Higher Butyrate Production Research Articles

347 Effects of Inoculation Period on the Nutritive Value of Pleurotus Ostreatus Treated Corn Stover

Abstract The objective of the study was to optimize and improve the feeding value of corn stover. Despite the documented ability of Pleurotus ostreatus to selectively delignify crop residues, it is not widely used for ruminant fiber digestion partly due to extended pretreatment time. Optimization of crop residues is influenced by enzymatic activities of fiber degrading enzymes and dependent on the stage of P. ostreatus growth. Thus, optimizing the inoculation period for lignin removal by P. ostreatus is important to improve and increase success on lignocellulose feed resources utilization. Therefore, we hypothesized a difference in nutritive value for different inoculation periods for P. ostreatus spent substrates. Two ruminally cannulated dairy cows were used in an in vitro study arranged in a 3 x 5 factorial design with 4 replicates to evaluate the effects of inoculation period [0 (control), 2, 4, 6 and 8 wk] on P. ostreatus spent substrates at 6, 24 and 48 h of fermentation on chemical composition, dry matter disappearance (DMd) and volatile fatty acid (VFA) production. Significant (P < 0.001) non-linear treatment effect across the inoculation period was observed in the treated samples (Table 1). An increase of 44.4 to 59.1% and 20.6 to 78.6% was noted for the crude protein and ash contents, respectively. Also, significant (P < 0.001) incubation time effect across the inoculation period resulted in a wide variation (22.6 - 755%) in the microbial mass with greater values for 2- and 4-wk treatments. Neutral detergent fiber and hemicellulose were significantly (P < 0.001) reduced with a range between 7.29 to 18.7% and 26.6 to 40.8%, respectively (Table 3). Inconsistent positive and negative outcomes across the periods in acid detergent fiber, lignin and cellulose resulted in significant (P < 0.001) 116 and 121% increment in non-fiber carbohydrates for 2 and 4-wk treatments, respectively. Consequently, both DMd and total VFA were significantly (P < 0.001) greater, ranging between 238 to 242% and 13.9 to 15.1% respectively for these treatments (Table 2). Additionally, the rumen fermentation pathway favored production of propionate with significant (P < 0.001) treatment and time effect that resulted in 7.46 and 8.30% increment in propionate production after 2 and 4 wk, respectively. Consequently, significant (P < 0.001) reduction was observed in acetate with 4.44 and 3.58%, and 11.7 and 11.1% in A:P ratio for 2 and 4 wk, respectively. High butyrate production was observed for 2 wk in comparison with 4 wk resulting in 18.3 and 9.86% increment, respectively, when compared with control (Table 4). The study showed that 2 wk of inoculation is sufficient to provide bio-transformed cow-calf feed resource from P. ostreatus treated corn stover, which is contrary to 30 days (4 wk) previously recommended.

Characterization of Short Chain Fatty Acids Produced by Selected Potential Probiotic Lactobacillus Strains.

Short-chain fatty acids (SCFAs), particularly butyrate, have received considerable attention with regard to their anti-cancer efficacy in delaying or preventing colorectal cancer. Several studies have reported that certain probiotic strains could produce SCFAs; however, different strains yielded different amounts of SCFAs. This study explored the ability to produce SCFAs of the following probiotic strains: Lacticaseibacillus paracasei SD1, Lacticaseibacillus rhamnosus SD4, Lacticaseibacillus rhamnosus SD11, and Lacticaseibacillus rhamnosus GG. L. paracasei SD1 and L. rhamnosus SD11 exhibited high butyrate production, particularly when the strains were combined. The functions of the SCFAs were further characterized; the SCFAs exerted a positive anti-cancer effect in the colon via various actions, including inhibiting the growth of the pathogens related to colon cancer, such as Fusobacterium nucleatum and Porphyromonas gingivalis; suppressing the growth of cancer cells; and stimulating the production of the anti-inflammatory cytokine IL-10 and antimicrobial peptides, especially human β-defensin-2. In addition, the SCFAs suppressed pathogen-stimulated pro-inflammatory cytokines, especially IL-8. The results of this study indicated that selected probiotic strains, particularly L. paracasei SD1 in combination with L. rhamnosus SD11, may serve as good natural sources of bio-butyrate, which may be used as biotherapy for preventing or delaying the progression of colon cancer.

