Probiotic Bacillus subtilis 29,784 improved weight gain and enhanced gut health status of broilers under necrotic enteritis condition.

The study investigated the benefit of a Bacillus subtilis probiotic (Bs 29,784) in necrotic enteritis (NE)-challenged broilers. Four treatments were performed with 312 male day-old Ross 308 reared in floor pens from day 0 to day 35: 2 groups fed control diet without or with NE challenge (CtrlNC and CtrlNE); 2 groups fed probiotic and antibiotic supplements in the control diet with NE challenge (ProNE and AntNE). Necrotic enteritis challenge procedures commenced with inoculation of Eimeria spp 1 mL/bird per os at day 9 and Clostridium perfringens EHE-NE18 (approximately 108 cfu/mL) 1 mL/bird per os at day 14 and day 15. Performance parameters were measured on day 16 and day 35. Lesion, cecal microbiota, and jejunal gene expression were analyzed on day 16. Necrotic enteritis challenge significantly suppressed the performance parameters compared with CtrlNC: 27% weight gain reduction, 11 points feed conversion ratio (FCR) increase at day 16, and 12% weight gain reduction, 5-point FCR increase at day 35. By day 35, ProNE and AntNE treatments enabled significantly higher weight gain (4 and 9%, respectively) than CtrlNE. Compared with CtlrNE and contrary to AntNE, ProNE treatment exhibited upregulation of genes coding for tight junctions proteins (CLDN1, JAM2, TJP1), cytokines (IL12, interferon gamma, TGFß), and Toll-like receptors (TLR5, TLR21) suggesting enhanced immunity and intestinal integrity. 16S NGS analysis of cecal microbiota at day 16 showed a decreased alpha diversity in challenged groups. Principal component analysis of operational taxonomic unit (OTU) abundance revealed that ProNE and AntNE grouped closely while both distantly from CtrlNC and CtrlNE, which were separately grouped, indicating the similar effects of ProNE and AntNE on the OTU diversity that were however different from both CtrlNC and CtrlNE. Microbiota analysis revealed an increase of genera Faecalibacterium, Oscillospira, and Butyricicoccus; and a decrease of genera Ruminococcus, Lactobacillus, and Bacteroides; and an increase of the Firmicutes-to-Bacteroidetes ratio in ProNE and AntNE groups compared with the CtlrNE group. It is concluded that Bs 29,784 may enable improved health of broiler chickens under NE conditions thus performance implications.

Bacillus subtilis 29784 induces a shift in broiler gut microbiome toward butyrate-producing bacteria and improves intestinal histomorphology and animal performance.

The study reports the effects of Bacillus subtilis 29784 on broiler performance. A total of 1,600 one-day-old Cobb 500 male broiler chicks received either a control diet or the same diet to which B. subtilis 29784 spores were added (1E8 CFU/kg of feed). The birds were slaughtered at 42 D of age. Ileal and cecal tissues and content were collected for histomorphological analysis and 16S rRNA gene sequencing, respectively. The inclusion of B. subtilis 29784 led to an increase of final body weight gain of broilers (+5.7%; P < 0.0001) and an improvement in feed conversion ratio (-5.4%; P < 0.0001). Higher feed efficiency in the Bacillus-fed group was correlated with a significant increase in intestinal microvilli length (+18% in ileum and +17% in cecum; P < 0.001). Among the differences revealed by 16S rRNA analysis, Ruminococcus, Lachnoclostridium, and Anaerostipes were found in higher relative abundance in Bacillus-treated birds at the cecal level. These bacterial genera include species that produce butyrate, the main source of energy for enterocytes and known to be an immune modulator. There was also a slight increase in the Butyrivibrio genus in the cecum, which is known to be an important player in the production of conjugated linoleic acid, also considered an anti-inflammatory compound. In conclusion, dietary supplementation of B. subtilis 29784 significantly improved the growth performance of broilers, likely through beneficial effects on microbiota and host.

Short-chain arabinoxylans prepared from enzymatically treated wheat grain exert prebiotic effects during the broiler starter period.

