen of sheep fed diets varying in type of forage as monitored by real-time PCR and automated ribosomal intergenic spacer analysis.

Four ruminally cannulated sheep were used in a crossover design to assess the postprandial changes of fiber-degrading microbes in the solid phase of the rumen of sheep fed 2 high-forage diets. The diets had forage:concentrate ratio of 70:30 (DM basis) and either alfalfa (Medicago sativa) hay (AL) or grass hay (GR) as forage (FOR). Sheep were fed twice daily, and samples from solid rumen digesta were taken at 0, 4, and 8 h after the morning feeding. Postprandial changes of DNA concentrations of all determined microbial populations were similar for the 2 diets. Samples taken at 4 h after feeding had lesser (P < 0.05) concentrations of total bacterial DNA determined with real-time PCR and bacterial diversity and greater (P < 0.05) protozoal DNA concentrations, relative abundance of fungal, Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus DNA compared with those taken at 0 and 8 h. No effect (P = 0.41 to 0.76) of FOR was detected either on concentrations of bacterial and protozoal DNA or the relative abundance of the 2 Ruminococcus DNA, but GR diet promoted greater (P < 0.001) relative abundance of F. succinogenes and fungal DNA compared with AL diet. Fibrobacter succinogenes was the most abundant (P < 0.05) of the 3 cellulolytic bacteria for both diets, with no differences (P < 0.05) between the 2 Ruminococcus species. Rumen pH and carboxymethylcellulase, Avicelase, and amylase activities were not affected (P = 0.15 to 0.69) by FOR, but xylanase activity was greater (P = 0.01) for GR diet. The influence of FOR on microbial communities in ruminal solid digesta was more evident in the first hours after feeding than at later times after feeding, which highlights the influence of sampling time when investigating dietary effects on rumen function and microbial populations.

Urinary excretion of purine derivatives, microbial protein synthesis, nitrogen use, and ruminal fermentation in sheep and goats fed diets of different quality.

The objective of this study was to compare N balance, microbial N flow (MNF) estimated from purine derivatives (PD) urinary excretion, and its variation when estimated using purine bases:N ratios in liquid associated bacteria (LAB) from models reported in the literature (MNF - response models) or measured ratios in liquid and solid-associated bacterial (SAB) pellets (MNF-LAB+SAB), diet digestibility, and rumen fermentation variables in sheep and goats fed 3 different practical, quality diets to study interspecies differences concerning N use as accurately as possible. Four mature female Merino sheep and 4 mature female Granadina goats, each fitted with a ruminal cannula, were used in 3 × 3 Latin square design with an extra animal. Two experimental diets had a forage-to-concentrate ratio of 70:30 (DM basis) with alfalfa hay (ALC) or grass hay (GRC) as forage, and the third diet contained 70% concentrate and 30% alfalfa hay (CAL). All animals were fed the diets at a daily rate of 56 g/kg BW(0.75) to minimize feed selection. Digestibility of nutrients was similar (P = 0.16 to 0.88) in the 2 species, but some animal species × diet interactions (P = 0.01 to 0.04) were detected. There were small differences between the fermentation patterns of both animal species. Goats showed decreased VFA concentrations (P = 0.005) and butyrate proportions (P = 0.04), and greater acetate proportions (P = 0.02) compared with sheep, whereas N intake and percentage of N intake excreted in feces were similar in both species (P = 0.58 and 0.15, respectively), the percentage excreted via the urine was greater in goats compared with sheep (P < 0.001). As a consequence, sheep had greater (P < 0.001) N retention than goats (averaged across diets, 32.6% and 16.1% of N intake, respectively). There were no differences (P = 0.95) between animal species in total PD excretion, but goats showed a greater excretion of allantoin (P = 0.01) and decreased excretion of xanthine (P = 0.008) and hypoxanthine (P = 0.007) compared with sheep. In general, differences between sheep and goats were more pronounced for the medium-quality diet (GRC) compared with those of high-quality diet (ALC and CAL). The greater urinary losses in goats would indicate a greater contribution of goats to N environmental contamination compared with sheep.

