Ruminal protein degradability of a range of tropical pastures

The rumen degradability parameters of the diet selected by two to four oesophageal-fistulated Brahman steers grazing a range of tropical pastures were determined by incubation of extrusa in nylon bags suspended in the rumen of rumen-fistulated (RF) Brahman steers. The effective protein degradability (Edg) was determined by measuring the rate of disappearance of neutral detergent insoluble nitrogen (NDIN) less acid detergent insoluble nitrogen (ADIN) in the incubated extrusa. Six to eight RF steers also grazed each of the pastures along with the oesophageal-fistulated steers, to allow determination of key rumen parameters and rumen particulate matter fractional outflow rates (FOR). The seven pastures studied included: native tropical grass (C4) pasture (major species Heteropogon contortus and Bothriochloa bladhii), studied in the early wet (NPEW), the wet/dry transition (NPT) and the dry (NPD) seasons; introduced tropical grass (C4) pasture (Bothriochloa insculpta), studied in the mid wet season (BB); the introduced tropical legumes (C3), Lablab purpureus (LL) and Clitoria ternatea (BP); and the temperate grass (C3) pasture, ryegrass (Lolium multiflorum, RG). Using the measured particle FOR values in calculations, the Edg estimates were very high for both C4 and C3 species: 0.82–0.91 and 0.95–0.98 g/g crude protein (CP), respectively. Substitution of an assumed FOR (kp = 0.02/h) for the measured values for each pasture type did not markedly affect estimates of Edg. However, C4 tropical grasses had much lower effective rumen degradable protein (ERDP) fractions (23–66 g/kg DM) than the C3 pasture species RG and LL (356 and 243 g/kg DM, respectively). This was associated with a lower potential degradability and degradation rate of organic matter (OM) in sacco, lower in vitro organic matter digestibility (IVOMD) and CP concentrations in the extrusa, and lower ammonia-N and branched-chain fatty acid concentrations in rumen fluid for the tropical grasses. As tropical grass pastures senesced, there was a decline in Edg, the ERDP and rumen undegradable protein (UDP) fractions, the potential degradability and degradation rate of OM and the IVOMD. These results provide useful data for estimating protein supply to cattle grazing tropical pastures.

In vitro detection and primary cultivation of bacteria producing materials inhibitory to ruminal methanogens.

A novel method for screening bacterial isolates for their potential to inhibit the growth of ruminal methanogenic Archaea was developed using a modification of the soft agar overlay technique, formally used for the isolation of lytic bacteriophages. This method may be used in the specific, hydrogen-rich conditions required for the growth of ruminal methanogenic Archaea.

Morphological diversity of ruminal bacteriophages from sheep and cattle

Large numbers of bacteriophages (2 x 10(7) to 1 x 10(8)/ml) were present in ruminal fluid from sheep and cattle. Twenty-six distinct types were identified and placed in three morphological groups; several phages possessed unusual structural features. The large numbers and diversity of phages observed indicates a possible role in bacterial lysis and hence in the population dynamics of the ruminal bacteria.

A lack of predatory interaction between rumen ciliate protozoa and Shiga-toxin producing Escherichia coli

Aims: To investigate interactions between rumen protozoa and Shiga toxin-producing Escherichia coli (STEC) and to ascertain whether it is likely that rumen protozoa act as ruminant hosts for STEC. Methods and Results: The presence of stx genes in different microbial fractions recovered from cattle and sheep rumen contents and faeces was examined using PCR. In animals shedding faecal STEC, stx genes were not detected in the rumen bacterial or rumen protozoal fractions. Direct interactions between ruminal protozoa and STEC were investigated by in vitro co-incubation. Rumen protozoa did not appear to ingest STEC, a STEC lysogen or non-STEC E. coli populations when co-incubated. Conclusions: The ruminal environment is unlikely to be a preferred habitat for STEC. Bacterial grazing by rumen protozoa appears to have little, if any, effect on STEC populations. Significance and Impact of the Study: This study indicates that ruminal protozoa are unlikely to be a major factor in the survival of STEC in ruminants. They appear as neither a host that protects STEC from the ruminal environment nor a predator that might reduce STEC numbers.