Dietary Fiber Modulates the Release of Gut Bacterial Products Preventing Cognitive Decline in an Alzheimer's Mouse Model.

Fiber intake is associated with a lower risk for Alzheimer´s disease (AD) in older adults. Intake of plant-based diets rich in soluble fiber promotes the production of short-chain fatty acids (SCFAs: butyrate, acetate, propionate) by gut bacteria. Butyrate administration has antiinflammatory actions, but propionate promotes neuroinflammation. In AD patients, gut microbiota dysbiosis is a common feature even in the prodromal stages of the disease. It is unclear whether the neuroprotective effects of fiber intake rely on gut microbiota modifications and specific actions of SCFAs in brain cells. Here, we show that restoration of the gut microbiota dysbiosis through the intake of soluble fiber resulted in lower propionate and higher butyrate production, reduced astrocyte activation and improved cognitive function in 6-month-old male APP/PS1 mice. The neuroprotective effects were lost in antibiotic-treated mice. Moreover, propionate promoted higher glycolysis and mitochondrial respiration in astrocytes, while butyrate induced a more quiescent metabolism. Therefore, fiber intake neuroprotective action depends on the modulation of butyrate/propionate production by gut bacteria. Our data further support and provide a mechanism to explain the beneficial effects of dietary interventions rich in soluble fiber to prevent dementia and AD. Fiber intake restored the concentration of propionate and butyrate by modulating the composition of gut microbiota in male transgenic (Tg) mice with Alzheimer´s disease. Gut dysbiosis wasassociated with intestinal damage and high propionate levels in control diet fed-Tg mice. Fiber-rich diet restored intestinal integrity and promoted the abundance of butyrate-producing bacteria. Butyrate concentration was associated with better cognitive performance in fiber-fed Tg mice. A fiber-rich diet may prevent the development of a dysbiotic microbiome and the related cognitive dysfunction in people at risk of developing Alzheimer´s disease.

Probiotic and Functional Characterization of Pediococcus acidilactici Isolated from Bhaati jaanr, Traditional Fermented Rice Porridge.

Traditional fermented foods are the ideal source of novel probiotic isolates which are known to have significant therapeutic benefits and play a vital role as bioprotective agents. Bhaati jaanr is an ethnic fermented rice beverage popularly consumed in sub-Himalayan regions. The strain UAMS was isolated from Bhaati jaanr based on high butyrate production and evaluated for the potential probiotic characteristics. MALDI-TOF MS and 16s rRNA gene sequencing revealed the identity of strains as Pediococcus acidilactici. The isolated strain exhibited high tolerance to gastric and bile stress, autoaggregation, hydrophobicity, and adherence to colon cells. Antibiotic susceptibility testing results showed the resistance of the isolated strain toward tested common antibiotics and the pathogenic determinants were absent in PCR-based detection. Moreover, the organism was able to inhibit the growth of Listeria, Salmonella, Staphylococcus, and Enterococcus species. The isolate was found to be a high butyrate producer along with other short-chain fatty acids and exhibited an anti-proliferative effect against colon cancer cells HT29 and SW480. Therefore, our study represents Pediococcus acidilactici UAMS as a potent putative probiotic with bioprotective abilities.