Comparison of fermentation of diets of variable composition and microbial populations in the rumen of sheep and Rusitec fermenters. I. Digestibility, fermentation parameters, and microbial growth.

Four ruminally and duodenally cannulated sheep and 8 Rusitec fermenters were used to determine the effects of forage to concentrate (F:C) ratio and type of forage in the diet on ruminal fermentation and microbial protein synthesis. The purpose of the study was to assess how closely fermenters can mimic the dietary differences found in vivo. The 4 experimental diets contained F:C ratios of 70:30 or 30:70 with either alfalfa hay or grass hay as the forage. Microbial growth was determined in both systems using (15)N as a microbial marker. Rusitec fermenters detected differences between diets similar to those observed in sheep by changing F:C ratio on pH; neutral detergent fiber digestibility; total volatile fatty acid concentrations; molar proportions of acetate, propionate, butyrate, isovalerate, and caproate; and amylase activity. In contrast, Rusitec fermenters did not reproduce the dietary differences found in sheep for NH(3)-N and lactate concentrations, dry matter (DM) digestibility, proportions of isobutyrate and valerate, carboxymethylcellulase and xylanase activities, and microbial growth and its efficiency. Regarding the effect of the type of forage in the diet, Rusitec fermenters detected differences between diets similar to those found in sheep for most determined parameters, with the exception of pH, DM digestibility, butyrate proportion, and carboxymethylcellulase activity. Minimum pH and maximal volatile fatty acid concentrations were reached at 2h and at 6 to 8h postfeeding in sheep and fermenters, respectively, indicating that feed fermentation was slower in fermenters compared with that in sheep. There were differences between systems in the magnitude of most determined parameters. In general, fermenters showed lower lactate concentrations, neutral detergent fiber digestibility, acetate:propionate ratios, and enzymatic activities. On the contrary, fermenters showed greater NH(3)-N concentrations, DM digestibility, and proportions of propionate, butyrate, isovalerate, valerate, and caproate. Values of efficiency of microbial growth were greater in fermenters compared with sheep for 70:30 diets, but they were lower for 30:70 diets. Differences between fermentation in sheep and fermenters can be mainly attributed to the lack of absorption in fermenters, differences in solid retention time, and compartmentalization in the Rusitec system. In general, the Rusitec system simulated more closely the in vivo fermentation of high-forage diets compared with high-concentrate diets.

Comparison of fermentation of diets of variable composition and microbial populations in the rumen of sheep and Rusitec fermenters. II. Protozoa population and diversity of bacterial communities.

Four ruminally and duodenally cannulated sheep and 8 Rusitec fermenters were used to determine the effects of dietary characteristics on microbial populations and bacterial diversity. The purpose of the study was to assess how closely fermenters can mimic the differences between diets found in vivo. The 4 experimental diets contained forage to concentrate (F:C) ratios of 70:30 (high forage; HF) or 30:70 (high concentrate; HC) with either alfalfa hay (A) or grass hay (G) as the forage. Total bacterial numbers were greater in the rumen of sheep fed HF diets compared with those fed HC diets, whereas the opposite was found in fermenters. The numbers of cellulolytic bacteria were not affected by F:C ratio in any fermentation system, but cellulolytic numbers were 2.7 and 1.8 times greater in sheep than in fermenters for HF and HC diets, respectively. Neither total bacterial nor cellulolytic numbers were affected by the type of forage in sheep or fermenters. Decreasing F:C ratio increased total protozoa and Entodiniae numbers in sheep by about 29 and 25%, respectively, but it had no effect in fermenters. Isotrichidae and Ophryoscolecinae numbers in sheep were not affected by changing F:C ratio, but both disappeared completely from fermenters fed HC diets. Total protozoa and Entodiniae numbers were greater in sheep fed A diets than in those fed G diets, whereas the opposite was found in fermenters. Results indicate that under the conditions of the present study, protozoa population in Rusitec fermenters was not representative of that in the rumen of sheep fed the same diets. In addition, protozoa numbers in fermenters were 121 and 226 times lower than those in the sheep rumen for HF and HC diets, respectively. The automated ribosomal intergenic spacer analysis of the 16S ribosomal DNA was used to analyze the diversity of liquid- and solid-associated bacteria in both systems. A total of 170 peaks were detected in the automated ribosomal intergenic spacer analysis electropherograms of bacterial pellets across the full set of 64 samples, from which 160 were detected in at least 1 individual from each system (sheep or fermenter). Diversity of liquid-associated bacterial pellets was greater with G diets in fermenters but seemed to be unaffected by diet in sheep. Bacterial diversity in solid-associated bacteria pellets was greater for G diets compared with A diets in sheep and fermenters. Different conditions in the fermenters compared with sheep rumen might have caused a selection of some bacterial strains.

