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.     

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.     

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.     

Markers for quantifying microbial protein synthesis in the rumen.

Measurement of ruminal microbial protein is necessary to quantify ruminal escape of dietary protein and microbial yields. Microbial markers used most widely have been the internal markers, diaminopimelic acid and nucleic acids (RNA, DNA, individual purines and pyrimidines, or total purines), and the external isotopic markers (e.g., 15N and 35S). Combined with digesta flow markers in ruminally and abomasally or intestinally cannulated ruminants, microbial yields can be estimated. An ideal marker system must account for both the bacterial and protozoal pools associated with both the fluid and particulate phases of digesta. No marker has proven completely satisfactory; hence, yield estimates are relative rather than absolute. Total purines represent robust microbial markers that should be adaptable by most investigators. Principal concerns about total purines relate to unequal purine: N ratios in protozoal and bacterial pools and to the need to assume that dietary purines are completely degraded in the rumen. A theoretically sounder, but more costly, method is continuous intraruminal infusion of 15N ammonium salts. However, 15N enrichments of bacterial and protozoal pools are not equal, so the basis for calculating microbial yield in faunated ruminants is uncertain. Urinary purine excretion may prove to be a noninvasive method for estimating microbial protein yields in intact dairy cows.     

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.     

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.     

Effect of inoculum preparation and dietary energy on microbial numbers and rumen protein degradation activity

Chilling of whole rumen contents prior to preparation of strained rumen fluid enriched with particle-associated microorganisms resulted in rumen inoculum with the highest microbial dry weight, total bacterial counts, and degradation rates for casein and soybean meal. Strained rumen fluid, whole rumen contents blended with strained rumen fluid, chilled strained rumen fluid, and strained rumen fluid plus particle-associated microorganisms were lower in bacterial counts and microbial dry weight. Except for strained rumen fluid plus particle-associated microorganisms, protein degradation rates were also lower. Three ruminally cannulated cows were used in a 3 X 3 Latin square experiment to determine the effect of diet on rumen microbial numbers and protein degradation rates. Cows were fed the following diets ad libitum: 1) 100% alfalfa hay (20.7% crude protein); 2) 63% alfalfa hay and 37% corn-soybean meal concentrate (18.2% crude protein); and 3) 37% alfalfa hay and 63% corn-soybean meal concentrate (15.1% crude protein). Diet 3 yielded rumen contents with the highest concentration of microbial dry weight, total and viable bacterial counts, total protozoal counts, and fractional degradation rates for casein and bovine serum albumin. However, degradation rate per unit microbial dry matter was not altered by diet.     

Apparent digestibility of nitrogen in rumen and whole tract of Friesian cattle fed direct-cut and wilted grass silages

Spring grass consisting of Lolium perene L. (81%), Poa pratensis L. (9%), and annual weeds (5%) was stored as direct-cut or as wilted silage and used in feeding trials to determine the effects of wilting on N utilization. Six mature Friesian cattle, fitted with rumen and simple duodenal cannulae, were fed the silages for ad libitum intake in a crossover design consisting of two 49-d periods. Wilting increased intake of organic matter and decreased ruminal apparent digestibility or organic matter and whole tract apparent digestibilities of organic matter, NDF, and total N. Rumen degradability of silage N was increased by wilting (.7 vs. .67). Direct-cut silage, in comparison with wilted silage, provide (g/d) less N intake (232 vs. 286) and lower duodenal flows of total N (215 vs. 293), non-NH3 N (202 vs. 280), microbial N (111 vs. 177), and total amino acids (1056.8 vs. 1342.7). Duodenal flows of NH3 N and undegraded N were not different between silages. Efficiency of bacterial N synthesis in rumen was higher for wilted than for direct-cut silage (32.3 vs. 21.4 g N/kg organic matter apparently digested in rumen). It was concluded that wilting increase silage intake, rumen bacterial synthetic efficiency, and duodenal flow of non-NH3 N in cattle.     

Nitrogen metabolism in the rumen

Protein metabolism in the rumen is the result of metabolic activity of ruminal microorganisms. The structure of the protein is a key factor in determining its susceptibility to microbial proteases and, thus, its degradability. Ruminal protein degradation is affected by pH and the predominant species of microbial population. Ruminal proteolytic activity decreases as pH decreases with high-forage dairy cattle-type rations, but not in high-concentrate beef-type rations. Accumulation of amino acid (AA) N after feeding suggests that AA uptake by rumen microorganisms could be the limiting factor of protein degradation in the rumen. In addition, there are several AA, such as Phe, Leu, and Ile, that are synthesized by rumen microorganisms with greater difficulty than other AA. The most common assessment of efficiency of microbial protein synthesis (EMPS) is determination of grams of microbial N per unit of rumen available energy, typically expressed as true organic matter or carbohydrates fermented. However, EMPS is unable to estimate the efficiency at which bacteria capture available N in the rumen. An alternative and complementary measure of microbial protein synthesis is the efficiency of N use (ENU). In contrast to EMPS, ENU is a good measurement for describing efficiency of N capture by ruminal microbes. Using EMPS and ENU, it was concluded that optimum bacterial growth in the rumen occurs when EMPS is 29 g of bacterial N/kg of fermented organic matter, and ENU is 69%, implying that bacteria would require about 1.31 x rumen-available N per unit of bacterial N. Because the distribution of N within bacterial cells changes with rate of fermentation, AA N, rather than total bacterial N should be used to express microbial protein synthesis.     

