Eukaryotic inhibitors or activators elicit responses to chemosensory compounds by ruminal isotrichid and entodiniomorphid protozoa

Our objectives were to evaluate potential signaling pathways regulating rumen protozoal chemotaxis using eukaryotic inhibitors potentially coordinated with phagocytosis as assessed by fluorescent bead uptake kinetics. Wortmannin (inhibitor of phosphoinositide 3-kinase), insulin, genistein (purported inhibitor of a receptor tyrosine kinase), U73122 (inhibitor of phospholipase C), and sodium nitroprusside (Snp, nitric oxide generator, activating protein kinase G) were preincubated with mixed ruminal protozoa for 3 h before assessing uptake of fluorescent beads and chemosensory behavior to glucose, peptides, and their combination; peptides were also combined with guanosine triphosphate (GTP; a chemorepellent). Entodiniomorphids were chemoattracted to both glucose and peptides, but chemoattraction to glucose was increased by Snp and wortmannin without effect on chemoattraction to peptides. Rate of fluorescent bead uptake by an Entodinium caudatum culture decreased when beads were added simultaneously with feeding and incubated with wortmannin (statistical interaction). Wortmannin also decreased the proportion of mixed entodiniomorphids consuming beads. Isotrichid protozoa exhibited greater chemotaxis to glucose but, compared with entodiniomorphids, were chemorepelled to peptides. Wortmannin increased chemotaxis by entodiniomorphids but decreased chemotaxis to glucose by isotrichids. Motility assays documented that Snp and wortmannin decreased net swimming speed (distance among 2 points per second) but not total swimming speed (including turns) by entodiniomorphids. Wortmannin decreased both net and total swimming behavior in isotrichids. Results mechanistically explain the isotrichid migratory ecology to rapidly take up newly ingested sugars and subsequent sedimentation back to the ventral reticulorumen. In contrast, entodiniomorphids apparently integrate cellular motility with feeding behavior to consume small particulates and thereby stay associated and pass with the degradable fraction of rumen particulates. These results extend findings from aerobic ciliate models to explain how rumen protozoa have adapted physiology for their specific ecological niches.     

Effects of wortmannin,sodium nitroprusside,insulin, genistein,and guanosine triphosphate on chemotaxis and cell growth of Entodinium caudatum, Epidinium caudatum, and mixed ruminal protozoa

The mechanisms by which ruminal protozoa sense and migrate toward nutrients are not fully understood. Chemotaxis by many diverse eukaryotic cells is mediated by phosphatidylinositol-3-kinase, which is highly conserved in receptor tyrosine kinase (RTK) signaling pathways and consistently inhibited by wortmannin. In experiment 1a, increasing the concentration of wortmannin inhibited cell growth nonlinearly at 24 h of a culture of the rumen protozoan Entodinium caudatum, but high variability prevented growth inhibition of Epidinium caudatum from reaching significance. In experiment 1b, increasing the insulin concentration recovered 24-h cell counts for both cultures, depending on wortmannin concentration. In experiment 2, addition of sodium nitroprusside (Snp; activator of protein kinase G for cilial beat reversal in nonrumen ciliate models) at 500 µM or wortmannin at 200 µM in beakers containing rumen fluid decreased random swimming by mixed entodiniomorphids into capillary tubes (inserted into beakers) containing saline. Both Snp and wortmannin increased chemotaxis into tubes containing glucose compared with the beaker control. For isotrichids, beaker treatments had no response. Glucose increased chemotaxis, but peptides decreased chemotaxis even when combined with glucose. In experiment 3, we assessed preincubation of genistein (a purported RTK blocker in nonrumen ciliate models) at 40 or 400 µM in beakers and guanosine triphosphate (GTP; a universal chemorepellent in nonrumen ciliate models, perhaps mediated through an RTK) at 10 or 100 µM combined with glucose in capillary tubes. Neither genistein nor GTP affected chemotaxis toward glucose for entodiniomorphids. However, GTP at 100 µM reduced chemotaxis toward glucose for isotrichids. After the animal is fed, isotrichids that are depleted in glycogen migrate to the dorsal area of the rumen, and the rapid uptake of sugars is enhanced through strong chemotaxis but can be reversed by peptides or GTP. In contrast, entodiniomorphids are less intensely chemoattracted to glucose than isotrichids but are chemoattracted to peptides. Entodiniomorphids’ chemoattraction appears to be integrated with slower but prolonged availability of energy from digesting starch and fiber.     

