Essential oil and monensin affect ruminal fermentation and the protozoal population in continuous culture.

The interaction of monensin and essential oil was hypothesized to suppress protozoa and methane production while maintaining normal rumen function. The objective of this study was to determine the effects of feeding monensin (MON) and CinnaGar (CIN, a commercial blend of cinnamaldehyde and garlic oil; Provimi North America, Brookville, OH) on ruminal fermentation characteristics. Continuous culture fermentors (n = 4) were maintained in 4 experimental periods in a 4 × 4 Latin square design. Four dietary treatments were arranged in a 2 × 2 factorial: (1) control diet, 37 g/d of dry matter (40 g/d at ∼92.5% dry matter) of a 50:50 forage:concentrate diet containing no additive; (2) MON at 11 g/909 kg of dry matter; (3) CIN at 0.0043% of dry matter; and (4) a combination of MON and CIN at the levels in (2) and (3). Treatment had no effects on protozoal populations, concentration of NH3N, total N flow of effluent, production of total volatile fatty acids, or flows of conjugated linoleic acid and total C18 fatty acids. The MON decreased acetate:propionate ratio and biohydrogenation of both total C18 and 18:1 cis-9 but increased protozoal generation time, concentration of peptide, and flow of 18:1 trans-11. The MON tended to decrease protozoal counts in effluent and flow of 18:0 but tended to increase propionate production. The CIN decreased true organic matter digestibility and protozoal N flow of effluent but increased nonammonia, nonmicrobial N flow. The CIN tended to decrease protozoal counts, microbial N flow, and neutral detergent fiber digestibility but tended to increase biohydrogenation of total C18, 18:2, and 18:3. The CIN tended to increase isovalerate production. The MON and CIN tended to interact for increased methane production and bacterial N flow. A second experiment was conducted to determine the effects of MON and CIN on protozoal nitrogen and cell volume in vitro. Four treatments included (1) control (feed only), (2) feed + 0.0043% dry matter CIN, (3) feed + 2.82 µM MON, and (4) feed + CIN + MON at the same levels as in (2) and (3). With no interactions, MON addition decreased percentage of protozoa that were motile and tended to decrease cell volume at 6 h. The CIN did not affect cell count or other indicators of motility or volume at either 3 or 6 h. Under the conditions of our study, we did not detect an additive response for MON and CIN to decrease protozoal counts or methane production. A 3-dimensional method is suggested to better estimate protozoal cell volume.

Chemotaxis toward carbohydrates and peptides by mixed ruminal protozoa when fed, fasted, or incubated with polyunsaturated fatty acids.

In contrast to the well-characterized chemotaxis and migratory behavior between the dorsal and ventral locations of the rumen by isotrichids, we hypothesized that chemotaxis toward soluble nutrients maintains entodiniomorphid protozoa in the particulate fraction. The objectives of these experiments were to compare the dose-responsive chemotaxis (1) toward different glucose concentrations when ruminal samples were harvested from fed versus fasted cows; (2) toward increasing concentrations of glucose compared with xylose when protozoa were harvested from a fed cow; (3) toward peptides of bacterial, protozoal, and soy origin; and (4) toward glucose when mixed ruminal protozoa were previously incubated for 0, 3, or 6h in the presence of emulsified polyunsaturated fatty acids (PUFA; Liposyn II, Hospira, Lake Forest, IL). In experiment 1, isotrichid protozoa decreased chemotaxis toward increasing glucose concentration when cows were fasted. Entodiniomorphids exhibited chemotaxis to similar concentrations of glucose as did isotrichids, but to a lesser magnitude of response. In experiment 2, xylose was chemotactic to both groups. Xylose might draw fibrolytic entodiniomorphid protozoa toward newly ingested feed. In contrast, even though isotrichids should not use xylose as an energy source, they were highly chemoattracted to xylose. In experiment 3, entodiniomorphids were not selectively chemoattracted toward bacterial or protozoal peptides compared with soy peptides. In experiment 4, despite isotrichid populations decreasing in abundance with increasing time of incubation in PUFA, chemotaxis to glucose remained unchanged. In contrast, entodiniomorphids recovered chemotaxis to glucose with increased time of PUFA incubation. Current results support isotrichid chemotaxis to sugars but also our hypothesis that a more moderate chemotaxis toward glucose and peptides explains how they swim in the fluid but pass from the rumen with the potentially digestible fraction of particulates.