Nitrate improves ammonia incorporation into rumen microbial protein in lactating dairy cows fed a low-protein diet

Generation of ammonia from nitrate reduction is slower compared with urea hydrolysis and may be more efficiently incorporated into ruminal microbial protein. We hypothesized that nitrate supplementation could increase ammonia incorporation into microbial protein in the rumen compared with urea supplementation of a low-protein diet fed to lactating dairy cows. Eight multiparous Chinese Holstein dairy cows were used in a crossover design to investigate the effect of nitrate or an isonitrogenous urea inclusion in the basal low-protein diet on rumen fermentation, milk yield, and ruminal microbial community in dairy cows fed a low-protein diet in comparison with an isonitrogenous urea control. Eight lactating cows were blocked in 4 pairs according to days in milk, parity, and milk yield and allocated to urea (7.0 g urea/kg of dry matter of basal diet) or nitrate (14.6 g of NO₃^?/kg of dry matter of basal diet, supplemented as sodium nitrate) treatments, which were formulated on 75% of metabolizable protein requirements. Nitrate supplementation decreased ammonia concentration in the rumen liquids (?33.1%) and plasma (?30.6%) as well as methane emissions (?15.0%) and increased dissolved hydrogen concentration (102%), microbial N (22.8%), propionate molar percentage, milk yield, and 16S rRNA gene copies of Selenomonas ruminantium. Ruminal dissolved hydrogen was positively correlated with the molar proportion of propionate (r = 0.57), and negatively correlated with acetate-to-propionate ratio (r = ?0.57) and estimated net metabolic hydrogen production relative to total VFA produced (r = ?0.58). Nitrate reduction to ammonia redirected metabolic hydrogen away from methanogenesis, enhanced ammonia incorporation into rumen microbial protein, and shifted fermentation from acetate to propionate, along with increasing S. ruminantium 16S rRNA gene copies, likely leading to the increased milk yield.     

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.     

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.     

Corn silage in dairy cow diets to reduce ruminal methanogenesis: Effects on the rumen metabolically active microbial communities

Methane produced by the methanogenic Archaea that inhabit the rumen is a potent greenhouse gas and represents an energy loss for the animal. Although several strategies have been proposed to mitigate enteric CH₄ production, little is known about the effects of dietary changes on the microbial consortia involved in ruminal methanogenesis. Thus, the current study aimed to examine how the metabolically active microbes are affected when dairy cows were fed diets with increasing proportions of corn silage (CS). For this purpose, 9 ruminally cannulated lactating dairy cows were used in a replicated 3 × 3 Latin square design and fed a total mixed ration (60:40 forage:concentrate ratio on a dry matter basis) with the forage portion being either alfalfa silage (0% CS), corn silage (100% CS), or a 50:50 mixture (50% CS). Enteric CH₄ production was determined using respiration chambers and total rumen content was sampled for the determination of fermentation characteristics and molecular biology analyses (cDNA-based length heterogeneity PCR, quantitative PCR). The cDNA-based length heterogeneity PCR targeting active microbes revealed similar bacterial communities in cows fed 0% CS and 50% CS diets, whereas important differences were observed between 0% CS and 100% CS diets, including a reduction in the bacterial richness and diversity in cows fed 100% CS diet. As revealed by quantitative PCR, feeding the 100% CS diet increased the number of total bacteria, Prevotella spp., Archaea, and methanogenic activity, though it reduced protozoal number. Meanwhile, increasing the CS proportion in the diet increased propionate concentration but decreased ruminal pH, CH₄ production (L/kg of dry matter intake), and concentrations of acetate and butyrate. Based on these microbial and fermentation changes, and because CH₄ production was reduced by feeding 100% CS diet, this study shows that the use of cDNA-based quantitative PCR to estimate archaeal growth and activity is not reliable enough to reflect changes in ruminal methanogenesis. A more robust technique to characterize changes in archaeal community structures will help to better understand the microbial process involved in ruminal methanogenesis and, hence, enabling the development of more effective dietary CH₄ mitigation strategies.     