Evaluation of bio-hydrogen production using rice straw hydrolysate extracted by acid and alkali hydrolysis

This study was conducted to investigate the properties of hydrolysates obtained from acid and alkali hydrolysis and to evaluate the feasibility of employing them for bio-hydrogen production. High sugar concentrations of 16.8 g/L and 13.3 g/L were present in 0.5% and 1.0% H2SO4 hydrolysates, respectively. However, H2SO4 hydrolysis resulted in large amounts of short-chain fatty acids (SCFAs) and furan derivatives, which were removed by detoxification. In bio-hydrogen production, 1.0% H2SO4 hydrolysate showed a 55.6 mL of highest hydrogen production and 1.14 mol-H2/mol-hexose equivalentadded of hydrogen yield. In control and 1.0% NaOH hydrolysate, 29.7 mL and 36.9 mL of hydrogen were produced, respectively. Interestingly, relatively high acetate and butyrate production resulted in lactate reduction. Also, NH4OH hydrolysate produced less than 10 mL of hydrogen. Thus, these results indicate that hydrogen production and metabolite distribution can vary depending on the sugars and by-product composition in the hydrolysate.

Butyrate production and purification by combining dry fermentation of food waste with a microbial fuel cell

This study developed and evaluated a high-purity butyrate producing bioprocess from food waste by combining dry fermentation (DF) with a microbial fuel cell (MFC). Acclimatization of a DF reactor with an enrichment culture resulted in high food waste degradation (VS removed, %) and butyrate production. A high VS degradation of 81%, butyrate concentration of up to 24 gCODbutyrate/L and butyrate yields of 497 gCODbutyrate/kg VSadded was obtained in the DF reactor. As a result, butyrate comprised 83% of all short chain fatty acids (SCFA) in the DF broth. Acetate (10%) and propionate (7%) comprised the rest of the SCFA. The butyrate composition was further purified by feeding the DF broth to a multi-electrode MFC enriched with anode respiring bacteria (ARB) such as Geobacter sp. (>55%). The ARB in the MFC removed acetate and propionate while purified butyrate was recovered in the MFC effluent. Butyrate purity in the MFC effluent reached as high as 99% at hydraulic retention time of 72 h. Along with butyrate purification, the MFC produced electric power in a range of 0.1–0.6 Wh/gCODbutyraterecovered (or 0.01–7.85 kWh/ton of food waste), demonstrating that MFCs can be an energy-positive butyrate purification bioprocess.

Interrogation of the perturbed gut microbiota in gouty arthritis patients through in silico metabolic modeling.

Recent studies have shown perturbed gut microbiota associated with gouty arthritis, a metabolic disease characterized by an imbalance between uric acid production and excretion. To mechanistically investigate altered microbiota metabolism associated with gout disease, 16S rRNA gene amplicon sequence data from stool samples of gout patients and healthy controls were computationally analyzed through bacterial community metabolic models. Patient‐specific community models constructed with the metagenomics modeling pipeline, mgPipe, were used to perform k‐means clustering of samples according to their metabolic capabilities. The clustering analysis generated statistically significant partitioning of samples into a Bacteroides‐dominated, high gout cluster and a Faecalibacterium‐elevated, low gout cluster. The high gout cluster was predicted to allow elevated synthesis of the amino acids D‐alanine and L‐alanine and byproducts of branched‐chain amino acid catabolism, while the low gout cluster allowed higher production of butyrate, the sulfur‐containing amino acids L‐cysteine and L‐methionine, and the L‐cysteine catabolic product H2S. By expanding the capabilities of mgPipe to provide taxa‐level resolution of metabolite exchange rates, acetate, D‐lactate and succinate exchanged from Bacteroides to Faecalibacterium were predicted to enhance butyrate production in the low gout cluster. Model predictions suggested that sulfur‐containing amino acid metabolism generally and H2S more specifically could be novel gout disease markers.

Anti-inflammatory effect of microbial consortia during the utilization of dietary polysaccharides