Carbohydrate-degrading multi-enzyme preparations (MEP) are used to improve broiler performances. Their mode of action is complex and not fully understood. In this study, we compared the effect of water-soluble fractions isolated at the pilot scale from wheat grain incubated with (WE) and without (WC) MEP. The fractions were incorporated in a wheat-based diet (0.1% w/w) to feed Ross PM3 broilers and compared with a non-supplemented control group (NC). The body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) until d 14 were determined. At d 14, ileal and cecal contents and tissue samples were collected from euthanized animals. The intestinal contents were used to measure the short-chain fatty acids (SCFA) concentration using gas chromatography and to determine the abundance and composition of microbiota using 16S sequencing. Villi length of ileal samples was measured, while L-cell and T-cell densities were determined using immuno-histochemistry. The MEP treatment increased the amount of water-soluble arabinoxylans (AX) and reduced their molecular weight while retaining their polymer behavior. The WE fraction significantly (P < 0.05) increased FI by 13.8% and BWG by 14.7% during the first wk post hatch when compared to NC. No significant effect on FCR was recorded during the trial. The WE increased the abundance of Enterococcus durans and Candidatus arthromitus in the ileum and of bacteria within the Lachnospiraceae and Ruminococcaceae families, containing abundant butyrate-producing bacteria, in the ceca. It also increased the concentration of SCFA in the ceca, decreased the T-lymphocyte infiltration in the intestinal mucosa, and increased the glucagon-like-peptide-2 (GLP-2)-producing L-cell density in the ileal epithelium compared with WC and NC. No significant effects were observed on villi length. These results showed that AX present in the WE fraction altered the microbiota composition towards butyrate producers in the ceca. Butyrate may be responsible for the reduction of inflammation, as suggested by the decrease in T-lymphocyte infiltration, which may explain the higher feed intake leading to improved animal growth.

Bacillus subtilis strain specificity affects performance improvement in broilers.

The study reports the effects on broiler performance of a newly isolated Bacillus subtilis strain, which is phylogenetically not closely related to already well-described strains of B. subtilis. In the first experiment, birds were reared in battery cages and exposed to C. perfringens. An increase in growth performance was observed with the strain when compared to the challenged animals. Three additional growth trials were conducted to 35 d of age, in different rearing conditions (genetic breeds, corn-soybean meal-based diet with or without animal proteins, in presence or absence of phytase, on fresh or used litter) to investigate the efficacy and the specificity of this new B. subtilis strain on the improvement of BWG and FCR of broilers in comparison with a B. subtilis-based DFM already used in the field. Whatever the rearing conditions tested, the new B. subtilis strain led to an average 3.2% improvement in feed conversion ratio or bodyweight. Comparatively, the commercial Bacillus strain significantly improved broiler performance in only one trial out of 3 with an average improvement reaching 2%. All these results indicate that this new B. subtilis strain consistently improves broiler performances.

A meta-analysis to assess the effect of the composition of dietary fat on α-tocopherol blood and tissue concentration in pigs.

A meta-analysis based on the results from 13 selected publications was performed to assess the effect of dietary fat supplementation (quantity and fatty acid composition) on α-tocopherol (TOL) concentration in 4 pig tissues (blood, liver, muscle, and adipose tissue). Dietary fat supplementation was defined by the quantity of fat added to the basal diet and its fatty acid profile. After standardization of tissue TOL concentration (as the dependent variable), statistical analyses were performed using multiple nonlinear regression, data partitioning, and partial least squares regression with 7 predictor variables including added vitamin E (VE), added fat, PUFA (% fat), MUFA (% fat), SFA (% fat), omega-3 fatty acids (-3; % fat), and omega-6 fatty acids (-6; % fat). The statistical analyses first showed that the VE level in the diet was the main factor that modulates tissue TOL concentration. The dose-response relationship followed a logarithmic curve, with a saturation of tissue TOL concentration in all the studied tissues. Moreover, the amount of dietary fat, at least up to 20%, was not linearly correlated with tissue TOL concentration, considering that the main fatty acid classes, MUFA and, to a lesser extent, SFA, were positively associated with tissue TOL concentrations. Finally, this study suggests that the inclusion of -3 fatty acids in the diet may decrease tissue and, more precisely, blood TOL concentration.

Kinetics of microbial methionine metabolism in continuous cultures administered different methionine sources.