Particle size and endosperm type of dry corn grain altered duodenal flow of B vitamins in lactating dairy cows

The objective of the experiment was to determine if factors such as endosperm type (floury vs. vitreous) and particle size (fine vs. medium) of dry corn grain, known to affect starch digestibility in the rumen, modify apparent ruminal synthesis and duodenal flow of B vitamins in lactating dairy cows. Eight lactating multiparous Holstein cows equipped with rumen and duodenal cannulas were assigned randomly to a treatment sequence according to a 2 × 2 factorial arrangement in duplicate 4 × 4 Latin square design experiment. Duration of each experimental period was 21 d. When expressed per unit of dry matter intake (DMI), floury treatments increased duodenal flow and apparent ruminal synthesis of niacin and folates but tended to increase apparent degradation of thiamin in the rumen. Duodenal flow of thiamin, riboflavin, niacin, folates, and vitamin B₁₂, expressed per unit of DMI, decreased with an increase in particle size. Similarly, apparent degradation of thiamin and riboflavin was greater and apparent synthesis of niacin, folates, and vitamin B₁₂ was reduced when cows were fed coarser dry corn grain particles. Neither endosperm type nor particle size had an effect on duodenal flow and apparent ruminal synthesis of vitamin B₆. Apparent ruminal syntheses, expressed per unit of DMI, of all studied B vitamins but thiamin were negatively correlated with apparent ruminal digestibility of neutral detergent fiber. Duodenal flow of microbial N was positively correlated with apparent ruminal synthesis of riboflavin, niacin, vitamin B₆, and folates. Under the conditions of the present experiment, except for thiamin, the effects of factors increasing starch digestibility of dry corn grain in the rumen on the amounts of B vitamins available for absorption by the dairy cow seem to be mediated through differences on ruminal digestibility of neutral detergent fiber and, to a lesser extent, on duodenal microbial N flow.     

Rumen digestion kinetics,microbial yield,and omasal flows of nonmicrobial,bacterial, and protozoal amino acids in lactating dairy cattle fed fermentation by-products or urea as a soluble nitrogen source

The objective of this study was to evaluate the effect of a fermentation by-product on rumen function, microbial yield, and composition and flows of nutrients from the rumen in high-producing lactating dairy cattle. Eight ruminally cannulated multiparous Holstein cows averaging (mean ± standard deviation) 60 ± 10 d in milk and 637 ± 38 kg of body weight were randomly assigned to 1 of 2 treatment sequences in a switchback design. Treatment diets contained (dry matter basis) 44% corn silage, 13% alfalfa silage, 12% ground corn, and 31% protein premix, containing either a control mix of urea and wheat middlings (CON) or a commercial fermentation by-product meal (Fermenten, Arm and Hammer Animal Nutrition, Princeton, NJ) at 3% diet inclusion rate (EXP). The trial consisted of three 28-d experimental periods, where each period consisted of 21 d of diet adaptation and 7 d of data and sample collection. A triple-marker technique and double-labeled ¹⁵N¹⁵N-urea were used to were used to measure protozoal, bacterial, and nonmicrobial omasal flow of AA. Rumen pool sizes and omasal flows were used to determine digestion parameters, including fractional rates of carbohydrate digestion, microbial growth, and yield of microbial biomass per gram of degraded substrate. Fermentation by-product inclusion in EXP diets increased microbial N and amino acid N content in microbes relative to microbes from CON cows fed the urea control. Microbial AA profile did not differ between diets. Daily omasal flows of AA were increased in EXP cows as a result of decreased degradation of feed protein. The inclusion of the fermentation by-product increased nonmicrobial AA flow in cows fed EXP versus CON. Average protozoal contribution to microbial N flow was 16.8%, yet protozoa accounted for 21% of the microbial AA flow, with a range of 8 to 46% for individual AA. Cows in this study maintained an average rumen pool size of 320 g of microbial N, and bacterial and protozoal pools were estimated at 4 different theoretical levels of selective protozoa retention. Fractional growth rate of all microbes was estimated to be 0.069 h^?1, with a yield of 0.44 g of microbial biomass per gram of carbohydrate degraded. Results indicated that fermentation by-product can increase omasal flow of AA while maintaining adequate rumen N available for microbial growth and protein synthesis. Simulations from a developmental version of the Cornell Net Carbohydrate and Protein System indicated strong agreement between predicted and observed values, with some areas key for improvement in AA flow and bacterial versus protozoal N partitioning.     