Effects of ruminal protozoa on methane emissions in ruminants—A meta-analysis

The effects of different ruminal protozoa (RP) on CH₄ emissions from ruminants were evaluated in a meta-analysis, using 64 publications reporting data from 79 in vivo experiments. Experiments included in the database reported CH₄ emissions (g/d) and total RP (TRP, log₁₀ cells/mL) from the same group of animals. The relationship between CH₄ emissions and RP (TRP, entodiniomorphids, and isotrichids), and TRP-, entodiniomorphid-, and isotrichid-based CH₄ emission prediction models, were evaluated as mixed models with experiment as a random effect and weighted by the reciprocal of the standard error of the mean and centered around one. Positive associations existed between TRP and isotrichids with CH₄ emissions but not between entodiniomorphids and CH₄ emissions. A reduction in CH₄ emissions was observed, averaging 7.96 and 4.25 g/d, per log unit reduction in TRP and isotrichid concentrations, respectively. Total RP and isotrichids were important variables in predicting CH₄ emissions from ruminants. Isotrichid CH₄ prediction model was more robust than the TRP, evidenciated by lower predicted sigma hat study (%), and error (%), and with higher concordance correlation coefficient. Both TRP and isotrichid models can accurately predict CH₄ emissions across different ruminant types, as shown by the low square root of the mean square prediction error, with 6.59 and 4.08% of the mean of root of the mean square prediction error in the TRP and isotrichid models, respectively. Our results confirm that isotrichids are more important than entodiniomorphids in methanogenesis. Distinguishing these 2 populations yielded a more robust CH₄ prediction model than combining them as total protozoa.     

Rumen microbial responses to supplemental nitrate. I. Yeast growth and protozoal chemotaxis in vitro as affected by nitrate and nitrite concentrations

Nitrates have been fed to ruminants, including dairy cows, as an electron sink to mitigate CH₄ emissions. In the NO₃^? reduction process, NO₂^? can accumulate, which could directly inhibit methanogens and some bacteria. However, little information is available on eukaryotic microbes in the rumen. Protozoa were hypothesized to enhance nitrate reductase but also have more circling swimming behavior, and the yeast Saccharomyces cerevisiae was hypothesized to lessen NO₂^? accumulation. In the first experiment, a culture of S. cerevisiae strain 1026 was evaluated under 3 growth phases: aerobic, anoxic, or transition to anoxic culture. Each phase was evaluated with a control or 1 of 3 isonitrogenous doses, including NO₃^?, NO₂^?, or NH₄⁺ replacing peptone in the medium. Gas head phase, NO₃^?, or NH₄⁺ did not influence culture growth, but increasing NO₂^? concentration increasingly inhibited yeast growth. In experiment 2, rumen fluid was harvested and incubated for 3 h in 2 concentrations of NO₃^?, NO₂^?, or sodium nitroprusside before assessing chemotaxis of protozoa toward glucose or peptides. Increasing NO₂^? concentration decreased chemotaxis by isotrichids toward glucose or peptides and decreased chemotaxis by entodiniomorphids but only toward peptides. Live yeast culture was inhibited dose-responsively by NO₂^? and does not seem to be a viable mechanism to prevent NO₂^? accumulation in the rumen, whereas a role for protozoal nitrate reductase and NO₂^? influencing signal transduction requires further research.     

The in-vitro metabolism of D,L-lactic acid by rumen microorganisms

Bacterial and protozoal fractions, isolated by centrifugation and filtration from the rumen of sheep given various diets, were used to examine the in-vitro metabolism of both the D(-) and L(+) isomers of lactic acid. The rate of disappearance of both the D- and L-lactic acid isomers in protozoal incubations was up to 15 × higher than the rate of disappearance in bacterial incubations, which was relatively constant at between 0.04 and 0.073 lactate per g protein per h for cells recovered from animals receiving various diets. However, the rate of lactate disappearance in the protozoal fraction varied from 0.133 g per g protein per h with a barley/hay diet to 1.12 g per g protein per h with a silage diet. L-Lactate disappeared more rapidly than the D-lactate in all protozoal incubations. It was confirmed that the disappearance of lactate associated with the protozoal fraction did not originate from adherent or associated bacteria. Further fractionation of the protozoal population by differential filtration showed that lactate only disappeared when incubated with entodiniomorphid protozoa and not with holotrich protozoa. Endogenous propionate and butyrate production by the entodiniomorphid ciliates was stimulated by 100% in the presence of lactate. It was calculated that on certain diets up to 30% of the volatile fatty acids formed from lactate could be protozoal in origin.     