The Met precursor 2-hydroxy-4-(methylthio) butanoic acid (HMB) is expected to be more extensively degraded in the rumen than its isopropyl ester (HMBi). A control and 3 isomolar treatments-0.097% dl-methionine, 0.11% HMBi (HMBi), and 0.055% HMBi plus 0.048% Met (Met + HMBi)-were dosed every 8h simultaneously with 3-times-daily feeding into continuous cultures. Starting on d 9, for 6 consecutive doses, both [1-(13)C]-l-Met and [methyl-(2)H3]-l-Met replaced part of the unlabeled dl-Met, [(13)C5]-dl-HMBi replaced a portion of the unlabeled dl-HMBi, and [1-(13)C]-l-Met plus [(13)C5]-dl-HMBi replaced a portion of the respective unlabeled doses for the Met + HMBi treatment. After the sixth dose (d 11), unlabeled Met or HMBi provided 100% of the doses to follow elimination kinetics of the labels in HMBi, free Met, and bacterial Met compartments. The free [1-(13)C]-l-Met recycled more and was recovered in bacterial Met to a lesser extent than was the free [methyl-(2)H3]-l-Met recycling and that was recovered in bacterial Met. Increasing HMBi inclusion (0, 50, and 100% substitution of the exogenously dosed Met on a molar equivalent basis) tended to increase HMBi escape from 54.7 to 71.3% for the 50 and 100% HMBi treatments, respectively. Despite HMBi substituting for and decreasing the dosage of Met, increasing HMBi increased accumulation of free Met in fermenter fluid. The HMBi (after de-esterification of the isopropyl group) presumably produces Met through the intermediate α-ketomethylthyiobutyrate with an aminotransferase that also has high affinity for branched-chain AA. We provide evidence that the HMBi-derived Met is likely released from bacterial cells and accumulates rather than being degraded, potentially as a result of lagging d-stereoisomer metabolism. More research is needed to evaluate racemization and metabolism of stereoisomers of HMBi, Met, and other AA in ruminal microbes.

Assessing the ruminal action of the isopropyl ester of 2-hydroxy-4-(methylthio) butanoic acid in continuous and batch cultures of mixed ruminal microbes.

In dairy rations, Met is often a limiting amino acid that is provided by rumen-undegradable protein and rumen-protected sources of Met. A Met precursor, 2-hydroxy-4-(methylthio) butanoic acid (HMB) has undergone considerable study for ruminal and postruminal metabolism, whereas its isopropyl ester (HMBi) has been evaluated primarily with respect to its supply of metabolizable Met rather than as a preformed source of Met for microbial metabolism. A control and 3 isomolar Met treatments-0.097% dl-Met, 0.048% dl-Met plus 0.055% HMBi (Met + HMBi treatment), and 0.11% HMBi-were pulse-dosed every 8h into continuous cultures simultaneously with feeding. Treatment had no effect on digestibilities of acid-detergent fiber or true organic matter. Digestibilities of neutral detergent fiber and hemicellulose were linearly decreased with increasing HMBi inclusion. Concentration of NH3-N tended to decrease linearly and quadratically, and NH3-N flow tended to decrease linearly, with increasing HMBi inclusion; in contrast, the proportion of bacterial N derived from NH3-N increased linearly. Peptide N increased linearly and tended to be affected quadratically (highest for the HMBi treatment). Acetate and propionate production both decreased with increasing HMBi, but acetate declined more such that acetate:propionate increased linearly. Isobutyrate production decreased, but isovalerate and valerate increased with increasing HMBi inclusion. Relative changes in population abundance were not detected by denaturing gradient gel electrophoresis. In the second study, which was done in batch culture, Met treatments consisted of control, 0.097% l-Met, 0.097% l-Met, 0.125% dl-HMBi, 0.098% dl-HMB, 0.250% dl-HMBi (2× HMBi), 0.049% dl-Met + 0.063% dl-HMBi (Met + HMBi), and 0.098% dl-HMB + 0.039% isopropanol. All of these Met treatments were unlabeled (i.e., at natural abundance of (13)C) but simultaneously dosed with equivalent dosages of [1-(13)C]-l-Met. All 8 treatments were inoculated with faunated or partially defaunated inocula. Protozoal abundance had minor effect on measurements. The unlabeled l-Met treatment had the lowest (13)C enrichment of Met in the microbial pellet followed by Met + HMBi and then d-Met or dl-HMB, which were lower than remaining treatments. The percentage of the [1-(13)C]-l-Met dose recovered in microbial Met was lowest for the l-Met treatment; intermediate for d-Met, dl-HMB (with or without isopropanol), and Met + HMBi treatments; and highest for HMBi, 2× HMBi, and control. Results suggest that racemization of d-Met lags behind l-Met. The similar conversions of the HMBi and 2× HMBi treatments compared with the control suggests a low degradation of HMBi to provide unlabeled Met to dilute the [1-(13)C]-l-Met dose for protein synthesis. The lack of treatment by time interaction suggests that these initial responses carried through during the 24h of incubation. The proportion of HMBi available to ruminal microbes can influence microbial metabolism, potentially through formation of l-Met.

