Currently used microbial markers cannot distinguish protozoal nitrogen (N) from bacterial N, thus limiting research on protozoal quantification in vivo by the lack of a repeatable, accurate marker for protozoal N. We report the development of a real-time PCR assay targeting the gene encoding 18S rDNA to quantify the amount of protozoal biomass in ruminal fluid and duodenal digesta. Protozoal cells were harvested from rumen fluid and concentrated for evaluation of recovery of rDNA in samples from the rumen and the duodenum. The DNA from concentrated cells was extracted with virtually 100% efficiency both before and after column purification. After serial spiking of protozoal cells into duodenal fluid over the entire range of quantification, the recovery was highly linear and constant at 81%. After serially spiking increasing quantities of protozoal rDNA into a constant volume of duodenal samples, nonlinear regression verified constant recovery of background rDNA in duodenal samples regardless of the ratio of target:nontarget rDNA. Recommendations for the procedure, including replication per sample, are described herein.

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.

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.

A lactating cow trial was conducted to study the effects of dietary addition of oregano leaf material (Origanum vulgare L.; OV; 0, control vs. 500 g/d) on ruminal fermentation, methane production, total tract digestibility, manure gas emissions, N metabolism, organoleptic characteristics of milk, and dairy cow performance. Eight primiparous and multiparous Holstein cows (6 of which were ruminally cannulated) were used in a crossover design trial with two 21-d periods. Cows were fed once daily. The OV material was top-dressed and mixed with a portion of the total mixed ration. Cows averaged 80 ± 12.5 d in milk at the beginning of the trial. Rumen pH, concentration of total and individual volatile fatty acids, microbial protein outflow, and microbial profiles were not affected by treatment. Ruminal ammonia-N concentration was increased by OV compared with the control (5.3 vs. 4.3mM). Rumen methane production, which was measured only within 8h after feeding, was decreased by OV. Intake of dry matter (average of 26.6 ± 0.83 kg/d) and apparent total tract digestibly of nutrients did not differ between treatments. Average milk yield, milk protein, lactose, and milk urea nitrogen concentrations were unaffected by treatment. Milk fat content was increased and 3.5% fat-corrected milk yield tended to be increased by OV, compared with the control (3.29 vs. 3.12% and 42.4 vs. 41.0 kg/d, respectively). Fat-corrected (3.5%) milk feed efficiency and milk net energy for lactation (NE(L)) efficiency (milk NE(L) ÷ NE(L) intake) were increased by OV compared with the control (1.64 vs. 1.54 kg/kg and 68.0 vs. 64.4%, respectively). Milk sensory parameters were not affected by treatment. Urinary and fecal N losses, and manure ammonia and methane emissions were unaffected by treatment. Under the current experimental conditions, supplementation of dairy cow diets with 500 g/d of OV increased milk fat concentration, feed and milk NE(L) efficiencies, and tended to increase 3.5% fat-corrected milk yield. The sizable decrease in rumen methane production with the OV supplementation occurred within 8h after feeding and has to be interpreted with caution due to the large within- and between-animal variability in methane emission estimates. The OV was introduced into the rumen as a pulse dose at the time of feeding, thus most likely having larger effect on methane production during the period when methane data were collected. It is unlikely that methane production will be affected to the same extent throughout the entire feeding cycle.

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.

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.

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.

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.