Assessment of ruminal bacterial populations and protozoal generation time in cows fed different methionine sources

Methionine supplemented as 2-hydroxy-4-(methylthio)-butanoic acid (HMB) has been suggested to alter bacterial or protozoal populations in the rumen. Our objective was to determine if source of Met would change microbial populations in the rumen and to compare those results to samples from the omasum. The ruminal and omasal samples were collected from cows fed control (no Met), dl-Met, HMB, or the isopropyl ester of HMB (HMBi; estimated 50% rumen protection) in a replicated 4 × 4 Latin square design. In one square, changes in protozoal populations were determined using microscopic counts and denaturing gradient gel electrophoresis (DGGE), whereas changes in bacterial populations were determined using DGGE and ribosomal intergenic spacer length polymorphism (RIS-LP). Neither the protozoal counts nor the DGGE banding patterns derived from protozoa were different among the dietary treatments or for ruminal vs. omasal samples. As revealed by both DGGE and RIS-LP, bacterial populations clustered by treatments in ruminal and especially in omasal samples. Using cows from both Latin squares, the flow of protozoal cells from the rumen was quantified by multiplying protozoal cell count in omasal fluid by the omasal fluid flow (using CoEDTA as a liquid flow marker) or was estimated by rumen pool size of cells multiplied by either the ruminal dilution rate of CoEDTA (after termination of CoEDTA dosing) or the passage rate of Yb-marked particles. Compared with the omasal fluid flow measurement (16.4 h), protozoal generation time was approximated much more closely using the particulate than the fluid passage rate from the rumen (generation times of 15.7 and 7.5 h, respectively). There seems to be minimal selective retention of protozoal genera in the rumen in dairy cattle fed every 2 h. Data support the validity of the omasal sampling technique under our conditions.     

Partitioning of amino acids flowing to the abomasum into feed, bacterial, protozoal, and endogenous fractions

We partitioned the flow of amino acids (AA) to the abomasum among rumen undegradable protein (RUP) and bacterial, protozoal, and endogenous fractions using four Holstein cows in midlactation that were equipped with ruminal and abomasal cannulas. A 2 x 2 factorial design with four diets, combinations of high or low ruminally degradable organic matter, and rumen degradable protein, was employed. Crude protein (CP) and AA contents of ruminal bacteria and protozoa and abomasal digesta were determined. Equations for the source compositions and in vivo flows of CP and 16 AA were then solved simultaneously with a linear program to estimate the contribution of RUP, bacterial, protozoal, and endogenous CP to AA flows. The flows of RUP and bacterial AA were not affected by diet. Low dietary RDP increased the flow of protozoal AA to the abomasum, but the ruminally degradable organic matter content of the diet did not affect protozoal AA flow. Across diets, RUP, bacterial, protozoal, and endogenous fractions provided 55, 33, 11, and <1% of the CP, and 62, 26, 12, and <1% of the AA that reached the abomasum. The linear program was a useful tool for partitioning AA that flows to the abomasum. The technique may also allow dietary effects on ruminal microbes and the AA profile of protein flowing to the duodenum to be better understood and perhaps manipulated.     

Ruminal nitrogen metabolism: perspectives for integration of microbiology and nutrition for dairy

Our objectives are to integrate current knowledge with a future perspective regarding how metagenomics can be used to integrate rumen microbiology and nutrition. Ruminal NH3-N concentration is a crude predictor of efficiency of dietary N conversion into microbial N, but as this concentration decreases below approximately 5 mg/dL (the value most often suggested to be the requirement for optimal microbial protein synthesis), blood urea N transfer into the rumen provides an increasing buffer against excessively low NH3-N concentrations, and the supply of amino N might become increasingly important to improve microbial function in dairy diets. Defaunation typically decreases NH3-N concentration, which should increase the efficiency of blood urea N and protein-derived NH3-N conversion into microbial protein in the rumen. Thus, we explain why more emphasis should be given toward characterization of protozoal interactions with proteolytic and deaminating bacterial populations. In contrast with research evaluating effects of protozoa on N metabolism, which has primarily been done with sheep and cattle with low dry matter intake, dairy cattle have greater intakes of readily available carbohydrate combined with increased ruminal passage rates. We argue that these conditions decrease protozoal biomass relative to bacterial biomass and increase the efficiency of protozoal growth, thus reducing the negative effects of bacterial predation compared with the beneficial effects that protozoa have on stabilizing the entire microbial ecosystem. A better understanding of mechanistic processes altering the production and uptake of amino N will help us to improve the overall conversion of dietary N into microbial protein and provide key information needed to further improve mechanistic models describing rumen function and evaluating dietary conditions that influence the efficiency of conversion of dietary N into milk protein.     