Dietary fat modulates dl-α-tocopheryl acetate (vitamin E) bioavailability in adult cockerels.

1. A trial was designed to assess the effect of fat supplementation (amount and type of fatty acids) on vitamin E bioavailability in adult cockerels. 2. A total of 60 birds were force-fed three different diets: a semi-purified diet without added fat (Control diet) or supplemented with 3% fat as linseed (Linseed diet) or hydrogenated coconut oil (Coconut diet). The three experimental diets were also supplemented with dl-α-tocopheryl acetate to provide 40 mg vitamin E per bird. 3. After one week of depletion, blood was collected from the wing vein before (baseline) and 6, 12, 24 and 96 h after the gavage. Plasma samples were analysed for their α-tocopherol, cholesterol and triglycerides concentrations. 4. Results showed that the addition of 3% fat in the experimental diet increased post-gavage plasma α-tocopherol response by 153% for Linseed diet and by 75% for Coconut diet (P < 0.0001) compared to the Control group. Furthermore, the plasma α-tocopherol response observed with the Linseed diet was 44% greater than that observed with the Coconut diet (P < 0.0001). There was no effect of treatments on either plasma triglycerides (P = 0.91) or cholesterol (P = 0.45) responses. 5. In conclusion, this study shows that the addition of 3% fat to the diet significantly increases dl-α-tocopheryl acetate bioavailability in adult cockerels. Supplementation of fat rich in unsaturated fatty acids also leads to a higher dl-α-tocopheryl acetate bioavailability than fat rich in saturated fatty acids.

Effect of the type of dietary triacylglycerol fatty acids on α-tocopherol concentration in plasma and tissues of growing pigs.

A study was performed in growing pigs to evaluate the efficacy of α-tocopherol (Tol) concentration in plasma, muscle, liver, and adipose tissue following dietary supplementation with vitamin E (VE) and various sources of fat. The trial involved 96 piglets weaned at an average of 28 d of age. Piglets were fed for 2 wk a semipurified diet not supplemented with VE. Piglets were then randomly assigned to 5 isoenergetic semipurified diets with 100 IU/kg VE as dl-α-tocopheryl acetate: a control (CTRL) diet (with no added fat) and 4 other diets containing either 3% linseed oil (LIN), 3% hydrogenated coconut oil (COC), 3% olive oil (OLI), or 3% safflower oil (SAF) representing diets rich in n-3 PUFA, SFA, MUFA, and n-6 PUFA, respectively. After 49 d of treatment, pigs were killed and blood, muscle (longissimus dorsi), adipose tissue, and whole liver (without gallbladder) were collected and analyzed for their Tol concentrations. For all tissues, LIN and SAF diets led to lower (P < 0.02) Tol concentrations as compared to the CTRL diet: -63 and -67%, respectively. α-Tocopherol concentrations in plasma, liver, and adipose tissue were greater (P < 0.001) in the COC group as compared to the CTRL group. The OLI diet led to greater (P < 0.01) liver Tol concentration (+92%) as compared to the CTRL diet but had no significant effect on plasma, muscle, and adipose tissue Tol concentrations. There were significant correlations (P < 0.001) between plasma, muscle, and liver Tol concentrations (r > 0.78). These results show that supplementation with PUFA markedly decreases Tol concentration in blood and tissues of growing pigs, whereas SFA increase Tol content in blood, liver, and adipose tissue. Monounsaturated fatty acids only increase liver Tol concentrations. Therefore, increasing the amount of fat in the diet (from <0.1 to approximately 3.5%) and the type of dietary fatty acids supplemented with VE are key factors with regards to VE concentration in plasma and tissue. The Tol:PUFA needs to be carefully considered to meet the VE pigs requirement and to ensure an optimal Tol meat enrichment.