Evaluation of rumen fatty acid hydrogenation intermediates and differences in bacterial communities after feeding wheat- or corn-based dried distillers grains to feedlot cattle

The effect of partially replacing rolled barley (86.6% of control diet) with 20% wheat dried distillers grains plus solubles (DDGS), 40% wheat DDGS, 20% corn DDGS, or 40% corn DDGS (dietary DM basis) on rumen fluid fatty acid (FA) composition and some rumen bacterial communities was evaluated using 100 steers (20 per treatment). Wheat DDGS increased the 11t-to 10t-18:1 ratio (P < 0.05) in rumen fluid and there was evidence that the conversion of trans-18:1 to 18:0 was reduced in the control and wheat DDGS diets but not in the corn DDGS diet. Bacterial community profiles obtained using denaturing gradient gel electrophoresis and evaluated by Pearson correlation similarity matrices were not consistent for diet and, therefore, these could not be linked to different specific rumen FA. This inconsistency may be related to the nature of diets fed (dominant effect of barley), limited change in dietary composition as the result of DDGS inclusion, large animal-to-animal variation, and possibly additional stress as a result of transport just before slaughter. Ruminal densities of a key fiber-digesting bacteria specie that produces 11t-18:1 from linoleic and linolenic acids (Butyrivibrio fibrisolvens), and a lactate producer originally thought responsible for production of 10t, 12c-18:2 (Megasphaera elsdenii) were not influenced by diet (P > 0.05).

Archaea in the foregut of macropod marsupials: PCR and amplicon sequence-based observations

Aims To investigate, using culture-independent techniques, the presence and diversity of methanogenic archaea in the foregut of kangaroos. Methods and Results DNA was extracted from forestomach contents of 42 kangaroos (three species), three sheep and three cattle. Four qualitative and quantitative PCR assays targeting the archaeal domain (16S rRNA gene) or the functional methanogenesis gene, mcrA, were used to determine the presence and population density of archaea in kangaroos and whether they were likely to be methanogens. All ruminal samples were positive for archaea, produced PCR product of expected size, contained high numbers of archaea and high numbers of cells with mcrA genes. Kangaroos were much more diverse and contradictory. Fourteen kangaroos had detectable archaea with numbers 10- to 1000-fold fewer than sheep and cattle. Many kangaroos that did not possess archaea were positive for the mcrA gene and had detectable numbers of cells with this gene and vice versa. DNA sequence analysis of kangaroos' archaeal 16S rRNA gene clones show that many methanogens were related to Methanosphaera stadmanae. Other sequences were related to non-methanogenic archaea (Thermoplasma sp.), and a number of kangaroos had mcrA gene sequences related to methane oxidising archaea (ANME). Conclusions Discrepancies between qualitative and quantitative PCR assays for archaea and the mcrA gene suggest that the archaeal communities are very diverse and it is possible that novel species exist. Significance and Impact of the Study Archaea (in general) were below detectable limits in many kangaroos, especially Red kangaroos; when present they are in lower numbers than in ruminants, and the archaea are not necessarily methanogenic. The determination of why this is the case in the kangaroo foregut could assist in reducing emissions from other ecosystems in the future.

Persistence of orally administered Megasphaera elsdenii and Ruminococcus bromii in the rumen of beef cattle fed a high grain (barley) diet