Modeling the distribution of ciliate protozoa in the reticulo-rumen using linear programming

The flow of ciliate protozoa from the reticulo-rumen is significantly less than expected given the total density of rumen protozoa present. To maintain their numbers in the reticulo-rumen, protozoa can be selectively retained through association with feed particles and the rumen wall. Few mathematical models have been designed to model rumen protozoa in both the free-living and attached phases, and the data used in the models were acquired using classical techniques. It has therefore become necessary to provide an updated model that more accurately represents these microorganisms and incorporates the recent literature on distribution, sequestration, and generation times. This paper represents a novel approach to synthesizing experimental data on rumen microorganisms in a quantitative and structured manner. The development of a linear programming model of rumen protozoa in an approximate steady state will be described and applied to data from healthy ruminants consuming commonly fed diets. In the model, protozoa associated with the liquid phase and protozoa attached to particulate matter or sequestered against the rumen wall are distinguished. Growth, passage, death, and transfer of protozoa between both pools are represented. The results from the model application using the contrasting diets of increased forage content versus increased starch content indicate that the majority of rumen protozoa, 63 to 90%, are found in the attached phase, either attached to feed particles or sequestered on the rumen wall. A slightly greater proportion of protozoa are found in the attached phase in animals fed a hay diet compared with a starch diet. This suggests that experimental protocols that only sample protozoa from the rumen fluid could be significantly underestimating the size of the protozoal population of the rumen. Further data are required on the distribution of ciliate protozoa in the rumen of healthy animals to improve model development, but the model described herein does indicate that the attached protozoal population is a significant component of the total rumen protozoal community.     

Rumen ciliate protozoa: effects on digestion in the stomach of sheep

Six wethers, each fitted with a rumen cannula and duodenal reentrant cannula, were used to study effects of ciliate protozoa on rumen digestion and metabolism. A corn: corn silage (1:1) diet was fed for two periods. During the first period, defaunation was attempted with nonyl phenol ethylene oxide. Defaunation was complete in three sheep and partial in the other three sheep in which a reduced population of a small Entodinium sp. was observed. During the second period the sheep were inoculated with ciliate protozoa, which established a large population in all animals. Apparent digestion in the stomach of organic matter and starch was higher when a large protozoal population was present. Amino acid flow through the duodenum was greater in defaunated animals. A large population of ciliates was associated with increases in both rumen ammonia and plasma urea but had a stabilizing effect on ruminal pH. Volatile fatty acids were higher in the defaunation period, but there were only small differences of molar proportions of the acids between the two periods. Effects of ciliate protozoa are related to animal performance.     

Ruminal metabolism of grass silage soluble nitrogen fractions

The present study was conducted to investigate ruminal N metabolism in dairy cows using ¹⁵N-labeled N sources and dynamic models. The data summarized in this study were obtained from 2 of 4 treatments whose effects were determined in a 4 × 4 Latin square design. Soluble N (SN) isolated from timothy grass silage labeled with ¹⁵N and ammonia N (AN) labeled with ¹⁵N were administered into the rumen contents of 4 ruminally cannulated dairy cows. Ruminal N pool sizes were determined by manual evacuation of rumen contents. The excess ¹⁵N-atom% was determined in N-fractions of rumen digesta grab samples that were collected frequently between 0 to 72 h and used to determine ¹⁵N metabolism in the rumen. Calculations of area under the curve ratios of ¹⁵N were used to estimate proportions of N fractions originating from precursor N pools. A model including soluble nonammonia N (SNAN), AN, bacterial N, and protozoal N pools was developed to predict observed values of ¹⁵N atomic excess pool sizes. The model described the pool sizes accurately based on small residuals between observed and predicted values. An immediate increase in ¹⁵N enrichment of protozoal N suggests physical attachment of bacteria pool to protozoa pool. The mean proportions of bacterial N, protozoal N, and feed N in rumen solid phase were 0.59, 0.20, and 0.21, respectively. These observations suggest that protozoal N accounted for 0.25 of rumen microbial N. About 0.90 of the initial dose of AN was absorbed or taken up by microbes within 2 h. Faster ¹⁵N enrichment of bacterial N with SN than with AN treatment indicates a rapid adsorption of SNAN to microbial cells. Additionally, the recovery of ¹⁵N as microbial and feed N flow from the rumen was approximately 0.36 greater for SN than for the AN treatment, indicating that SNAN was more efficiently used for microbial growth than AN. The present study indicated that about 0.15 of microbial N flowing to the duodenum was of protozoal origin and that 0.95 of the protozoal N originated from engulfed bacterial N. The kinetic variables indicated that 0.125 of SNAN escaped ruminal degradation, which calls into question the use of in situ estimations of protein degradation to predict the flow of rumen undegradable protein.     