Wheat-barley-rye- or corn-fed growing pigs respond differently to dietary supplementation with a carbohydrase complex.

Thirty-six pigs (22 kg of BW) were used to evaluate a carbohydrase preparation, with xylanase and ß-glucanase as main activities, added to either wheat-barley-rye- (WBR) or corn-based diets on performance, intestinal environment, and nutrient digestibility. Pigs were offered 1 of 4 different dietary treatments for 27 d according to a factorial arrangement of treatments (a 2 × 2) with 2 cereal types (WBR or corn) and 2 levels of supplemental carbohydrase (0 or 0.01%). Pig growth and feed intake were individually measured every week until the end of the experiment when pigs were slaughtered to obtain samples of digesta and tissues. Cereal type affected performance only during wk 1, in which WBR improved ADG (590 vs. 440 g/d; P = 0.008) and G:F (0.61 vs. 0.43; P = 0.045) compared with corn. The WBR also increased the viscosity of the digestive contents in stomach (1.95 vs. 1.23 mPa·s; P = 0.001) and ileum (6.53 vs. 2.80 mPa·s; P = 0.001) and resulted in greater cecal starch digestibility (95.7 vs. 93.9%; P = 0.012). However, trends for a reduction in digestibility were observed for glucose in the nonstarch polysaccharide (NSP) fraction in the ileum (64.4 vs. 75.8%; P = 0.074) and galactose in the NSP fraction in the cecum (1.4 vs. 1.8%; P = 0.055). The use of the enzyme preparation increased ADFI during wk 2 (1,328 vs. 1,215 g/d; P = 0.028), and increased villus height (423 vs. 390 µm; P = 0.045) and tended to reduce relative pancreas weight (0.16 vs. 0.17% BW; P = 0.079) at d 27. The enzyme also improved cecal starch digestibility (95.5 vs. 94.1%; P = 0.043) and tended to improve ileal energy digestibility (61.3 vs. 53.7%; P = 0.090) and cecal glucose digestibility in the NSP fraction (76.0 vs. 54.5%; P = 0.055). However, it reduced the cecal digestibility of mannose in the NSP fraction (27.0 vs. 50.5%; P = 0.016). Interactions (P < 0.05) between cereal type and enzyme supplementation were observed for ADG and G:F during wk 2, BW and ADG during wk 3, and BW and ADFI over the whole trial; and also for villus-height-to-crypt-depth ratio and for cecal DM digestibility. In all instances, whereas the added enzyme had no effect in the case of the corn diet, improvements were observed with WBR. In conclusion, the multi-enzyme tested had different effects depending on the type of cereal present in the diet.

Effect of feeding different sources of rumen-protected methionine on milk production and N-utilization in lactating dairy cows.

Objectives of this study were to quantify production responses of lactating dairy cows to supplying absorbable Met as isopropyl-2-hydroxy-4-(methylthio)-butanoic acid (HMBi), or rumen-protected Met (RPM, Smartamine M; Adisseo, Alpharetta, GA) fed with or without 2-hydroxy-4-(methylthio)-butanoic acid (HMB), and to determine whether Met supplementation will allow the feeding of reduced dietary crude protein (CP). Seventy cows were blocked by parity and days in milk into 14 blocks and randomly assigned within blocks to 1 of the 5 dietary treatments based on alfalfa and corn silages plus high-moisture corn: 1 diet with 15.6% CP and no Met source (negative control); 3 diets with 15.6% CP plus 0.17% HMBi, 0.06% RPM + 0.10% HMB, or 0.06% RPM alone; and 1 diet with 16.8% CP and no Met supplement (positive control). Assuming that 50% of ingested HMBi was absorbed from the gastrointestinal tract and 80% of the Met in RPM was absorbed at intestine, the HMBi and RPM supplements increased metabolizable Met supply by 9 g/d and improved the Lys:Met ratio from 3.6 to 3.0. After a 2-wk covariate period during which all cows received the same diet, cows were fed test diets continuously for 12 wk. Diet did not affect dry matter intake (mean ± SD, 25.0±0.3 kg/d), body weight gain (0.59±0.2 kg/d), or milk yield (41.7±0.6 kg/d). However, feeding HMBi increased yield of energy-corrected milk and milk content of protein and solids-not-fat. Moreover, trends were observed for increased milk fat content and yield of fat and true protein on all 3 diets containing supplemental Met. Apparent N efficiency (milk N/N intake) was highest on the RPM treatment. Feeding 16.8% CP without a Met source elevated milk urea N and urinary excretion of urea N and total N and reduced apparent N efficiency from 34.5 to 30.2%, without improving production. Overall results suggested that feeding HMBi or RPM would give similar improvements in milk production and N utilization.