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 24h 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 500µM or wortmannin at 200µM 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 400µM in beakers and guanosine triphosphate (GTP; a universal chemorepellent in nonrumen ciliate models, perhaps mediated through an RTK) at 10 or 100µM combined with glucose in capillary tubes. Neither genistein nor GTP affected chemotaxis toward glucose for entodiniomorphids. However, GTP at 100µM 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.

Interaction of unsaturated fat or coconut oil with monensin in lactating dairy cows fed 12 times daily. I. Protozoal abundance, nutrient digestibility, and microbial protein flow to the omasum.

Monensin (tradename: Rumensin) should reduce the extent of amino acid deamination in the rumen, and supplemental fat should decrease protozoal abundance and intraruminal N recycling. Because animal-vegetable (AV) fat can be biohydrogenated in the rumen and decrease its effectiveness as an anti-protozoal agent, we included diets supplemented with coconut oil (CNO) to inhibit protozoa. In a 6 × 6 Latin square design with a 2 × 3 factorial arrangement of treatments, 6 rumen-cannulated cows were fed diets without or with Rumensin (12 g/909 kg) and either no fat (control), 5% AV fat, or 5% CNO. The log10 concentrations (cells/mL) of total protozoa were not different between control (5.97) and AV fat (5.95) but were decreased by CNO (4.79; main effect of fat source). Entodinium and Dasytricha decreased as a proportion of total cells from feeding CNO, whereas Epidinium was unchanged in total abundance and thus increased proportionately. Total volatile fatty acid concentration was not affected by diet, but the acetate:propionate ratio decreased for CNO (1.85) versus control (2.95) or AV fat (2.58). Feeding CNO (23.8%) decreased ruminal neutral detergent fiber digestibility compared with control (31.1%) and AV fat (30.5%). The total-tract digestibility of NDF was lower for CNO (45.8%) versus control (57.0%) and AV fat (54.6%), with no difference in apparent organic matter digestibility (averaging 69.8%). The omasal flows of microbial N and non-ammonia N were lower for CNO versus control and AV fat, but efficiency of microbial protein synthesis was not affected. The dry matter intake was 4.5 kg/d lower with CNO, which decreased milk production by 3.1 kg/d. Main effect means of dry matter intake and milk yield tended to decrease by 0.7 and 1.2 kg/d, respectively, when Rumensin was added. Both percentage and production of milk fat decreased for CNO (main effect of fat source). An interaction was observed such that AV decreased milk fat yield more when combined with Rumensin. Using large amounts of supplemental fat, especially CNO, to decrease abundance of protozoa requires further research to characterize benefits versus risks, especially when combined with Rumensin.

Effects of dietary protein concentration and coconut oil supplementation on nitrogen utilization and production in dairy cows.

The objective of this study was to investigate the effect of metabolizable protein (MP) deficiency and coconut oil supplementation on N utilization and production in lactating dairy cows. The hypothesis of the study was that a decrease in ruminal protozoal counts with coconut oil would increase microbial protein synthesis in the rumen, thus compensating for potential MP deficiency. The experiment was conducted for 10 wk with 36 cows (13 primiparous and 23 multiparous), including 6 ruminally cannulated cows. The experimental period, 6 wk, was preceded by 2-wk adaptation and 2-wk covariate periods. Cows were blocked by parity, days in milk, milk yield, and rumen cannulation and randomly assigned to one of the following diets: a diet with a positive MP balance (+44 g/d) and 16.7% dietary crude protein (CP) concentration (AMP); a diet deficient in MP (-156 g/d) and 14.8% CP concentration (DMP); or DMP supplemented with approximately 500 g of coconut oil/head per day (DMPCO). Ruminal ammonia tended to be greater and plasma urea N (20.1, 12.8, and 13.1 mg/dL, for AMP, DMP, and DMPCO diets, respectively) and milk urea N (12.5, 8.3, and 9.5mg/dL, respectively) were greater for AMP compared with DMP and DMPCO. The DMPCO diet decreased total protozoa counts (by 60%) compared with DMP, but had no effect on the methanogens profile in the rumen. Total tract apparent digestibility of dry matter and CP was decreased by DMP compared with AMP. Fiber digestibility was lower for both DMP and DMPCO compared with AMP. Urinary N excretion was decreased (by 37%) by both DMP and DMPCO compared with AMP. The DMP and DMPCO diets resulted in greater milk N efficiency compared with AMP (32.0 and 35.1 vs. 27.6%, respectively). Milk yield was decreased by both DMP and DMPCO compared with AMP (36.2, 34.4, and 39.3 kg/d, respectively) and coconut oil supplementation suppressed feed intake and caused milk fat depression. Coconut oil supplementation decreased short-chain fatty acid (C4:0, C6:0, and C8:0) concentration and increased medium-chain (C12:0 and C14:0) and total trans fatty acids in milk. Overall, the MP-deficient diets decreased N losses, but could not sustain milk production in this study. Coconut oil decreased feed intake and similar to DMP, suppressed fiber digestibility. Despite decreased protozoal counts, coconut oil had no effect on the methanogen population in the rumen.