Evaluation of a real-time PCR assay quantifying the ruminal pool size and duodenal flow of protozoal nitrogen

We have recently developed a real-time polymerase chain reaction (PCR) assay to quantify copies of the genes encoding protozoal 18S rRNA. The assay includes procedures for isolating and concentrating protozoal cells from the rumen for use as a standard to convert 18S rRNA gene copies to a biomass basis. The current objectives were to 1) determine the degree of reduction of bacterial contamination in the protozoal standard, 2) determine if protozoal standards derived from ruminal fluid are appropriate for predicting duodenal flows, and 3) evaluate the assay's determined values for protozoal N in the rumen and flowing to the duodenum compared with independent measurements. Our protozoal collection method reduced non-associated bacterial contamination by 33-fold, the contamination of which could otherwise significantly bias RNA (microbial marker) and N percentages of concentrated protozoal fractions. Based on denaturing gradient gel electrophoresis, the use of protozoal cells isolated from ruminal fluid appears appropriate for use in quantitative assays determining protozoal N flow postruminally. Using real-time PCR, protozoal N was determined to be 4.8 and 12.7% of the rumen microbial N pool and 5.9 and 11.9% of the duodenal flow of microbial N on diets containing low (16%) or high (21%) forage neutral detergent fiber, respectively, which were comparable with independent measures and expectations.     

Effects of bentonite and monensin on selected elements in the stomach and liver of fauna-free and faunated sheep.

Forty-eight rams, originating from a fauna-free flock, were divided into three groups of 16 and fed a corn silage-based diet that was unsupplemented (control) or included bentonite or monensin supplements. Eight rams in each group were inoculated with a mixed population of ruminal protozoa; the other rams remained free of protozoa throughout the 110-d experiment. The rams had free access to drinking water and assigned diet. All rams were killed at the end of the experiment, and ruminal and abomasal contents and livers were removed and sampled. Protozoal numbers in ruminal fluid of faunated rams were lower for groups fed bentonite or monensin supplements than for the control group. Bentonite decreased and monensin increased ruminal pH. The ruminal solubilities of Cu, Zn, and Mg were decreased by the presence of ruminal protozoa, but those of Fe, Mn, and Ca were not affected. Bentonite supplement decreased, and monensin supplement increased, the ruminal solubilities of Cu, Zn, and Mg. Protozoa increased the abomasal solubilities of Fe, but the other elements were not affected. Liver concentrations of Cu were decreased by bentonite and increased by monensin, but protozoa decreased the liver concentrations of both Cu and Mg. Liver concentration of Zn was affected by a monensin x protozoa interaction and that of Mg by a bentonite x protozoa interaction. It was concluded that chronic Cu poisoning could be accelerated by dietary supplements of monensin in sheep without ruminal microfauna, and the dietary Cu bioavailability could be decreased by dietary supplements of bentonite in sheep with a normal population of protozoa in the rumen.     