Influence of detachment procedure and diet on recovery of solid-associated bacteria from sheep ruminal digesta and representativeness of bacterial isolates as assessed by automated ribosomal intergenic spacer analysis-polymerase chain reaction.

Six ruminally and duodenally cannulated sheep were used in a partially replicated 4 x 4 Latin square experiment designed to evaluate the efficiency of 3 detachment procedures (DP) to recover solid-associated bacteria (SAB) from ruminal digesta. The 4 experimental diets contained forage to concentrate (F:C) ratios of 70:30 or 30:70 with either alfalfa hay or grass hay as the forage. Bacterial biomass was labeled with 15NH4Cl. The DP were 1) MET: digesta was incubated at 38 degrees C for 15 min with saline solution (0.9% NaCl) containing 0.1% methylcellulose under continuous shaking; 2) STO: digesta was mixed with cold saline solution and homogenized with a stomacher for 5 min at 230 rpm; 3) FRE: digesta was immediately frozen at -20 degrees C for 72 h, thawed at 4 degrees C, mixed with saline solution and subjected to STO procedure. Common to all treatments was storing at 4 degrees C for 24 h after the treatment, homogenization, filtration, and resuspension of digesta 2 times in the treatment solutions. The automated ribosomal intergenic spacer analysis of the 16S ribosomal DNA was used to analyze the similarity between bacterial communities attached to the digesta and those in the pellet obtained after each DP. There were no significant F:C x DP or forage x DP interactions for any variable. On average, STO treatment detached 65.8% of SAB from ruminal digesta, about 1.2 and 1.5 times more than FRE and MET treatments, respectively. Total recovery of SAB in STO pellets (48.9%) was greater compared with FRE (31.7%) and MET (33.1%), values being greater for high-forage compared with high-concentrate diets. Similarity index between the bacteria attached to digesta and those in the pellets were lower for FRE (48.2%) compared with MET (54.1%) and STO (54.1%), which suggests that FRE could have destroyed cell integrity of some bacterial species, thus reducing the bacterial diversity present in the pellets. The STO method was the most effective removing SAB from digesta, but only a moderate similarity between the bacterial communities attached to digesta and those recovered in the bacterial pellets was obtained. Values of duodenal microbial flow estimated using SAB as reference bacteria were greater with FRE compared with STO and MET, but all DP detected similar differences between diets, and therefore did not influence the interpretation of results.

Effects of dilution rate and retention time of concentrate on efficiency of microbial growth, methane production, and ruminal fermentation in Rusitec fermenters.