Interactions of monensin with dietary fat and carbohydrate components on ruminal fermentation and production responses by dairy cows.

Variation in milk fat percentage resulting from monensin supplementation to lactating dairy cows could be due to altered ruminal fermentation with interactions of monensin with ruminal biohydrogenation of fat and ruminal carbohydrate availability. The objective of the study was to determine the effects of feeding monensin as Rumensin (R) in diets differing in starch availability (ground or steam-flaked corn), effective fiber (long or short alfalfa hay, LAH or SAH), and 4% fat (F) from distillers grains, roasted soybeans, and an animal-vegetable blend on ruminal fermentation characteristics and milk production in lactating dairy cows. Six ruminally cannulated lactating Holstein cows were used in a balanced 6×6 Latin square design with 21-d periods. The cows were fed 6 diets: (1) C=control diet with ground corn and LAH, (2) CR=C plus R, (3) CRFL=CR plus F, (4) CRFS=ground corn, R, F, and SAH, (5) SRFL=steam-flaked corn, R, F, and LAH, and (6) SRFS=steam-flaked corn, R, F, and SAH. Mean particle size of LAH was 5.00 mm and 1.36 mm for SAH. All diets were formulated to have 21% forage NDF and 40% NFC. The R tended to decrease DMI, decreased milk fat yield, and numerically lowered milk fat percentage (3.41 vs. 2.98%). Addition of F to R diets did not affect milk fat percentage. By feeding diets containing R and F, SAH tended to increase milk fat percentage for the ground-corn diet, but SAH tended to decrease milk fat percentage with steam-flaked corn (CRFL+SRFS vs. CRFS+SRFL). The steam-flaked corn increased total-tract NDF digestibility (CRFL + CRFS vs. SRFL+SRFS; 51.1 vs. 56%). Addition of F with R decreased total VFA concentration and increased rumen pH. Fat addition with R decreased rumen NH3N and MUN (12.8 vs. 13.9 mg/dL), and SFC decreased NH3N concentration compared with ground corn. Although R caused milk fat depression, addition of F did not further exacerbate milk fat depression. Fatty acid analysis did not implicate any particular biohydrogenation intermediate as the causative factor for the milk fat depression.

Effect of Saccharomyces cerevisiae fermentation product on ruminal fermentation and nutrient utilization in dairy cows.

The goal of this experiment was to investigate the effect of yeast culture (Saccharomyces cerevisiae) on rumen fermentation, nutrient utilization, and ammonia and methane emission from manure in dairy cows. Eight ruminally cannulated Holstein cows were allocated to 2 dietary treatments in a crossover design. Treatments were control (no yeast culture) and XP (yeast culture, fed at 56 g/head per day; XP, Diamond V Mills Inc., Cedar Rapids, IA). Dry matter intake, milk yield, milk composition, and body weight were similar between treatments. Milk urea nitrogen concentration was also not affected by treatment. Rumen pH was similar between the control and XP treatments, but rumen ammonia concentration tended to be lower with XP than with the control. Treatment had no effect on concentrations of total or individual volatile fatty acids, protozoal counts, polysaccharide-degrading activities (except amylase activity that tended to be increased by XP), or methane production in the rumen. Urinary N losses did not differ significantly between treatments, but allantoin and total purine derivative excretions and the estimated microbial N outflow from the rumen tended to be increased by XP compared with the control treatment. Total-tract apparent digestibility of dietary nutrients was not affected by XP. Milk fatty acid composition was also not altered by XP supplementation. Cumulative (253 h) ammonia and methane emissions from manure, measured in a steady-state gas emission system, were slightly decreased by XP. Overall, the yeast culture tested had little effect on ruminal fermentation, digestibility, or N losses, but tended to reduce rumen ammonia concentration and increase microbial protein synthesis in the rumen, and decreased ammonia and methane emissions from manure.

Effect of lauric acid and coconut oil on ruminal fermentation, digestion, ammonia losses from manure, and milk fatty acid composition in lactating cows.

