Fenja Klevenhusen

Grain-rich diets differently alter ruminal and colonic abundance of microbial populations and lipopolysaccharide in goats

High grain feeding has been associated with ruminal pH depression and microbial dysbiosis in cattle. Yet, the impact of high grain feeding on the caprine rumen and hindgut microbial community and lipopolysaccharide (LPS) release is largely unknown. Therefore, the objective was to investigate the effect of increasing dietary levels of barley grain on the microbial composition and LPS concentrations in the rumen and colon of goats. Effects were compared with respect to the responses of ruminal and colonic pH and short-chain fatty acid (SCFA) generation. Growing goats (n = 5-6) were fed diets containing 0, 30, or 60% coarsely ground barley grain for 6 weeks. Ruminal ciliate protozoa were counted with Bürker counting chamber, and quantitative PCR was used to compare bacterial populations. Increasing dietary grain level linearly increased (P < 0.05) ruminal numbers of entodiniomorphids. With the 60% grain diet, there was a reduction in ruminal abundance of the genus Prevotella and Fibrobacter succinogenes, whereas the ruminal abundance of Lactobacillus spp. increased compared to the 0 and 30% grain diets (P < 0.05). In the colon, abundance of the genus Prevotella and F. succinogenes increased (P < 0.05) in goats fed the 60% grain diet compared to those fed the other diets. Colonic abundance of Clostridium cluster I was related to the presence of grain in the diet. Ruminal LPS concentration decreased (P < 0.05) in response to the 60% grain diet, whereas its colonic concentration increased in response to the same diet (P < 0.05). Present results provide first insight on the adaptive response of rumen protozoa and rumen and colonic bacterial populations to increasing dietary levels of grain in goats. Although luminal pH largely affects microbial populations, fermentable substrate flow to the caprine hindgut may have played a greater role for colonic bacterial populations in the present study.

Feeding barley grain-rich diets altered electrophysiological properties and permeability of the ruminal wall in a goat model

High-producing ruminants are commonly fed large amounts of concentrate to meet their high energy demands for rapid growth or high milk production. However, this feeding strategy can severely impair rumen functioning, leading to subacute ruminal acidosis. Subacute ruminal acidosis might have consequences for electrophysiological properties by changing the net ion transfer and permeability of ruminal epithelia, which may increase the uptake of toxic compounds generated in the rumen into the systemic circulation. The objective of the present study was to investigate the effects of excessive barley feeding on the electrophysiological and barrier functions of the ruminal epithelium and serum inflammation and ketogenesis markers after a long-term feeding challenge, using growing goats as a ruminant model. A feeding trial was carried out with growing goats allocated to 1 of the 3 groups (n=5-6 animals/group), with diets consisting exclusively of hay (control diet) or hay with 30 or 60% barley grain. Samples of the ventral ruminal epithelium were taken after euthanasia and instantly subjected to Ussing chamber experiments, where electrophysiological properties of the epithelium were measured in parallel with the permeability of marker molecules of different sizes [fluorescein 5(6)-isothiocyanate and horseradish peroxidase] from luminal to apical side. Additionally, ruminal fluid and blood samples were taken at the beginning of the experiment as well as shortly before euthanasia. Ruminal fluid samples were analyzed for volatile fatty acids and pH, whereas blood samples were analyzed for lipopolysaccharide, serum amyloid A, and β-hydroxybutyrate. Electrophysiological data indicated that barley feeding increased the epithelial short-circuit current compared with the control. Tissue conductance also increased with dietary barley inclusion. As shown with both marker molecules, permeability of ruminal epithelia increased with barley inclusion in the diet. Despite a lowered ruminal pH associated with increased volatile fatty acids (such as propionate and butyrate) concentrations as well as altered epithelial properties in response to high-grain feeding, no signs of inflammation became apparent, as blood serum amyloid A concentrations remained unaffected by diet. However, greater amounts of grain in the diet were associated with a quadratic increase in lipopolysaccharide concentration in the serum. Also, increasing the amounts of barley grain in the diet resulted in a tendency to quadratically augment serum concentrations of β-hydroxybutyrate and, hence, the alimentary ketogenesis. Further studies are needed to clarify the role of barley inclusion in the development of subacute ruminal acidosis in relation to ruminal epithelial damage and the translocation of toxic compounds in vivo.