The objective of this study was to investigate the effects of 2 dilution rates (DL) and 2 concentrate retention times (RT) on microbial growth, methane production, and fermentation of a 30:70 alfalfa hay:concentrate diet in Rusitec fermenters maintained at similar pH. The DL were 3.78 (low DL, LDL) and 5.42%/h (high DL, HDL), and concentrate RT was either 24 h (T24) or 48 h (T48). Forage RT was 48 h in all fermenters. Apparent disappearance of diet DM and NDF was greater in HDL fermenters compared with LDL fermenters, but there was a significant DL x concentrate RT interaction, showing that the effect of DL was more pronounced in T48 compared with T24 fermenters. Methane production was not affected by DL, but was greater in T48 compared with T24 fermenters, which was consistent with the increased fiber degradation in T48 fermenters. Increasing DL augmented volatile fatty acid production and molar proportions of propionate, isovalerate, and valerate, and reduced those of caproate, but no effects were observed on acetate, butyrate, and isobutyrate proportions. Increasing concentrate RT resulted in greater volatile fatty acid production and proportions of acetate, butyrate, and caproate, but reduced those of propionate, valerate, and isovalerate. Ammonia-N production was not affected by concentrate RT, but was greater at HDL compared with LDL. Microbial growth was not affected by DL, but microbial growth efficiency was lower in HDL compared with LDL fermenters. Concentrate RT affected microbial growth and its efficiency, with both being greater in T48 compared with T24 fermenters. Carboxymetylcellulase and xylanase activities in ruminal fluid were greater in HDL compared with LDL fermenters, but were not affected by concentrate RT. There were DL x concentrate RT interactions for diet apparent disappearance, molar proportions of propionate, butyrate, isovalerate, and caproate, and acetate:propionate ratio, indicating that effects of DL on these variables were influenced by concentrate RT. The results would indicate that using higher DL and shorter concentrate RT than those typically used in Rusitec fermenters would contribute to improving the simulation of in vivo fermentation of high-concentrate diets.

Microbial protein synthesis, ruminal digestion, microbial populations, and nitrogen balance in sheep fed diets varying in forage-to-concentrate ratio and type of forage.

Six ruminally and duodenally cannulated sheep were used in a partially replicated 4 x 4 Latin square to evaluate the effects of 4 diets on microbial synthesis, microbial populations, and ruminal digestion. The experimental diets had forage to concentrate ratios (F:C; DM basis) of 70:30 (HF) or 30:70 (HC) with alfalfa hay (A) or grass hay (G) as forage and were designated as HFA, HCA, HFG, and HCG. The concentrate was based on barley, gluten feed, wheat middlings, soybean meal, palmkern meal, wheat, corn, and mineral-vitamin premix in the proportions of 22, 20, 20, 13, 12, 5, 5, and 3%, respectively (as-is basis). Sheep were fed the diets at a daily rate of 56 g/kg of BW(0.75) to minimize feed selection. High-concentrate diets resulted in greater (P < 0.001) total tract apparent OM digestibility compared with HF diets, but no differences were detected in NDF digestibility. Ruminal digestibility of OM, NDF, and ADF was decreased by increasing the proportion of concentrate, but no differences between forages were detected. Compared with sheep fed HF diets, sheep receiving HC diets had less ruminal pH values and acetate proportions, but greater butyrate proportions. No differences among diets were detected in numbers of cellulolytic bacteria, but protozoa numbers were less (P = 0.004) and total bacteria numbers tended (P = 0.08) to be less for HC diets. Carboxymethylcellulase, xylanase, and amylase activities were greater for HC compared with HF diets, with A diets showing greater (P = 0.008) carboxymethylcellulase activities than G diets. Retained N ranged from 28.7 to 37.9% of N intake and was not affected by F:C (P = 0.62) or the type of forage (P = 0.31). Microbial N synthesis and its efficiency was greater (P < 0.001) for HC diets compared with HF diets. The results indicate that concentrates with low cereal content can be included in the diet of sheep up to 70% of the diet without detrimental effects on ruminal activity, microbial synthesis efficiency, and N losses.

Evaluation of procedures for detaching particle-associated microbes from forage and concentrate incubated in Rusitec fermenters: efficiency of recovery and representativeness of microbial isolates.