When cattle are fed grain, acidotic ruminal conditions and decreased efficiency in starch utilisation can result from the rapid production and accumulation of lactic acid in the rumen. The efficacy of drenching cattle with Megasphaera elsdenii and Ruminococcus bromii to improve animal performance was investigated. A feedlot trial was undertaken with 80 Bos indicus crossbred steers (initial liveweight 347.1 (s.d. 31.7) kg) in 10 pens in a randomised complete block design. An empty-pen-buffer was maintained between treated (inoculated) and untreated (control) groups to avoid transfer of inoculant bacteria to the control steers. Inoculated steers were orally drenched with M. elsdenii YE34 and R. bromii YE282, and populations increased rapidly over 3-14 days. The steers were fed for a total of 70 days with commercial, barley-based, feedlot rations. High growth rates (1.91 kg per day) were achieved throughout the experiment in both the inoculated and control steers. Intakes averaged 21.3 g dry matter (DM) per kg liveweight per day. There was probably no acidosis achieved in this trial following challenge (i.e. no change in pH occurred). There were no differences in any production or carcass measurements between the control and inoculated steers overall. However, the control group acquired dense ruminal populations of M. elsdenii by Day 14, while R. bromii populations established at high densities within the first 2 weeks but then declined and were undetectable by Day 50. R. bromii appears to be only transiently dominant, and once its dominance waned, it appeared that Ruminobacter spp. established in the rumen. Ruminobacter spp. became dominant between 14 and 28 days in all the steers examined and persisted through to the end of the study. These Ruminobacter spp. may be of future interest in the development of probiotics for grain-fed cattle.

Effect of varying the proportion of molasses in the diet on intake, digestion and microbial protein production by steers

The present experiment was conducted to determine the efficiency of microbial protein production in the rumen and intake by cattle fed high-molasses diets. Intake and microbial crude protein (MCP) production were measured along with the concentration of rumen ammonia-nitrogen (N) and volatile fatty acids (VFA), pH and the rate of digestion of roughage in the rumen. Eight Brahman crossbred steers weighing 211 ± 19.3 (± s.d.) kg were used in a double 4 × 4 Latin square design. Steers were allocated to one of four total mixed rations: control (pangola hay only), 25M (25% molasses/urea mix + 75% hay), 50M (50% molasses/urea + 50% hay), and 75M (75% molasses/urea + 25% hay). The production and efficiency of production of MCP (EMCP) of the diet increased quadratically as the level of molasses in the diet increased. The EMCP from the molasses/urea mix was estimated as 166 g MCP/kg digestible organic matter (DOM), a relatively high value. Intake of dry matter (DM) and DOM increased quadratically, reaching a peak when molasses was ~50% (as fed) of the ration. Digestibility of DM increased quadratically and that of neutral detergent fibre decreased linearly with increasing level of molasses in the diet. Molasses inclusion in the diet had no effect on rumen pH, ammonia and VFA concentration in the rumen fluid, plasma urea-N, urine pH or ruminal fractional outflow rate of ytterbium-labelled particles and Cr-EDTA. It was concluded that a diet with a high level of molasses (>50%) and supplemented with adequate N had high EMCP, and that low MCP production was not a factor limiting intake or performance of cattle consuming high-molasses diets.

Effect of varying the proportion of molasses in the diet on intake, digestion and microbial protein production by steers

The present experiment was conducted to determine the efficiency of microbial protein production in the rumen and intake by cattle fed high-molasses diets. Intake and microbial crude protein (MCP) production were measured along with the concentration of rumen ammonia-nitrogen (N) and volatile fatty acids (VFA), pH and the rate of digestion of roughage in the rumen. Eight Brahman crossbred steers weighing 211 ± 19.3 (± s.d.) kg were used in a double 4 x 4 Latin square design. Steers were allocated to one of four total mixed rations: control (pangola hay only), 25M (25% molasses/urea mix + 75% hay), 50M (50% molasses/urea + 50% hay), and 75M (75% molasses/urea + 25% hay). The production and efficiency of production of MCP (EMCP) of the diet increased quadratically as the level of molasses in the diet increased. The EMCP from the molasses/urea mix was estimated as 166 g MCP/kg digestible organic matter (DOM), a relatively high value. Intake of dry matter (DM) and DOM increased quadratically, reaching a peak when molasses was ∼50% (as fed) of the ration. Digestibility of DM increased quadratically and that of neutral detergent fibre decreased linearly with increasing level of molasses in the diet. Molasses inclusion in the diet had no effect on rumen pH, ammonia and VFA concentration in the rumen fluid, plasma urea-N, urine pH or ruminal fractional outflow rate of ytterbium-labelled particles and Cr-EDTA. It was concluded that a diet with a high level of molasses (>50%) and supplemented with adequate N had high EMCP, and that low MCP production was not a factor limiting intake or performance of cattle consuming high-molasses diets.