Transfer of linoleic and linolenic acid from feed to milk in cows fed isoenergetic diets differing in proportion and origin of concentrates and roughages.

The transfer of ingested alpha-linolenic acid (ALA) and linoleic acid (LA) determines the nutritional quality of milk, but the factors determining this transfer are unclear. The present experiment investigated the influence of roughage to concentrate proportions and the effect of concentrate types on milk fat composition. Respectively, six lactating dairy cows were fed one of three isoenergetic (5.4+/-0.05 MJ net energy for lactation/kg dry matter; DM) and isonitrogenous (215+/-3.5 g crude protein/kg DM) diets, consisting of ryegrass hay only (33 g fatty acids/kg DM; ALA-rich, no concentrate), maize (straw, whole maize pellets and gluten; 36 g fatty acids/kg DM; LA-rich; 560 g concentrate/kg DM), or barley (straw and grain plus soybean meal; 19 g fatty acids/kg DM; LA-rich; 540 g concentrate/kg DM). The fatty acid composition of feeds and resulting milk fat were determined by gas chromatography. The ALA concentration in milk fat was highest (P<0.001) with the hay-diet, but the proportionate transfer of ALA from diet to milk was lower (P<0.001) than with the maize- or barley-diets. The LA concentration in milk fat was highest with the maize-diet (P<0.05, compared with hay) but relative transfer rate was lower (P=0.01). The transfer rates of ALA and LA were reciprocal to the intake of individual fatty acids which thus contributed more to milk fat composition than did roughage to concentrate proportions. The amount of trans-11 18:1 in milk fat was lowest with the barley-diet (P<0.001) and depended on the sum of ALA and LA consumed. The milk fat concentration of cis-9, trans-11 18:2 (rumenic acid) was more effectively promoted by increasing dietary LA (maize) than ALA (hay). Amounts of 18:0 secreted in milk were four (maize) to seven (hay) times higher than the amounts ingested. This was suggestive of a partial inhibition of biohydrogenation in the maize-diet, possibly caused by the high dietary LA level.

Transient feeding of a concentrate-rich diet increases the severity of subacute ruminal acidosis in dairy cattle.

The objective of this study was to investigate the effect of the pattern of concentrate-rich feeding on subacute ruminal acidosis (SARA), its severity, and the corresponding changes in VFA concentration. Eight rumen-cannulated Holstein cows were assigned to a 2 × 2 crossover design with 2 SARA challenge models and 2 experimental runs ( = 8 per treatment). Each run lasted for 40 d, consisting of a 6-d baseline, a 6-d gradual grain adaptation, and a 28-d SARA challenge period. The 2 SARA challenge models were transient (TRA) and persistent (PER) SARA. Initially, all cows were subjected to a forage-only diet (baseline) and gradually switched to 60% concentrate (DM basis). Then, cows in the PER model were continuously challenged for 28 d, whereas cows in the TRA model had a 7-d break from the SARA diet and were fed the forage-only diet after the first 7 d of SARA challenge. Thereafter, the TRA cows were rechallenged with the SARA diet. Wireless ruminal pH sensors were used to obtain ruminal pH profiles and temperature over the experimental period. For the determination of VFA, free ruminal liquid (FRL) and particle-associated ruminal liquid (PARL) were collected once for the baseline and twice (d 20 and 40 for the PER model) or 3 times (d 13, 30, and 40 for the TRA model) during SARA, each time at 0, 4, and 8 h after the morning feeding. Cows in both models experienced SARA albeit with day-to-day variation. From the start until the first 7-d SARA, cows of both models had similar pH profiles, but during the rechallenge, SARA was more severe in the TRA model than in the PER model based on lower daily mean ruminal pH (5.93 vs. 6.15; SEM 0.058) and double the amount of time at pH < 5.8 (497 vs. 278 min; SEM 68.61, < 0.05). Mean ruminal temperature was raised during SARA compared with the baseline (38.9 vs. 38.7°C; SEM 0.057, < 0.001). Concentrations of VFA increased with increasing time after feeding ( < 0.001). In general, SARA challenge (d 40 vs. the baseline), but not the challenge model, altered VFA concentrations and profile of both FRL and PARL by increasing the amounts of propionate and butyrate, whereas total VFA concentration was less affected. Proportions of VFA shifted over the duration of SARA challenge with more propionate but less acetate and butyrate proportions with advancing days of SARA challenge, leading to the values of the last SARA day being different from the earlier days ( < 0.05). In conclusion, the TRA condition led to the higher severity of SARA, but factors beyond feed intake and VFA alterations seemed to play a role.