Technical note: effect of removal of microbial cells by centrifugation on peptide and alpha-amino nitrogen concentrations in ruminal fluid

We evaluated the effect of centrifuging rumen fluid prior to analysis on concentrations of alpha-amino N (AAN) and peptides. Rumen fluid was collected from steers fed grain-based diets at either various times after feeding or after dosing the rumens with solubilized casein. Fluid was either directly processed for peptide analysis by acidifying 10 ml of rumen fluid with 0.5 ml of 70% (wt/wt) perchloric acid, or first centrifuged at 500 x g for 20 min to remove protozoa and then at 30,000 x g for 15 min to remove bacterial cells prior to further processing. By removing microbial cells, intracellular AAN and peptides were not included in subsequent analyses. Concentrations of AAN were determinedusing an automated trinitrobenzene sulfonic acid assay, and peptides were determined as the increase in AAN following acid hydrolysis of the samples. When casein was not dosed, removal of microbial cells prior to analysis decreased concentrations of both AAN andpeptides, and the decrease was greater for AAN (2.2 mM)than for peptides (1.2 mM). Dosing with casein led to much higher concentrations of ruminal peptides and AAN. After casein dosing, decreases in AAN and peptidecon-centrations due to prior centrifugation (2.1 mM and 1.0 mM for AAN a nd pept ides, respectively) were similar to the decreases observed before the casein dosing. Results suggest that the contribution of intracellular AAN and peptides to the concentrations in ruminal fluid are relatively constant across broad ranges of dietary protein supply for cattle fed corn-based diets.     

Microbial transaminase activities and their relationship with bovine rumen metabolites.

Two each adult male crossbred cattle and murrah buffalo were fed a diet of alfalfa hay, chopped wheat straw, and concentrate mixture. Total rumen transaminase activity of cattle was higher than that of buffalo. Rumen protozoal fractions showed higher total transaminase activity than bacterial fractions in both ruminant species. Besides generally studied glutamate oxalacetate transaminase and glutamate pyruvate transaminase, a large number of other microbial transaminases also have been detected in the rumen of both the ruminant species. Bacterial fractions of rumen liquor were devoid of transaminases utilizing tryptophan, threonine, and lysine as their substrates. Ruminal ammonia and nonprotein nitrogen were correlated positively with microbial transaminases in both species. Transamination reactions may be important for assimilation of ruminal ammonia to cellular proteins.     

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.     

Effect of incremental urea supplementation of a conventional corn silage-based diet on ruminal ammonia concentration and synthesis of microbial protein

One primiparous and 3 multiparous lactating Holstein cows fitted with ruminal and duodenal cannulas were used in a 4 × 4 Latin square design to determine the efficacy of adding urea to a corn silage-based diet on ruminal fermentation and microbial protein synthesis. Dietary treatments were 0, 0.3, 0.6, and 0.9% urea in diet dry matter (DM); urea was manually top dressed and incorporated into the ration. The basal diet contained (DM basis) 52% forage (with 61% of forage provided as corn silage) and 48% concentrate ingredients. The basal diet was formulated to meet National Research Council (NRC, 2001) requirements for energy and all nutrients except rumen-degradable protein (RDP) and metabolizable protein. Experimental periods lasted 14 d with the first 9 d for adaptation. The basal diet, without urea addition, contained 9.2% RDP in DM and had a predicted RDP balance of −167 g/d (NRC, 2001). There were no effects of dietary treatment on ruminal true digestibility of organic matter or ruminal apparent digestibility of neutral detergent fiber and acid detergent fiber. Total ruminal volatile fatty acid concentrations increased linearly with increasing urea level. Feeding increasing amounts of urea quadratically increased rumen ammonia N concentrations (9.0, 11.9, 12.8, and 17.4 mg/dL at 0, 0.3, 0.6, and 0.9% urea supplementation, respectively), passage of microbial N, and microbial N in duodenal digesta as a percentage of nonammonia N. The results of this study indicate that there were some positive effects of adding urea to the described lactating dairy cow diet, and that microbial protein synthesis was maximized at an average ruminal ammonia N concentration of 12.8 mg/dL when urea was added at 0.6% in diet DM.     

Development of a quantitative real-time PCR method to enumerate total bacterial counts in ready-to-eat fruits and vegetables

A newly developed real-time PCR assay rapidly quantifies the total bacterial numbers in contaminated ready-to-eat vegetables and fruits compared with the standard plate count method. Primers targeting the rpoB gene, which encodes for the beta subunit of the bacterial RNA polymerase and which is common to most bacterial species, was used instead of the 16S rRNA gene, which has multiple copies and varies among bacterial species. A primer pair specific for rpoB was confirmed to amplify rpoB in a wide range of bacterial species after we assessed 49 strains isolated from five kinds of fruits and vegetables. We purchased fruits and vegetables from retail shops and enumerated the bacteria associated with them by use of real-time PCR and compared this to the number found by the culture method. We found a high correlation between the threshold PCR cycle number when compared with the plate count culture number. The real-time PCR assay developed in this study can enumerate the dominant bacterial species in ready-to-eat fruits and vegetables.