Rumen protozoa are rich in polyunsaturated fatty acids due to the ingestion of chloroplasts

Within this study, we investigated whether the polyunsaturated fatty acids (PUFA)-rich nature of rumen protozoa is a consequence of ingestion of PUFA-rich chloroplasts. Four Hereford × Friesian steers were offered hay [low 18:3 (n-3) and low chlorophyll concentration] followed by freshly cut perennial ryegrass [high 18:3 (n-3) and high chlorophyll concentration] for 16 days. On the 14th and 16th days, rumen protozoa as well as attached and planktonic bacteria were fractionated 1 h before (−1 h), 2 and 6 h postfeeding, and their fatty acid concentrations determined. Protozoa fractionated from fresh grass-fed steers were richer ( P <0.05) in PUFA, except conjugated linoleic acid, for all time points compared with those from hay-fed steers. Protozoal density was higher ( P <0.05) for grass compared with hay. Entodinomorphid abundance was 3.4 times higher on fresh grass ( P <0.01) compared with hay. Confocal microscopy and transmission electron microscopy confirmed that Epidinium spp. were commonly saturated with intracellular cytoplasmic chloroplasts. These data suggest that engulfment of chloroplasts is a major contributor to the high 18:3 (n-3) concentration of protozoa.     

Feeding level and individual metabolic rate affect δ13C and δ15N values in carp: implications for food web studies

Stable isotope analyses are often used to calculate relative contributions of multiple food sources in an animals diet. One prerequisite for a precise calculation is the determination of the diet-tissue fractionation factor. Isotopic ratios in animals are not only affected by the composition of the diet, but also by the amount of food consumed. Previous findings regarding the latter point are controversial. As stable isotope analyses have often been used to investigate aquatic food webs, an experiment with carp (Cyprinus carpio L.) was carried out to test the influence of the feeding level and individual metabolic rate on ¹³C and ¹⁵N values of the whole body. After an initial phase, 49 carp were assigned randomly to four groups and fed the same diet at different levels for 8 weeks. For 15 fish, the energy budget was determined by indirect calorimetry. Feed and individual fish were analysed for their proximate composition, gross energy content and ¹³C and ¹⁵N values. ¹³C and ¹⁵N values differed significantly at different feeding levels. While ¹³C values of the lipids and ¹⁵N values decreased with increasing feeding rate, ¹³C values of the lipid-free matter showed a non-linear pattern. Data obtained from fish held in the respirometric system revealed a relationship between ¹³C values and the percentage retention of metabolizable energy. Our results show that reconstructing the diets of fish from the isotopic ratios when the feeding level and individual metabolic rates are unknown would introduce an error into the data used for back-calculation of up to 1 for both ¹³C and ¹⁵N values and may have substantial effects on the results of calculated diets. As other workers have pointed out, the development and application of stable isotopes to nutritional ecology studies is a field in its infancy and gives rise to erroneous, misleading results without nutritional, physiological and ecological knowledge.     

A neutral model of transcriptome evolution

Microarray technologies allow the identification of large numbers of expression differences within and between species. Although environmental and physiological stimuli are clearly responsible for changes in the expression levels of many genes, it is not known whether the majority of changes of gene expression fixed during evolution between species and between various tissues within a species are caused by Darwinian selection or by stochastic processes. We find the following: (1) expression differences between species accumulate approximately linearly with time; (2) gene expression variation among individuals within a species correlates positively with expression divergence between species; (3) rates of expression divergence between species do not differ significantly between intact genes and expressed pseudogenes; (4) expression differences between brain regions within a species have accumulated approximately linearly with time since these regions emerged during evolution. These results suggest that the majority of expression differences observed between species are selectively neutral or nearly neutral and likely to be of little or no functional significance. Therefore, the identification of gene expression differences between species fixed by selection should be based on null hypotheses assuming functional neutrality. Furthermore, it may be possible to apply a molecular clock based on expression differences to infer the evolutionary history of tissues.     