Technical note: Evaluation of a real-time wireless pH measurement system relative to intraruminal differences of digesta in dairy cattle

The aim of this study was to evaluate the accuracy and precision of indwelled wireless sensors relative to intrareticuloruminal differences in dairy cows transitioned from a forage to a high-concentrate diet. A feeding trial was performed with 8 rumen-cannulated Holstein cows. The cows were stepwise switched from 0 to 60% concentrate in the diet and fed 5 wk. Samples from the free ruminal liquid (FRL) from the ventral rumen and from the particle-associated ruminal liquid (PARL) in the rumen mat were manually taken at 0, 4, and 8 h after the morning feeding on d 0, 7, 14, and 34 of the experiment through the ruminal cannula to measure pH in FRL and PARL using a pH electrode. Additionally indwelling reticular wireless pH sensors were used to measure reticular pH every 10 min throughout the experiment. Precision and accuracy properties as a measure of reproducibility of the methods were statistically evaluated. Data showed significant differences among pH readings of indwelling sensors and pH measurements taken by means of a conventional electrode in both FRL and PARL (P<0.05). These differences became more evident when 60% concentrate diet was fed. Across all experimental days, the pH of the FRL was greatest and the pH reported by indwelling sensors intermediate, whereas the pH of PARL was lowest. The concordance correlation coefficient (CCC) analysis revealed a high agreement between indwelling sensors and FRL (CCC=0.709) but a low agreement with the pH of PARL (CCC=0.495). In conclusion, the study indicated that wireless sensors can satisfactorily reflect the pH of FRL but poorly reflect that of PARL.

Experimental validation of the Intergovernmental Panel on Climate Change default values for ruminant-derived methane and its carbon-isotope signature.

Two aspects regarding the ruminants contribution to global methane (CH4) emissions were investigated: (i) testing the accuracy of the Intergovernmental Panel on Climate Change default values for dairy cows fed different diet types and differing slurry storage temperatures; and (ii) providing carbon-isotope (C-isotope) signature data to contribute information on the characteristics of ruminant-derived CH4 as global source. The experimental diets, fed to 18 dairy cows, were separated into forage-only (hay, C3 plant) and forage-concentrate diets (barley, C3 plant; maize, C4 plant). Accumulated slurry was stored at either 14 or 27C. The hay diet had the highest CH4 conversion rate (Ym 7.9%). Negligible amounts of CH4 were emitted from slurries stored at low temperature. No diet effect was found at 27C (~33 L/kg volatile solids). The isotope ratios of enteric CH4 averaged 67.7‰ (C3 plants) and 57.4‰ (C4; maize). High temperature slurry storage resulted in different enrichment factors eCO2-CH4 for maize (33.2‰) and hay (35.9‰). Compared with the Intergovernmental Panel on Climate Change default values for Ym and slurry CH4 emission the results gained in the present experiment were higher and lower, respectively. Slurry-derived CH4 was less depleted in 13 C than

Pyrosequencing reveals shifts in the bacterial epimural community relative to dietary concentrate amount in goats.