Three detachment procedures (DP) were evaluated for their ability to remove particle-associated microbes from digesta in Rusitec fermenters fed a 30:70 alfalfa hay:concentrate diet. Forage and concentrate were incubated in separate nylon bags, and incubation residues were treated independently. Microbial biomass was labeled with (15)NH(4)Cl. Treatments were 1) MET: residues were incubated at 38 degrees C for 15 min with saline solution (0.9% NaCl) containing 0.1% methylcellulose with continuous shaking; 2) STO: residues were mixed with cold saline solution and homogenized with a stomacher for 5 min at 230 revolutions per min; and 3) FRE: residues were immediately frozen at -20 degrees C for 72 h, thawed at 4 degrees C, mixed with saline solution, and subjected to STO procedure. Common to all treatments was storing at 4 degrees C for 24 h after the treatment, homogenization, filtration, and resuspension of residues 2 times in the treatment solutions. Microbial pellets were obtained by centrifugation, and microbial removal was estimated indirectly by measuring removal of (15)N. The PCR-single-stranded conformation polymorphism analysis of the 16S ribosomal DNA was used to analyze the similarity between microbial communities attached to the substrate and those in the pellet obtained after each DP. There were no feed x DP interactions (P = 0.16 to 0.96) for any variable, except for N content in microbial pellets (P = 0.02). Detaching efficiency (P = 0.004) and total recovery (P = 0.01) were affected by DP, with STO showing the greatest values (mean values across substrates of 64.1% for detaching efficiency and 58.3% for total recovery) and MET the least values (57.0 and 51.8%). Similarity index between the microbes attached to substrates and those in the pellets were affected (P = 0.02) by DP, with MET showing greater (P < 0.02) values (84.0 and 86.4% for forage and concentrate, respectively) than FRE (72.5 and 67.8%) and STO having intermediate values (77.1 and 82.4%). There were no differences (P = 0.70) among particle-associated microbe pellets in their N content, but MET pellets had greater (P < 0.05) (15)N enrichments than those obtained by STO and FRE. Although STO was the most effective method to detach ruminal microbes from concentrate and forage, MET produced pellets with the greatest similarity to the microbial communities attached to the substrates and therefore could be considered the most appropriate DP method for treating digesta from Rusitec fermenters.

Comparison of microbial fermentation of high- and low-forage diets in Rusitec, single-flow continuous-culture fermenters and sheep rumen.

Eight Rusitec and eight single-flow continuous-culture fermenters (SFCCF) were used to compare the ruminal fermentation of two diets composed of alfalfa hay and concentrate in proportions of 80 : 20 (F80) and 20 : 80 (F20). Results were validated with those obtained previously in sheep fed the same diets. Rusitec fermenters were fed once daily and SFCCF twice, but liquid dilution rates were similar in both types of fermenters. Mean values of pH over the 12 h postfeeding were higher (P < 0.001) in Rusitec than in SFCCF, with diet F80 showing higher values (P < 0.001) in both types of fermenters. Concentrations of total volatile fatty acids (VFA) were higher (P < 0.001) in SFCCF than in Rusitec, and in both systems were higher (P = 0.002) for diet F20 than for diet F80. There were significant differences between systems in the proportions of the main VFA, and a fermentation system × diet interaction (P < 0.001) was detected for all VFA with the exception of valerate. No differences (P = 0.145) between the two types of fermenters were detected in dry matter (DM) digestibility, but NDF, microbial N flow and its efficiency were higher (P = 0.001) in SFCCF compared to Rusitec. Whereas pH values and VFA concentrations remained fairly stable through the day in both in vitro systems, pH dropped and VFA increased shortly after feeding in sheep rumen reaching the minimum and maximal values, respectively, about 4 h after feeding. Both in vitro systems detected differences between diets similar to those found in sheep for liquid dilution rate, pH values, DM digestibility, microbial N flow and growth efficiency. In contrast, acetate/propionate ratios were lower for diet F20 than for F80 in sheep rumen (2.73 and 3.97) and SFCCF (3.07 and 4.80), but were higher for diet F20 compared to F80 (4.29 and 3.40) in Rusitec, with values considered to be unphysiological for high-concentrate diets. In vivo NDF digestibility was affected (P = 0.017) by diet, but no differences between diets (P > 0.05) were found in any in vitro system. A more precise control of pH in both types of fermenters and a reduction of concentrate retention time in Rusitec could probably improve the simulation of in vivo fermentation.