This experiment (replicated 3 x 3 Latin square design) was conducted to investigate the effects of lauric acid (LA) or coconut oil (CO) on ruminal fermentation, nutrient digestibility, ammonia losses from manure, and milk fatty acid (FA) composition in lactating cows. Treatments consisted of intraruminal doses of 240 g of stearic acid/d (SA; control), 240 g of LA/d, or 530 g of CO/d administered once daily, before feeding. Between periods, cows were inoculated with ruminal contents from donor cows and allowed a 7-d recovery period. Treatment did not affect dry matter intake, milk yield, or milk composition. Ruminal pH was slightly increased by CO compared with the other treatments, whereas LA and CO decreased ruminal ammonia concentration compared with SA. Both LA and CO decreased protozoal counts by 80% or more compared with SA. Methane production rate in the rumen was reduced by CO compared with LA and SA, with no differences between LA and SA. Treatments had no effect on total tract apparent dry matter, organic matter, N, and neutral detergent fiber digestibility coefficients or on cumulative (15 d) in vitro ammonia losses from manure. Compared with SA, LA and CO increased milk fat 12:0, cis-9 12:1, and trans-9 12:1 content and decreased 6:0, 8:0, 10:0, cis-9 10:1, 16:0, 18:0, cis 18:1, total 18:2, 18:3 n-3 and total polyunsaturated FA concentrations. Administration of LA and 14:0 (as CO) in the rumen were apparently transferred into milk fat with a mean efficiency of 18 and 15%, respectively. In conclusion, current data confirmed that LA and CO exhibit strong antiprotozoal activity when dosed intraruminally, an effect that is accompanied by decreases in ammonia concentration and, for CO, lowered methane production. Administration of LA and CO in the rumen also altered milk FA composition.

Investigating unsaturated fat, monensin, or bromoethanesulfonate in continuous cultures retaining ruminal protozoa. I. Fermentation, biohydrogenation, and microbial protein synthesis.

Methane is an end product of ruminal fermentation that is energetically wasteful and contributes to global climate change. Bromoethanesulfonate, animal-vegetable fat, and monensin were compared with a control treatment to suppress different functional groups of ruminal prokaryotes in the presence or absence of protozoa to evaluate changes in fermentation, digestibility, and microbial N outflow. Four dual-flow continuous culture fermenter systems were used in 4 periods in a 4 x 4 Latin square design split into 2 subperiods. In subperiod 1, a multistage filter system (50-microm smallest pore size) retained most protozoa. At the start of subperiod 2, conventional filters (300-microm pore size) were substituted to efflux protozoa via filtrate pumps over 3 d; after a further 7 d of adaptation, the fermenters were sampled for 3 d. Treatments were retained during both subperiods. Flow of total N and digestibilities of NDF and OM were 18, 16, and 9% higher, respectively, for the defaunated subperiod but were not different among treatments. Ammonia concentration was 33% higher in the faunated fermenters but was not affected by treatment. Defaunation increased the flow of nonammonia N and bacterial N from the fermenters. Protozoal counts were not different among treatments, but bromoethanesulfonate increased the generation time from 43.2 to 55.6 h. Methanogenesis was unaffected by defaunation but tended to be increased by unsaturated fat. Defaunation did not affect total volatile fatty acid production but decreased the acetate:propionate ratio; monensin increased production of isovalerate and valerate. Biohydrogenation of unsaturated fatty acids was impaired in the defaunated fermenters because effluent flows of oleic, linoleic, and linolenic acids were 60, 77, and 69% higher, and the ratio of vaccenic acid:unsaturated FA ratio was decreased by 34% in the effluent. This ratio was increased in both subperiods with the added fat diet, indicating an accumulation of intermediates of biohydrogenation. However, the flow of 18:2 conjugated linoleic acid was unaffected by defaunation or by treatments other than added fat. The flows of trans-10, trans-11, and total trans-18:1 fatty acids were not affected by monensin or faunation status.

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.

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 microM) 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 microM). The 18S rDNA copies per milligram of nucleic acids were least for Ophryoscolex compared with the other cultures. When averaged over cultures (no culture x 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.

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 x 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.

Biohydrogenation of fatty acids and digestibility of fresh alfalfa or alfalfa hay plus sucrose in continuous culture.