Ecological balance in the rumen is highly sensitive to concentrate-rich diets. Yet the effects of these feeding practices on the caprine bacterial epimural microbiome (CBEM), a microbial community with putative important physiological functions in the rumen, are largely unexplored. This study aimed to investigate the effect of dietary concentrate amount on ruminal CBEM. Seventeen growing goats were fed diets with 0 [n=5; 6.2MJ of metabolizable energy (ME)/d], 30 (n=6; 7.3MJ of /d), or 60% (n=6; 10.2MJ of ME/d) concentrate for 6 wk. Two hours after their last feeding, goats were euthanized and tissue samples of the ventral rumen wall were collected, washed in phosphate-buffered saline to detach loosely attached bacteria, and stored at -20°C for further processing. Genomic DNA was isolated from thawed rumen mucosa samples and used for Roche/454 Life Science (Branford, CT) 16S rRNA gene amplicon pyrosequencing yielding 122,458 reads. Pyrosequencing data were clustered into 1,879 operational taxonomic units (OTU; 0.03 distance level). Pyrosequencing revealed Proteobacteria, Bacteroidetes, Firmicutes, and Spirochaetes as the most abundant phyla (97.7%). Compared with the 30% group, both the 60 and 0% concentrate groups harbored significantly more Firmicutes and SR1, respectively. On an OTU level, a Bergeriella-related OTU was most abundant in the CBEM, followed by 2 Campylobacter OTU, which responded differently to diets: 1 OTU was significantly increased whereas the other significantly decreased with highest concentrate amount in the diet. At the genus level, the 0% concentrate group harbored increased Kingella-like sequences compared with the other feeding groups. Furthermore, the 0% concentrate group tended to have more Bergeriella than the 30 and 60% concentrate groups. The genus Bergeriella was significantly decreased in the 60% feeding group compared with the other diets. In conclusion, this is the first report of CBEM using deep-sequencing methods on the genus and OTU level, and our study revealed major shifts in the CBEM in response to concentrate-rich diets with potential health relevance in goats.

Diallyl disulphide and lovastatin: effects on energy and protein utilisation in, as well as methane emission from, sheep

Currently research on feed supplementation with natural compounds to improve energy and protein utilisation and to mitigate the greenhouse gas methane in ruminants is intensively pursued. Two compounds, diallyl disulphide (DADS), an important component of garlic oil, and lovastatin, an inhibitor of a key enzyme of methanogenic Archaea, were selected on the basis of their in vitro anti-methanogenic potential. In three 23-day experimental runs, six sheep received hay and concentrate in a duplicate 3×3 Latin square design. The concentrate was either not supplemented or supplemented with either 4 g DADS or 80 mg lovastatin per kg of total dietary dry matter. There were no refusals of concentrate for any treatment. Respiratory measurements were conducted on experimental days 7/8 (Period 1) and days 17/18 (Period 2). Relative to the control, digestibility of neutral detergent fibre (NDF) tended to increase (p = 0.09) with DADS by 14%. This was associated with an increased (p = 0.07) body energy retention of the animals. Effects on nitrogen balance and ruminal fermentation traits were never significant. No influence of supplements or period was found on total daily CH4 production which averaged at 28.6 g per sheep. However, across both periods the amount of CH4 produced per kg NDF digested was lower (−8%; p = 0.02) with DADS than without supplementation, and the lovastatin treatment ranged in between. In conclusion, the study demonstrated a certain potential of DADS to improve fibre digestion and body energy retention and to limit CH4 formation in relation to digestible fibre intake, while lovastatin remained ineffective.