Investigating unsaturated fat, monensin, or bromoethanesulfonate in continuous cultures retaining ruminal protozoa. II. Interaction of treatment and presence of protozoa on prokaryotic communities

Increasing the consistency of responses to reduce emissions of ruminal methane and nitrogenous wastes into the environment using microbial inhibitors requires an accurate assessment of microbial community profiles. In addition to direct inhibition of methanogens by feed additives, protozoa are often targeted for inhibition because their close physical association with endo- and ectosymbionts stimulates methanogenesis in the rumen. In this study, we first modified a continuous culture system to maintain a diverse protozoal population (faunated subperiod) and then selectively effluxed them without using any chemical agents (defaunated subperiod). In both subperiods, unsaturated fat (potentially inhibitory to ciliate protozoa, methanogens, and gram-positive bacteria), monensin (assumed to inhibit gram-positive bacteria), and bromoethanesulfonate (BES; a potent inhibitor of methanogens) were used to suppress the respective functional groups of microorganisms. Changes in microbial populations were determined using denaturing gradient gel electrophoresis, followed by cloning and DNA sequencing of the excised bands. Neither monensin nor unsaturated fat consistently affected methanogen populations under our conditions in either the faunated or defaunated subperiods. When BES was administered, bands presumptively linked to protozoa-associated methanogens in the faunated subperiod disappeared in the defaunated subperiod. However, there was no noticeable adaptation of the sensitive methanogens to BES. The effect of dietary treatments on bacterial populations in the fermenters was harder to ascertain because of the overriding period effect caused by a different inoculum in each period. Defaunation selectively decreased the intensity of bands associated with ruminococci and clostridia but seemed to increase some Butyrivibrio and related populations. Presence of protozoa influenced both bacterial and archaeal populations, probably by selective predation, competition for substrate, or through symbiotic interactions.     

Effect of sodium laurate on ruminal fermentation and utilization of ruminal ammonia nitrogen for milk protein synthesis in dairy cows

A crossover design trial with 4 ruminally and duodenally cannulated lactating dairy cows was conducted to study the effect of sodium laurate on ruminal fermentation, nutrient digestibility, and milk yield and composition. The daily dose of sodium laurate (0, control or 240 g/cow, LA) was divided in 2 equal portions and introduced directly into the rumen through the cannula before feedings. Ruminal samples (29 in 114 h) were analyzed for fermentation variables and protozoal counts. Sodium laurate had no effect on ruminal pH and total and individual volatile fatty acids concentrations. Ruminal ammonia concentration, ammonia N pool size, and the irreversible loss of ammonia N were unaffected by treatment. Compared to control, protozoal counts were reduced by 91% by LA. Carboxymethylcellulase and xylanase activities of ruminal fluid were decreased (by 40 and 36%, respectively), and amylase activity was not affected by LA compared with control. Flow of microbial N to the duodenum was reduced by LA. Dry matter intake and apparent total tract digestibility of dry matter, organic matter, crude protein, neutral detergent fiber, and acid detergent fiber were not different between the 2 treatments. Milk yield, fat-corrected milk yield, milk fat and protein concentrations and yields, and milk urea N content were not affected by treatment. Sodium laurate did not affect transfer of ruminal ammonia-15N into bacterial or milk protein. In conclusion, LA at approximately 0.3% of the rumen weight reduced ruminal protozoal population and had a negative effect on fibrolytic activities of ruminal fluid and microbial protein flow to the intestine. Treatment had no other significant effects on ruminal fermentation, total tract digestibility, or transfer of ruminal ammonia-15N into milk protein.     

In vitro response to EPA,DPA, and DHA: Comparison of effects on ruminal fermentation and biohydrogenation of 18-carbon fatty acids in cows and ewes

The modulation of milk fat nutritional quality through fish oil supplementation seems to be largely explained by the action of n-3 very long chain polyunsaturated fatty acids (PUFA) on ruminal biohydrogenation (BH) of C18 fatty acids (FA). However, relationships among this action, disappearance of those PUFA in the rumen, and potential detrimental consequences on ruminal fermentation remain uncertain. This study compared the effect of 20:5n-3 (eicosapentaenoic acid; EPA), 22:5n-3 (docosapentaenoic acid; DPA), and 22:6n-3 (docosahexaenoic acid; DHA) on rumen fermentation and BH of C18 FA and was conducted simultaneously in cows and sheep to provide novel insights into interspecies differences. The trial was performed in vitro using batch cultures of rumen microorganisms with inocula collected from cannulated cows and ewes. The PUFA were added at a dose of 2% incubated dry matter, and treatment effects on ruminal C18 FA concentrations, PUFA disappearances, and fermentation parameters (gas production, ammonia and volatile FA concentrations, and dry matter and neutral detergent fiber disappearances) were examined after 24 h of incubation. A principal component analysis suggested that responses to PUFA treatments explained most of the variability; those of ruminant species were of lower relevance. Overall, EPA and DHA were equally effective for inhibiting the saturation of trans-11 18:1 to 18:0 and had a similar influence on ruminal fermentation in cows and sheep (e.g., reductions in gas production and acetate:propionate ratio). Nevertheless, DHA further promoted alternative BH pathways that lead to trans-10 18:1 accumulation, and EPA seemed to have specific effects on 18:3n-3 metabolism. Only minor variations attributable to DPA were observed in the studied parameters, suggesting a low contribution of this FA to the action of marine lipids. Although most changes due to the added PUFA were comparable in bovine and ovine, there were also relevant specificities, such as a stronger inhibition of 18:0 formation in cows and a greater increase in 18:3n-3 metabolites in sheep. No direct relationship between in vitro disappearance of the incubated PUFA and effect on BH (in particular, inhibition of the last step) was found in either cows or ewes, calling into question a putative link between extent of disappearance and toxicity for microbiota. Conversely, an association between the influence of these PUFA on ruminal lipid metabolism and fermentation may exist in both species. In vivo verification of these findings would be advisable.     