Influence of direct-fed fibrolytic enzymes on diet digestibility and ruminal activity in sheep fed a grass hay-based diet.

Six rumen-fistulated Merino sheep were used in a crossover design experiment to evaluate the effects of an exogenous fibrolytic enzyme preparation (12 g/d; ENZ), delivered directly into the rumen, on diet digestibility, ruminal fermentation, and microbial protein synthesis. The enzyme contained endoglucanase and xylanase activities. Sheep were fed a mixed grass hay:concentrate (70:30; DM basis) diet at a daily rate of 46.1 g/kg of BW(0.75). Samples of grass hay were incubated in situ in the rumen of each sheep to measure DM and NDF degradation. The supplementation with ENZ did not affect diet digestibility (P = 0.30 to 0.66), urinary excretion of purine derivatives (P = 0.34), ruminal pH (P = 0.46), or concentrations of NH(3)-N (P = 0.69) and total VFA (P = 0.97). In contrast, molar proportion of propionate were greater (P = 0.001) and acetate:propionate ratio was lower (P < 0.001) in ENZ-supplemented sheep. In addition, ENZ supplementation tended to increase (P = 0.06) numbers of cellulolytic bacteria at 4 h after feeding. Both the ruminally insoluble potentially degradable fraction of grass hay DM and its fractional rate of degradation were increased (P = 0.002 and 0.05, respectively) by ENZ treatment. Supplementation with ENZ also increased (P = 0.01 to 0.02) effective and potential degradability of grass hay DM and NDF. Ruminal fluid endoglucanase and xylanase activities were greater (P < 0.001 and 0.03, respectively) in ENZ-supplemented sheep than in control animals. It was found that ENZ supplementation did not affect either exoglucanase (P = 0.12) or amylase (P = 0.83) activity. The results indicate that supplementing ENZ directly into the rumen increased the fibrolytic activity and stimulated the growth of cellulolytic bacteria without a prefeeding feed-enzyme interaction.

Methane production and substrate degradation by rumen microbial communities containing single protozoal species in vitro.

AIMS: To assess the effect of protozoal species on rumen fermentation characteristics in vitro. METHODS AND RESULTS: Entodinium caudatum, Isotricha intestinalis, Metadinium medium, and Eudiplodinium maggii from monofaunated wethers and mixed protozoa from conventional wethers were obtained by centrifugation, re-suspended at their normal densities in rumen fluid supernatants from defaunated or conventional wethers and incubated in vitro. The presence of protozoa increased the concentration of ammonia and altered the volatile fatty acids balance with more acetate and butyrate produced at the expense of propionate. Differences among species were observed, notably in the production of methane, which increased with E. caudatum as compared to other ciliates and to defaunated and mixed protozoa treatments (P < 0.05). The increased methanogenesis was not correlated to protozoal biomass indicating that the metabolism of this protozoan and/or its influence on the microbial ecosystem was responsible for this effect. CONCLUSIONS: Entodinium caudatum stimulated the production of methane, a negative effect that was reinforced by a concomitant increase in protein degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: Comparison of individual species of protozoa highlighted the particular influence of E. caudatum on rumen fermentation. Its elimination (targeted defaunation) from the rumen could reduce methane production without affecting feed degradation.

Influence of exogenous fibrolytic enzymes and fumarate on methane production, microbial growth and fermentation in Rusitec fermenters.

Two incubation runs were conducted with Rusitec fermenters to investigate the effects of three additive treatments (mixed fibrolytic enzymes from Trichoderma longibrachiatum (FE), disodium fumarate (FUM) and both additives (MIX)) on rumen microbial growth and fermentation of a grass hay:concentrate (600 : 400 g/kg DM) substrate. Each fermenter received daily 20 g substrate DM. Application rate (per g substrate DM) was 34.3 endoglucanase, 0.57 exoglucanase, 24.7 xylanase and 5.51 amylase units for FE and 30 mg fumarate for FUM. MIX fermenters received both additives. Both FE and MIX increased (P 0.05). Supplementing with FUM increased (P 0.05) any other variable, thus suggesting that observed effects were due to fermentation of FUM itself. The lack of effects of FUM and the absence of differences between FE and MIX on most of the measured variables would indicate that beneficial effects found in MIX fermenters were mainly due to the action of FE. Combining FE and FUM as feed additives under the conditions of the present experiment did not further improve rumen fermentation, compared to FE alone.

