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.

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.