Quantitative effects of cell internalization of two types of ultrasmall superparamagnetic iron oxide nanoparticles at 4.7 T and 7 T.

PURPOSE: MRI coupled with the intravenous injection of ultrasmall superparamagnetic particles of iron oxides (USPIOs) is a promising tool for the study of neuroinflammation. Quantification of the approximate number of magnetically labelled macrophages may provide an effective and efficient method for monitoring inflammatory cells. The purpose of the present study was to characterise the relaxation properties of macrophages labelled with two types of USPIOs, at 4.7 T and 7 T. METHODS: USPIO-labelled bone-marrow-derived macrophage phantoms were compared with phantoms of free dispersed USPIOs with the same global iron concentration, using multi-parametric (T1, T2 and T2) quantitative MRI. The same protocol was then evaluated in living mice after intracerebral injection of iron-labelled macrophages vs free iron oxide. RESULTS: A linear relationship was observed among R1, R2 and R2 values and iron concentration in vitro at 4.7 T and at 7 T. At a given field, T1 and T2 relaxivities of both types of USPIOs decreased following internalisation into macrophages, while T2 relaxivities increased. CONCLUSION: There was fair overall agreement between the theoretical number of injected cells and the number estimated from T2 quantification and in vitro calibration curves, supporting the validity of the present in vitro calibration curves for in vivo investigation.

Lactobacillus by-products inhibit the growth and virulence of uropathogenic Escherichia coli.

Uropathogenic strains of Escherichia coli (UPEC) can colonize the vagina and cause infections within the entire urogenital tract, including those associated with urinary tract devices. Lactobacilli typically dominate the vaginal microbiota in healthy women, and studies have shown that they can inhibit UPEC growth and vaginal colonization. However, little is known regarding the mechanisms behind these effects. Using a luciferase-based reporter construct, gradients of lactic acid, hydrogen peroxide and spent culture supernatants (SCS) from urogenital probiotic strains Lactobacillus rhamnosus GR-1 and L. reuteri RC-14 were examined for their effects on growth and virulence factor expression in UPEC isolate C1212. In a dose- and pH- dependent manner, lactic acid, hydrogen peroxide and Lactobacillus SCS all strongly inhibited UPEC C1212 growth and increased the promoter activity of outer membrane proteins (OMPs) A and X, two porins normally upregulated in response to UPEC membrane stress. Lactic acid and the culture supernatants also downregulated the promoter activity of the major subunits of type 1 and P fimbriae, critical adherence factors within the urogenital tract. Our findings indicate that compounds secreted by lactobacilli likely protect the urogenital tract from UPEC colonization and infection by inhibiting growth, inducing stress and downregulating proteins critical for host attachment.

Short communication: effects of 2-hydroxy-4-(methylthio) butanoic acid isopropyl ester on milk production and composition of lactating Holstein dairy cows.

Sixteen multiparous Holstein cows were used to determine the effects of 2-hydroxy-4-(methylthio) butanoic acid isopropyl ester (HMBi: 0 vs. 1.26 g/kg of total ration dry matter (DM) and dietary crude protein (CP) concentration [14.7% (low) vs. 16.9% (standard), DM basis] on milk yield and composition using a replicated 4 x 4 Latin square design experiment with 4-wk periods. Cows were fed ad libitum a total mixed ration with a 1:1 forage-to-concentrate ratio (DM basis), and diets provided an estimated 6.71 and 1.86% lysine and methionine, respectively, in metabolizable protein for the low-protein diet and 6.74 and 1.82% in the standard protein diet. Dry matter intake, milk yield, and composition were measured during wk 4 of each period. There were no effects on DM intake, which averaged 24.7 kg/d. There was an interaction between dietary CP and HMBi for milk yield and 3.5% fat-corrected milk (FCM). Feeding HMBi decreased milk and FCM yield when fed with the low-CP diet but did not affect milk or FCM yield when fed with the standard CP diet. Feeding HMBi increased milk protein concentration regardless of diet CP concentration and increased milk protein yield when added to the standard CP diet but not the low-CP diet. The positive effect of HMBi on milk protein yield was only observed at the standard level of dietary CP, suggesting other factors limited the response to HMBi when dietary protein supply was restricted.