Effects of exogenous cellulase supplementation on microbial growth and ruminal fermentation of a high-forage diet in Rusitec fermenters.

Two incubation runs were carried out with a Rusitec system to investigate the effects of 2 exogenous pure cellulases on ruminal microbial growth and fermentation of a 70:30 grass hay:concentrate (DM basis) substrate. The substrate was sprayed with buffer (control; pH = 6.5), a cellulase from Trichoderma longibrachiatum (TRI), a cellulase from Aspergillus niger (ASP), or a 1:1 mixture of both cellulases (MIX) 24 h before being placed in the fermenters. Enzymes were applied at a rate of 30 endoglucanase units/g of substrate DM. Treating the substrate with enzymes reduced substrate NDF and ADF content (P < 0.001 to P = 0.002) and increased DM, NDF, and ADF disappearance after 6 and 24 h of incubation (P < 0.001 to P = 0.004) but not after 48 h of incubation. Daily VFA production was increased (P = 0.004) by 15, 9, and 15% for TRI, ASP, and MIX, respectively, with half of the increase being due to production of acetate. All enzyme treatments augmented (P = 0.009) methane production, but none of them altered the methane:VFA ratio (P = 0.70). There were no differences (P = 0.80) among treatments in the daily flow of solid-associated microorganisms, as measured using 15N as a microbial marker. Although the TRI and MIX treatments increased (P < 0.05) the daily flow of liquid-associated microorganisms and the proportion of microbial N in the solid residue after 48 h of incubation, no effects were observed (P = 0.92 and P = 0.95, respectively) for the ASP treatment. The results show that the TRI and MIX treatments enhanced in vitro fermentation by increasing substrate fiber degradation, VFA production, and ruminal microbial growth. The lack of differences between TRI and MIX in most of the measured variables indicates that treating the substrate with a mixture of both cellulases did not further improve the effects of the TRI treatment.

Effects of malate on diet digestibility, microbial protein synthesis, plasma metabolites, and performance of growing lambs fed a high-concentrate diet.

The objective of this study was to evaluate the effects of malate supplementation on growth rate, feed efficiency, and diet digestibility in growing lambs. Twenty-four Merino lambs with a mean BW of 15.3 +/- 0.22 kg were divided into 3 homogenous groups. Each group was randomly allocated to 1 of 3 malate (16% disodium malate:84% calcium malate) levels: 0 (control), 4 (MAL-4), or 8 (MAL-8) g/kg of concentrate. Lambs were fed concentrate and barley straw ad libitum for 35 d. After a 20-d period, diet digestibility was determined, and microbial N flow at the duodenum was estimated from the urinary excretion of purine derivatives. Blood samples were taken on d 0, 20, and 35. On d 35, lambs were slaughtered and ruminal fluid samples were collected. There were no effects (P = 0.18 to P = 0.95) of malate on concentrate or straw intake, ADG, carcass yield, and apparent digestibility of OM, CP, NDF, or ADF. Malate supplementation did not influence (P = 0.80) the daily urinary excretion of total purine derivatives, and therefore there were no treatment effects (P = 0.77) on estimated microbial N flow at the duodenum. No differences (P > 0.05) among treatments were observed for plasma concentrations of glucose, cholesterol, triglycerides, urea-N, lactate, or VFA, but malate addition increased (P = 0.003) the molar proportion of butyrate in ruminal fluid (4.29, 6.14, and 5.45% of total VFA for control, MAL-4 and MAL-8, respectively). The use of malate as a feed additive under the conditions of the current study did not influence diet intake or digestion, and consequently did not improve lamb performance.