Programming rumen bacterial communities in newborn Merino lambs

Establishment of the rumen microbiome can be affected by both early-life dietary measures and rumen microbial inoculation. This study used a 2 × 3 factorial design to evaluate the effects of inclusion of dietary fat type and the effects of rumen inoculum from different sources on ruminal bacterial communities present in early stages of the lambs’ life. Two different diets were fed ad libitum to 36 pregnant ewes (and their lambs) from 1 month pre-lambing until weaning. Diets consisted of chaffed lucerne and cereal hay and 4% molasses, with either 4% distilled coconut oil (CO) provided as a source of rumen-active fat or 4% Megalac® provided as a source of rumen-protected fat (PF). One of three inoculums was introduced orally to all lambs, being either (1) rumen fluid from donor ewes fed the PF diet; (2) rumen fluid from donor ewes fed CO; or (3) a control treatment of MilliQ-water. After weaning at 3 months of age, each of the six lamb treatment groups were grazed in spatially separated paddocks. Rumen bacterial populations of ewes and lambs were characterised using 454 amplicon pyrosequencing of the V3/V4 regions of the 16S rRNA gene. Species richness and biodiversity of the bacterial communities were found to be affected by the diet in ewes and lambs and by inoculation treatment of the lambs. Principal coordinate analysis and analysis of similarity (ANOSIM) showed between diet differences in bacterial community groups existed in ewes and differential bacterial clusters occurred in lambs due to both diet and neonatal inoculation. Diet and rumen inoculation acted together to clearly differentiate the bacterial communities through to weaning, however the microbiome effects of these initial early life interventions diminished with time so that rumen bacterial communities showed greater similarity 2 months after weaning. These results demonstrate that ruminal bacterial communities of newborn lambs can be altered by modifying the diet of their mothers. Moreover, the rumen microbiome of lambs can be changed by diet while they are suckling or by inoculating their rumen, and resulting changes in the rumen bacterial microbiome can persist beyond weaning.

Probiotic Bacillus amyloliquefaciens Strain H57 Improves the Performance of Pregnant and Lactating Ewes Fed a Diet Based on Palm Kernel Meal