The pattern of biohydrogenation of fatty acids from fresh alfalfa or alfalfa hay supplemented with 3 concentrations (0, 4, and 8%) of sucrose was studied at a constant pH of 6.2. Four continuous culture fermenters were used in a 4 x 4 Latin square design to test the hypothesis that fresh forage would increase flow of vaccenic acid (VA) from the fermenters compared with the same forage in hay form and that this difference would be diminished by adding sucrose to the hay diet by changing the bacterial community profile. Effluent was collected from each of the 4 fermenters during the last 3 d of each 10-d period. Nutrient digestibility, volatile fatty acids (VFA), and fatty acids in the effluent were measured. Flow of bacterial organic matter (OM) and neutral and acid detergent fiber and acid detergent fiber digestibilities were higher for fresh alfalfa than alfalfa hay. True OM digestibility of alfalfa hay tended to linearly decrease with sucrose supplementation. However, microbial efficiency and flow of bacterial OM (g/d) linearly increased with sucrose addition. There was no change in total VFA concentration; however, proportion of acetate linearly decreased and proportion of butyrate linearly increased with sucrose addition. Fresh alfalfa increased total biohydrogenation of fatty acids compared with than hay. Vaccenic acid flow (mg/d) was much higher for fresh alfalfa compared with alfalfa hay (216 vs. 41) and VA was the predominant 18:1 isomer, followed by trans-13 18:1; however, sucrose had no effect on VA flow. The percentage of VA (of total trans-18:1) was not different between fresh alfalfa and hay, whereas percentage of trans-10 18:1 was much lower for fresh alfalfa. Therefore, the ratio of VA to trans-10 18:1 was higher for fresh alfalfa. Flow of trans-12 18:1 linearly increased, whereas flows of cis-12 and total cis-18:1 had quadratic responses to sucrose supplementation. Total biohydrogenation and biohydrogenation of linoleic and linolenic acids linearly decreased with sucrose; however, there was no effect of sucrose on total trans fatty acid flow. Sucrose may be more detrimental to the last step of biohydrogenation of VA. The effects of sucrose on biohydrogenation and concentration of VFA may have been caused by a shift in microbial population by mechanisms that are independent of pH.

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 2-hydroxy-4-(methylthio) butanoic acid (HMB) on microbial growth in continuous culture.

2-Hydroxy-4-(methylthio) butanoic acid (HMB) positively affects milk composition and yield, potentially through ruminal actions. Four continuous culture fermenters were used to determine the optimal concentration of HMB for digestibility of organic matter (OM), neutral detergent fiber (NDF), acid detergent fiber (ADF), and hemicellulose and synthesis of microbial N. A highly degradable mix of hay and grain was used as a basal diet to simulate a typical lactation diet. Three concentrations of HMB (0, 0.055, and 0.110%) and one concentration of dl-Met (0.097%) were infused into the fermenters according to a 4 x 4 Latin square design. Digesta samples were collected during the last 3 d of each of the four 10-d experimental periods. Digestibility of OM, hemicellulose, and NDF was largely insensitive to treatment. Digestibility of ADF showed a quadratic effect to supplementation of HMB, with 0.055% having lower digestibility than 0 or 0.110%. Total production of VFA was not influenced by HMB supplementation, but differences in concentration and production of individual VFA were seen. Isobutyrate increased linearly with increasing HMB supplementation. Propionate concentration decreased linearly with increased HMB supplementation, but propionate production showed a quadratic trend (P = 0.13). A higher concentration of acetate was detected for dl-Met compared with the highest HMB concentration. There were trends (P < 0.15) for dl-Met to decrease the production of isobutyrate and to lower the concentration of butyrate when compared with HMB. Microbial efficiency was not different among treatments. The proportion of bacterial N produced from NH3-N decreased linearly with increasing HMB, and bacteria receiving dl-Met synthesized more N from NH3-N than those receiving HMB. These data suggest that supplementation of HMB may have a sparing effect on branched chain volatile fatty acids because the fatty acids are not needed to provide carbon for synthesis of valine, isoleucine and leucine with ammonia. Comparisons of bacterial community structure in the fermenter effluent samples using PCR amplicons containing the ribosomal intergenic spacer region and its flanking partial 16S ribosomal RNA gene showed no distinct banding patterns, though treatments tended to group together. Both Met and HMB affect the rumen microbial population, but Met supplied as dl-Met does not act identically to that supplied as HMB.

Technical note: Specific PCR amplification of protozoal 18S rDNA sequences from DNA extracted from ruminal samples of cows.

A protozoa-specific primer (P-SSU-342f) was designed and paired with a eukarya-specific primer to amplify a 1,360-bp fragment of DNA encoding protozoal small subunit (SSU) ribosomal RNA from ruminal fluid of cows fed a mixed forage:grain diet or alfalfa hay. Sequencing of clones showed that P-SSU-342f is specific to ruminal protozoa and, with slight modifications, the primer will be useful for ecological studies of ruminal protozoa.