Influence of fish oil on ruminal biohydrogenation of C18 unsaturated fatty acids.

Dietary cis-9, trans-11-conjugated linoleic acid (CLA) is generally thought to be beneficial for human health. Fish oil added to ruminant diets increases the CLA concentration of milk and meat, an increase thought to arise from alterations in ruminal biohydrogenation of unsaturated fatty acids. To investigate the mechanism for this effect, in vitro incubations were carried out with ruminal digesta and the main biohydrogenating ruminal bacterium, Butyrivibrio fibrisolvens. Linoleic acid (LA) or alpha-linolenic acid (LNA) was incubated (1.67 g/l) with strained ruminal digesta from sheep receiving a 50:50 grass hay-concentrate ration. Adding fish oil (up to 4.17 g/l) tended to decrease the initial rate of LA (P=0.025) and LNA (P=0.137) disappearance, decreased (P<0.05) the transient accumulation of conjugated isomers of both fatty acids, and increased (P<0.05) the accumulation of trans-11-18:1. Concentrations of EPA (20:5n-3) or DHA (22:6n-3), the major fatty acids in fish oil, were low (100 mg/l or less) after incubation of fish oil with ruminal digesta. Addition of EPA or DHA (50 mg/l) to pure cultures inhibited the growth and isomerase activity of B. fibrisolvens, while fish oil had no effect. In contrast, similar concentrations of EPA and DHA had no effect on biohydrogenation of LA by mixed digesta, while the addition of LA prevented metabolism of EPA and DHA. Neither EPA nor DHA was metabolised by B. fibrisolvens in pure culture. Thus, fish oil inhibits ruminal biohydrogenation by a mechanism which can be interpreted partly, but not entirely, in terms of its effects on B. fibrisolvens.

A xylanase produced by the rumen anaerobic protozoan Polyplastron multivesiculatum shows close sequence similarity to family 11 xylanases from gram-positive bacteria.

We report for the first time the cloning and characterisation of a protozoal enzyme involved in plant cell wall polysaccharide degradation. A cDNA library was constructed from the ruminal protozoan Polyplastron multivesiculatum and a stable clone expressing xylanase activity was isolated. The encoded enzyme belongs to the glycoside hydrolase family 11, and phylogenetic analysis indicates a closer relationship with catalytic domains from Gram-positive bacteria than the other fibrolytic eukaryotes from the rumen, the anaerobic fungi.

Influence of sampling site on concentrations and carbohydrate-degrading enzyme activities of protozoa and bacteria in the rumen.

Four ruminally cannulated cows were used to assess the distribution of the concentrations and carbohydrate-degrading enzyme activities of the liquid-associated protozoa (LAP) and solid-associated bacteria (SAB) in the rumen. The cows were fed diets (7 kg of DM/d) of 100% hay (Diet F) or 60% barley plus 40% hay (Diet C) in a 2 x 2 crossover experimental design. Samples of ruminal digesta were collected successively from the dorsal (DS), ventral (VS), and anterior (AS) sacs 1h before and 3 h after the morning feeding on two sampling days with a 2-d interval. Irrespective of diet and sampling time, the greatest proportion of entodiniomorphs, representing the main population of protozoa, was found in the DS (40% mean; P < .05). Low pH values observed in the DS (P < .05) indicated higher fermentative activity in this site than in the other parts of the rumen. Protozoa may contribute more than previously thought to the high digestive potential present at the top of the rumen that has classically been attributed to bacteria. The specific activity of plant cell wall polysaccharide-degrading enzymes in the LAP was correspondingly greater (P < .05) for DS than for VS or AS. For the two diets and sampling times, specific activity of fibrolytic enzymes in the SAB tended to be less in the upper than in the lower parts of the rumen, and less in the VS than in the AS. This tendency became significant (P < .05) for total fibrolytic enzyme activity. Differences in bacterial colonization of particles among the three sampling sites may explain such differences in fibrolytic activity of the SAB. Data suggest a spatio-temporal complementary action of the bacteria and protozoa in ruminal plant cell wall degradation, at least with the barley diet, for which the number of protozoa was highest.