Effects of ethyl-3-nitrooxy propionate and 3-nitrooxypropanol on ruminal fermentation,microbial abundance,and methane emissions in sheep

The aim of this work was to investigate the effect of feeding ethyl-3-nitrooxy propionate (E3NP) and 3-nitrooxypropanol (3NP), 2 recently developed compounds with potential antimethanogenic activity, in vitro and in vivo in nonlactating sheep on ruminal methane production, fermentation pattern, the abundance of major microbial groups, and feed degradability. Three experiments were conducted, 1 in vitro and 2 in vivo. The in vitro batch culture trial (experiment 1) tested 2 doses of E3NP and 3NP (40 and 80 μL/L), which showed a substantial reduction of methane production (up to 95%) without affecting concentration of volatile fatty acids (VFA). The 2 in vivo trials were conducted over 16 d (experiment 2) and 30 d (experiment 3) to study their effects in sheep. In experiment 2, 6 adult nonpregnant sheep, with permanent rumen cannula and fed alfalfa hay and oats (60:40), were treated with E3NP at 2 doses (50 and 500 mg/animal per day). After 7, 14, and 15 d of treatment, methane emissions were recorded in respiration chambers and rumen fluid samples were collected for VFA analysis and quantification of bacterial, protozoal, and archaeal numbers by real-time PCR. Methane production decreased by 29% compared with the control with the higher dose of E3NP on d 14 to 15. A decrease in the acetate:propionate ratio was observed without detrimental effects on dry matter intake. In experiment 3, 9 adult nonpregnant sheep, with permanent rumen cannula and fed with alfalfa hay and oats (60:40), were treated with E3NP or 3NP at one dose (100 mg/animal per day) over 30 d. On d 14 and d 29 to 30, methane emissions were recorded in respiration chambers. Rumen fluid samples were collected on d 29 and 30 for VFA analysis and quantification of bacterial, protozoal, and archaeal numbers by real-time PCR. In addition, on d 22 and 23, samples of oats and alfalfa hay were incubated in the rumen of sheep to determine dry matter ruminal degradation over 24 and 48 h, respectively; no effect was observed (78.6, 78.3, and 78.8% of alfalfa and 74.2, 74.0, and 70.6% of oats in control, E3NP, and 3NP groups, respectively). A reduction in methane production was observed for both additives at d 14 and d 29 to 30. In both treatments, the acetate:propionate ratio was significantly decreased. Likewise, total concentrations of the analyzed microbial groups in the rumen showed no difference among treatments and doses for both experiments. Both tested compounds showed promise as methane inhibitors in the rumen, with no detrimental effects on fermentation or intake, which would need to be confirmed in lactating animals.     

Rumen ciliated protozoa decrease generation time and adjust 18S ribosomal DNA copies to adapt to decreased transfer interval, starvation, and monensin