The dynamics of major fibrolytic microbes and enzyme activity in the rumen in response to short- and long-term feeding of Sapindus rarak saponins

AIMS: To investigate the short- and long-term effects of an extract of Sapindus rarak saponins (SE) on the rumen fibrolytic enzyme activity and the major fibrolytic micro-organisms. METHODS AND RESULTS: Two feeding trials were conducted. In the short-term trial, four fistulated goats were fed a basal diet containing sugar cane tops and wheat pollard (65:35, w/w) and were supplemented for 7 days with SE at a level of 0.6 g kg(-1) body weight. Rumen liquor was taken before, during and after SE feeding. In the long-term trial, 28 sheep were fed the same basal diet as the goats and were supplemented for 105 days with 0.24, 0.48 and 0.72 g kg(-1) body mass of the extract. Rumen liquor was taken on days 98 and 100. Protozoal numbers were counted under the microscope. Cell wall degradation was determined by enzyme assays and the major fibrolytic micro-organisms were quantified by dot blot hybridization. Sapindus extract significantly depressed rumen xylanase activity in both trials and carboxymethylcellulase activity in the long-term trial (P < 0.01). Fibrobacter sp. were not affected by the SE in both trials, while ruminococci and the anaerobic fungi showed a short-term response to the application of saponins. Protozoal counts were decreased only in the long-term trial with sheep. CONCLUSION: These data suggest that there is an adaptation of Ruminococcus albus, Ruminococcus flavefaciens and Chytridiomycetes (fungi) to saponin when fed over a long period. The fact that no correlation between the cell wall degrading enzyme activities and the cell wall degrading micro-organisms was observed suggests that the organisms tracked in this experiment are not the only key players in ruminal cell wall degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: Sapindus rarak saponins partially defaunate the rumen flora. Their negative effect on cell wall degradation, however, is not related to rumen organisms currently recognized as the major cell wall degrading species. The adaptation of microbes in the long-term feeding experiment suggests that the results from short-term trial on the ruminal microbial community have to be interpreted carefully.     

Does Dietary Mitigation of Enteric Methane Production Affect Rumen Function and Animal Productivity in Dairy Cows?

It has been suggested that the rumen microbiome and rumen function might be disrupted if methane production in the rumen is decreased. Furthermore concerns have been voiced that geography and management might influence the underlying microbial population and hence the response of the rumen to mitigation strategies. Here we report the effect of the dietary additives: linseed oil and nitrate on methane emissions, rumen fermentation, and the rumen microbiome in two experiments from New Zealand (Dairy 1) and the UK (Dairy 2). Dairy 1 was a randomized block design with 18 multiparous lactating cows. Dairy 2 was a complete replicated 3 x 3 Latin Square using 6 rumen cannulated, lactating dairy cows. Treatments consisted of a control total mixed ration (TMR), supplementation with linseed oil (4% of feed DM) and supplementation with nitrate (2% of feed DM) in both experiments. Methane emissions were measured in open circuit respiration chambers and rumen samples were analyzed for rumen fermentation parameters and microbial population structure using qPCR and next generation sequencing (NGS). Supplementation with nitrate, but not linseed oil, decreased methane yield (g/kg DMI; P<0.02) and increased hydrogen (P<0.03) emissions in both experiments. Furthermore, the effect of nitrate on gaseous emissions was accompanied by an increased rumen acetate to propionate ratio and consistent changes in the rumen microbial populations including a decreased abundance of the main genus Prevotella and a decrease in archaeal mcrA (log₁₀ copies/ g rumen DM content). These results demonstrate that methane emissions can be significantly decreased with nitrate supplementation with only minor, but consistent, effects on the rumen microbial population and its function, with no evidence that the response to dietary additives differed due to geography and different underlying microbial populations.     