Treatment of grain with organic acids at 2 different dietary phosphorus levels modulates ruminal microbial community structure and fermentation patterns in vitro

Recent data indicate positive effects of treating grain with citric (CAc) or lactic acid (LAc) on the hydrolysis of phytate phosphorus (P) and fermentation products of the grain. This study used a semicontinuous rumen simulation technique to evaluate the effects of processing of barley with 50.25 g/L (wt/vol) CAc or 76.25 g/L LAc on microbial composition, metabolic fermentation profile, and nutrient degradation at low or high dietary P supply. The low P diet [3.1g of P per kg of dry matter (DM) of dietary P sources only] was not supplemented with inorganic P, whereas the high P diet was supplemented with 0.5 g of inorganic P per kg of DM through mineral premix and 870 mg of inorganic P/d per incubation fermenter via artificial saliva. Target microbes were determined using quantitative PCR. Data showed depression of total bacteria but not of total protozoa or short-chain fatty acid (SCFA) concentration with the low P diet. In addition, the low P diet lowered the relative abundance of Ruminococcus albus and decreased neutral detergent fiber (NDF) degradation and acetate proportion, but increased the abundance of several predominantly noncellulolytic bacterial species and anaerobic fungi. Treatment of grain with LAc increased the abundance of total bacteria in the low P diet only, and this effect was associated with a greater concentration of SCFA in the ruminal fluid. Interestingly, in the low P diet, CAc treatment of barley increased the most prevalent bacterial group, the genus Prevotella, in ruminal fluid and increased NDF degradation to the same extent as did inorganic P supplementation in the high P diet. Treatment with either CAc or LAc lowered the abundance of Megasphaera elsdenii but only in the low P diet. On the other hand, CAc treatment increased the proportion of acetate in the low P diet, whereas LAc treatment decreased this variable at both dietary P levels. The propionate proportion was significantly increased by LAc at both P levels, whereas butyrate increased only with the low P diet. Treatments with CAc or LAc reduced the degradation of CP and ammonia concentration compared with the control diet at both P levels. In conclusion, the beneficial effects of CAc and LAc treatment on specific ruminal microbes, fermentation profile, and fiber degradation in the low P diet suggest the potential for the treatment to compensate for the lack of inorganic P supplementation in vitro. Further research is warranted to determine the extent to which the treatment can alleviate the shortage of inorganic P supplementation under in vivo conditions.

Evidence of In Vivo Absorption of Lactate and Modulation of Short Chain Fatty Acid Absorption from the Reticulorumen of Non-Lactating Cattle Fed High Concentrate Diets

Short-chain fatty acids (SCFAs) and lactate are endproducts of rumen fermentation and important energy sources for the host ruminant. Because their rapid accumulation results in ruminal acidosis, enhancement of the absorption of SCFA and lactate across reticuloruminal wall is instrumental in increasing energy supply and preventing ruminal acidosis in cattle. This study investigated whether the reticuloruminal absorption of SCFAs and lactate was altered by different strategies of high concentrate feeding. Eight rumen-cannulated, non-lactating Holstein cows were fed a forage-only diet (baseline) and then gradually adapted over 6 d to a 60% concentrate level. Thereafter, this concentrate-rich diet was fed for 4 wk either continuously (Con; n = 8) or interruptedly (Int; n = 8). Absorption of SCFAs and lactate was determined in vivo from the experimental buffer introduced into the washed reticulorumen. The buffer contained acetate, propionate, butyrate and lactate at a concentration of 60, 30, 10 and 5 mmol/L, respectively and Cr-EDTA as a marker for correcting ruminal water fluxes. The reticuloruminal absorption after 35 and 65 min of buffer incubation was measured at the baseline, after 1 wk of 60% concentrate feeding in the interrupted model (Int-1) and after 4 wk of concentrate feeding in both feeding models (Int-4 and Con-4). Data showed that the absorption rates of individual and total SCFAs during the first 35 min of incubation of Con-4 were highest (~1.7 times compared to baseline), while Int-1 and Int-4 were similar to respective baseline. Lactate was not absorbed during forage-only baseline and 1-wk concentrate feeding, but after 4-wk feeding of concentrates in both models. In conclusion, SCFAs absorption across the reticulorumen of non-lactating cattle was enhanced by the 4-wk continuous concentrate feeding, which seems to be more advantageous in terms of rumen acidosis prevention compared to the interrupted feeding model. The study provides evidence of lactate absorption across the reticulorumen of non-lactating cattle after both continuous and interrupted 4-wk concentrate feeding.