Defaunation studies have documented decreased ammonia concentrations associated with reduced microbial protein recycling and wastage of dietary protein, whereas many methods to suppress protozoa can reduce feed intake or depress ruminal organic matter or fiber digestibility. Therefore, more research is needed to optimize dietary conditions that improve protozoal growth and ruminal outflow relative to autolysis and recycling. Response in growth rate to ruminal outflow was simulated by abrupt changes in transfer interval of batch cultures, and substrate availability was evaluated by feeding without or with abrupt addition of monensin, which was postulated to inhibit digestive vacuole function. In experiment 1, Entodinium caudatum, a mix of Entodinium species, Epidinium caudatum, or Ophryoscolex caudatus cultures rapidly adjusted their generation times to approach respective changes in transfer interval from 3 to 2 or 1 d (cultures were always fed at 24-h intervals). Monensin (0.25 μM) consistently delayed this response. To evaluate a metabolic upshift associated with feeding or a downshift associated with substrate depletion, experiment 2 used real-time PCR to quantify protozoal 18S rRNA gene (rDNA) copies that were expressed relative to cell numbers or to the cellular constituents N and nucleic acids after feeding without or with monensin (0.5 μM). The 18S rDNA copies per milligram of nucleic acids were least for Ophryoscolex compared with the other cultures. When averaged over cultures (no culture × treatment interaction), 18S rDNA copies per unit of nucleic acids decreased at 16 h when cultures were starved but increased with feeding unless monensin uncoupled availability of consumed substrate. Rumen protozoal growth increased in response to decreased transfer interval in experiment 1. Substrate availability appeared to initiate metabolic responses preparing for cell growth, explaining how cultures could rapidly adjust to decreasing transfer interval in experiment 2. Because feeding was not coupled with transfer in experiment 2, however, a metabolic control probably arrested cell division to prevent overgrowth relative to substrate availability.     

Short communication: Gelatinization and enzymatic hydrolysis characteristics relevant to digestion and analysis of glycogen granules isolated from ruminal protozoa

Glycogen is an α-glucan produced by rumen microbes from various feed carbohydrates. It may be digested ruminally or intestinally to provide nutrients. The physicochemical and enzymatic hydrolysis characteristics of microbial glycogen have not been described in detail, but do influence its conversion to absorbable nutrients in vivo, its nutritional comparability with plant starch sources, and its accurate analysis in vitro. The objectives of this study were to determine presence or absence of a gelatinization response and to describe enzymatic digestion characteristics of glycogen granules isolated from ruminal protozoa obtained from lactating dairy cows. Protozoal glycogen granules were determined to be 98.3% α-glucan. Granules displayed gelatinization, the breaking of hydrogen bonds between molecules or branches, at 65°C compared with purified wheat and corn starches, which initiated gelatinization at 50 and 65°C, respectively. Digestion of ungelatinized samples with amyloglucosidase for 2 h at 39°C showed approximately 3-fold greater hydrolysis to glucose for protozoal glycogen (25.2% of dry matter; DM) than for wheat (9.9% of DM) or corn (8.2% of DM) starches. Based on enzymatic digestion results, protozoal glycogen may be more readily digested than intact corn or wheat starches and should be gelatinized or the hydrogen bonds otherwise disrupted to allow more complete recovery in enzymatic analysis.     

Microbial Protein Synthesis in Rumens of Cows Fed Dried Whole Whey

Two rumen-fistulated Holstein cows, weighing approximately 550 kg, were in an experiment with switchback design to evaluate effects of consuming large amounts (38% of total ration dry matter) of dried whole whey on synthesis of microbial protein in the rumen. Cows were fed total mixed rations of (dry matter) 45% corn silage, 10% alfalfa hay, and 45% concentrate mix. The concentrate mix was primarily corn and soybean meal (control) or 85% dried whole whey. Dry matter intakes averaged 16.4 and 15.3 kg/day for control and whey diets. Diaminopimelic acid nitrogen as percent of bacterial nitrogen was similar for both diets (.61 and .63% for control and whey diets). Likewise, aminoethylphosphonic acid nitrogen as percent of protozoal nitrogen was similar for both diets (.17 and .19% for control and whey diets). For the control diet, total ruminal nitrogen was estimated to be 45% bacterial and 27% protozoal. Bacteria and protozoa accounted for 52 and 22% of the total ruminal nitrogen in the cows fed the whey diet. Ruminal fluid volume (33.8 and 39.2 liters for control and dried whey diets) and dilution rates (10.2 and 12.8% h) were higher for dried whey. Ruminal ammonia (5.0 and 3.4 mg/dl) was lower for dried whey. Butyrate (16.5 and 24.4 moles/100 moles total volatile fatty acids) was higher, whereas propionate was lower (32.4 and 23.2 moles/100 moles total volatile fatty acids) when cows were fed dried whey. Bacterial synthesis appeared to be increased when cows were fed a diet containing large amounts of dried whey.