Probiotic supplements are single or mixed strain cultures of live microorganisms that benefit the host by improving the properties of the indigenous microflora (Seo et al 2010). In a pilot study at the University of Queensland, Norton et al (2008) found that Bacillus amyloliquefaciens Strain H57 (H57), primarily investigated as an inoculum to make high-quality hay, improved feed intake and nitrogen utilisation over several weeks in pregnant ewes. The purpose of the following study was to further challenge the potential of H57 -to show it survives the steam-pelleting process, and that it improves the performance of ewes fed pellets based on an agro-industrial by-product with a reputation for poor palatability, palm kernel meal (PKM), (McNeill 2013). Thirty-two first-parity White Dorper ewes (day 37 of pregnancy, mean liveweight = 47.3 kg, mean age = 15 months) were inducted into individual pens in the animal house at the University of Queensland, Gatton. They were adjusted onto PKM-based pellets (g/kg drymatter (DM): PKM, 408; sorghum, 430; chick pea hulls, 103; minerals and vitamins; Crude protein, 128; ME: 11.1MJ/kg DM) until day 89 of pregnancy and thereafter fed a predominately pelleted diet incorporating with or without H57 spores (10 9 colony forming units (cfu)/kg pellet, as fed), plus 100g/ewe/day oaten chaff, until day 7 of lactation. From day 7 to 20 of lactation the pelleted component of the diet was steadily reduced to be replaced by a 50:50 mix of lucerne: oaten chaff, fed ad libitum, plus 100g/ewe/day of ground sorghum grain with or without H57 (10 9 cfu/ewe/day). The period of adjustment in pregnancy (day 37-89) extended beyond expectations due to some evidence of mild ruminal acidosis after some initially high intakes that were followed by low intakes. During that time the diet was modified, in an attempt to improve palatability, by the addition of oaten chaff and the removal of an acidifying agent (NH4Cl) that was added initially to reduce the risk of urinary calculi. Eight ewes were removed due to inappetence, leaving 24 ewes to start the trial at day 90 of pregnancy. From day 90 of pregnancy until day 63 of lactation, liveweights of the ewes and their lambs were determined weekly and at parturition. Feed intakes of the ewes were determined weekly. Once lambing began, 1 ewe was removed as it gave birth to twin lambs (whereas the rest gave birth to a single lamb), 4 due to the loss of their lambs (2 to dystocia), and 1 due to copper toxicity. The PKM pellets were suspected to be the cause of the copper toxicity and so were removed in early lactation. Hence, the final statistical analysis using STATISTICA 8 (Repeated measures ANOVA for feed intake, One-way ANOVA for liveweight change and birth weight) was completed on 23 ewes for the pregnancy period (n = 11 fed H57; n = 12 control), and 18 ewes or lambs for the lactation period (n = 8 fed H57; n = 10 control). From day 90 of pregnancy until parturition the H57 supplemented ewes ate 17 more DM (g/day: 1041 vs 889, sed = 42.4, P = 0.04) and gained more liveweight (g/day: 193 vs 24.0, sed = 25.4, P = 0.0002), but produced lambs with a similar birthweight (kg: 4.18 vs 3.99, sed = 0.19, P = 0.54). Over the 63 days of lactation the H57 ewes ate similar amounts of DM but grew slower than the control ewes (g/day: 1.5 vs 97.0, sed = 21.7, P = 0.012). The lambs of the H57 ewes grew faster than those of the control ewes for the first 21 days of lactation (g/day: 356 vs 265, sed = 16.5, P = 0.006). These data support the findings of Norton et al (2008) and Kritas et al (2006) that certain Bacillus spp. supplements can improve the performance of pregnant and lactating ewes. In the current study we particularly highlighted the capacity of H57 to stimulate immature ewes to continue to grow maternal tissue through pregnancy, possibly through an enhanced appetite, which appeared then to stimulate a greater capacity to partition nutrients to their lambs through milk, at least for the first few weeks of lactation, a critical time for optimising lamb survival. To conclude, H57 can survive the steam pelleting process to improve feed intake and maternal liveweight gain in late pregnancy, and performance in early lactation, of first-parity ewes fed a diet based on PKM.

Responses to various protein and energy supplements by steers fed low-quality tropical hay. 1. Comparison of response surfaces for young steers

Response curves were established for different supplements, offered at intakes ranging from 0 to 20 g/kg liveweight (W).day to young Bos indicus crossbred steers fed low-quality Rhodes grass (Chloris gayana) hay ad libitum in two pen experiments. Supplements included protein meals of varying rumen-degradability (cottonseed meal (CSM) or fishmeal), as well as ‘energy sources’ comprising grains of high and low ruminal starch degradability (barley and sorghum) and a highly fermentable sugar source (molasses), with all diets adjusted for rumen-degradable nitrogen and mineral content. Unsupplemented steers gained 0.08 and 0.15 kg/day, in Experiments 1 and 2, respectively. Growth of steers increased linearly with intake of ‘energy source’ supplements in increasing order of molasses, sorghum and barley (all differences P < 0.05). Steer growth rate also increased linearly with fishmeal, albeit over a narrow intake range (0–4.1 g/kg W.day), whereas the response with CSM was asymptotic, showing a steep response at low intake before levelling at ~1.2 kg/day. All supplement types were associated with a linear reduction in hay intake by the steers (energy substitution) where the reduction was greater (P < 0.05) for barley and molasses (not different) than for sorghum (P < 0.05), and for fishmeal compared with CSM (P < 0.05). In concurrent metabolism studies with the same rations, organic matter digestibility of the total ration (561–578 g/kg DM, unsupplemented) was increased linearly by barley and molasses (both P < 0.05) but was unaffected by CSM and sorghum supplements. The efficiency of microbial protein synthesis in steers increased linearly, from 91 g microbial crude protein/kg digestible organic matter (unsupplemented), in both molasses and CSM-supplemented steers, with the trend for a higher response to molasses (P = 0.05), and appeared most closely related to digestible organic matter intake. The response curves from these studies provide the practical framework upon which to formulate rations for cattle grazing low-quality forages.