Effects of feed intake on enteric methane emissions from sheep fed fresh white clover (Trifolium repens) and perennial ryegrass (Lolium perenne) forages

Published analyses of enteric methane (CH₄) emissions from sheep and cattle show an inverse relationship between feed intake and CH₄ yield (g CH₄/kg dry matter (DM) intake), which suggests opportunities for reducing CH₄ emissions from feed eaten and per unit of animal production. Most relationships between feed intake and CH₄ yield have been based on animals fed conserved feeds, especially silages and grains. Our research is a series of experiments with fresh white clover (Trifolium repens) and perennial ryegrass (Lolium perenne; ryegrass) forages fed to sheep at a range of feed intake levels. This study was comprised of four experiments where good quality freshly harvested white clover or ryegrass were fed to sheep over a three-fold range in DM intake, and CH₄ emissions were measured in respiration chambers for two consecutive days in each experiment. Measurements were made from 16 sheep in Experiment 1 (fed at 1.6 × metabolizable energy requirements for maintenance; ME_m), 28 sheep in Experiment 2 (at 0.8 and 2.0 × ME_m), eight sheep and two measurement periods in Experiment 3 (at 1.6 × ME_m), and 30 sheep in Experiment 4 (fed at 0.8, 1.2, 1.6, 2.0 and 2.5 × ME_m). Prior to each experiment, sheep had a 10 d acclimatization period to diets. Apparent digestibility was measured over 7 d from sheep in Experiments 1, 3 and 4, along with collection of rumen digesta for volatile fatty acid (VFA) determination. Although CH₄ yields differed when sheep were fed white clover or ryegrass at similar intakes, the differences were inconsistent and mean values similar across all experiments. This, and a similar structure of all experiments, enabled combined analysis of data from all four experiments using the restricted maximum likelihood (REML) procedure to estimate effects of feed intake level on digestibility, digestible nutrient intake, gas emissions, and VFA concentrations in the rumen. The REML analysis showed that when DM intake increased from 0.40 to 1.60 kg/d, the predicted responses were an increase in CH₄ production (g/d) of 187% (12.4–35.6 g/d; P<0.001), and a decline in CH₄ yield of 21% (25.6–20.2 g/kg DM intake; P<0.001). High feed intake levels were associated with increased molar proportions (mM of total VFA) of propionate from 0.17 to 0.21 (P=0.038). Single and multiple regressions were completed on the data from all experiments, with organic matter (OM) intake predicting 0.87 of the variation in CH₄ production, and molar proportion of propionate predicting 0.60 of the variation in CH₄ yield. Increasing feed intakes by 1 kg/d of DM reduced CH₄ yield by 4.5 g/kg DM intake. Plant chemical composition was weakly related to CH₄ yield. High intakes of fresh forages will lower CH₄ yield from fermentation, but effects of feed composition on CH₄ emissions were minor. The interaction between effects of feed intake and rumen function requires further investigation to understand relationships with CH₄ emissions.     

Effect of Quillaja saponaria saponins and Yucca schidigera plant extract on growth of Escherichia coli

Escherichia coli K-12 was exposed to Quillaja saponaria saponins from various commercial firms (Sigma, Roth and Nor-feed) and to an extract of Yucca schidigera plant powder (DK Sarsaponin 30) at different concentrations (0·05–1·0% w/v). A concentration-dependent response was observed. Quillaja saponaria saponins from Sigma increased growth up to 0·1% (w/v) level, whereas Nor-feed and Roth saponins produced maximum growth at a much higher level (0·5 and 0·75%, w/v, respectively). These results suggest that quillaja saponins from various sources differ in their biological activity, although all three saponins had the same content of vanillin-sulphuric acid reactive moieties. The lyophilized water extract from the DK Sarsaponin powder showed maximum growth at 0·1% (w/v) level. The levels at which maximum growth was observed did not change on subjecting the quillaja or yucca saponins to heat treatment in an autoclave (121 °C for 30 min). All the saponins and the plant extract increased growth of Escherichia coli up to a certain concentration and thereafter decreased growth. In spite of the decreased growth at higher levels of saponins, it was higher compared to the control (without saponin) up to levels of 1% (w/v) for all saponins except Quillaja saponins from Sigma, for which the growth was lower at levels of 0·25% (w/v) and higher. Saponins have the potential to modulate microbial growth in natural and artificial fermenters.