The gut microbiome has a significant impact on host health, especially at the metabolic level. Dietary compounds arriving at the colon have a large influence on the composition of the gut microbiome. High fiber diets have been associated to health benefits that are mediated in great part by short chain fatty acids (SCFA). Gut microbial interactions are relevant for the utilization of complex carbohydrates in the gut microbiome. In this work we characterized the utilization of two dietary polysaccharides by combinations of representative adult gut microbes, and the impact of their activities on a cellular inflammation model. Paired combinations of Bifidobacterium adolescentis, Bacteroides dorei, Lactobacillus plantarum, Escherichia coli and Clostridium symbiosum were grown in inulin or xylan as carbon source. Their relative abundance, substrate consumption and major SCFAs produced were determined. Higher cell growth was observed during inulin consumption, and B. adolescentis and L. plantarum were dominant in co-cultures. The co-culture of B. dorei and C. symbiosum was dominant in xylan. In several cases the combined bacterial growth was lower in co-cultures than monocultures, with a few exceptions of synergistic growth between microorganisms. Inulin fermentation resulted in larger acetate and lactate concentrations, and several combinations grown in xylan containing C. symbiosum were characterized by high amounts of butyrate. These microbial consortia were scaled to batch bioreactor fermentations reaching high cell densities and similar profiles to co-culture experiments. Interestingly, a microbial combination producing high amounts of butyrate was able to reduce IL-8 expression in HT-29 cells co-incubated with TNFα. In summary, this work shows that microbial interactions during the utilization of dietary polysaccharides are complex and substrate dependent. Moreover, certain combinations deploy potent anti-inflammatory effects, which are independent of individual microbial growth, and could be mediated in part by higher butyrate production.

Impact of dietary pattern of the fecal donor on in vitro fermentation properties of whole grains and brans

Because diet influences gut microbiota composition and function, the purpose of this study was to determine how fecal donor diet impacts in vitro fermentation properties of whole grain flours and brans from corn, oats, rye, and wheat. Samples were fermented with fecal microbiota from subjects with similar energy intakes but differing in intakes of several beneficial nutrients (G1>G2). Shifts in the microbiota during fermentation were a function of diet group and time. Fecal microbiota from G1 subjects showed less decrease in diversity during fermentation and these microbiotas showed higher carbohydrate utilization and butyrate production compared with microbiota from G2 subjects. More carbohydrates were fermented from whole grains than brans. Rye induced high carbohydrate fermentability and butyrate production accompanied by low ammonia production, but only when using fecal microbiota from G1 subjects. Thus, diet quality influences the ability of the microbiota to ferment carbohydrates, differentiate among grains, and produce butyrate.

In vitro batch cultures of gut microbiota from healthy and ulcerative colitis (UC) subjects suggest that sulphate-reducing bacteria levels are raised in UC and by a protein-rich diet

Imbalances in gut microbiota composition during ulcerative colitis (UC) indicate a role for the microbiota in propagating the disorder. Such effects were investigated using in vitro batch cultures (with/without mucin, peptone or starch) inoculated with faecal slurries from healthy or UC patients; the growth of five bacterial groups was monitored along with short-chain fatty acid (SCFA) production. Healthy cultures gave two-fold higher growth and SCFA levels with up to ten-fold higher butyrate production. Starch gave the highest growth and SCFA production (particularly butyrate), indicating starch-enhanced saccharolytic activity. Sulphate-reducing bacteria (SRB) were the predominant bacterial group (of five examined) for UC inocula whereas they were the minority group for the healthy inocula. Furthermore, SRB growth was stimulated by peptone presumably due to the presence of sulphur-rich amino acids. The results suggest raised SRB levels in UC, which could contribute to the condition through release of toxic sulphide.

In vitro fermentation of various pectin substrates by human fecal microbiota

Metabolic activity of the gut microbiota is formation of short chain fatty acids (SCFAs). SCFA contribute to human healthy by serving as an energy source for intestinal epithelial cells and inhibiting pathogenic growth by lowering pH in the intestinal lumen. The aims of this study were to assess the short chain fatty acids production of various pectin samples (High Methoxy Pectin (HMP), Sugar Beet Pectin (SBP), and Soy pectin (SOY)) and to determine the composition of gut microbiota. Fructooligosaccharide (FOS, known prebiotic) was used as a control. Fresh feces were collected from 4 healthy men and incubated anaerobically with various substrates at 37°C. Culture aliquots were taken at 0, 6, 12, 24, and 30 h. SCFAs were measured by GC. FOS resulted in higher butyrate production and decreased the production of acetate compared with other pectin treatments (P<0.05). Compared with other treatments, incubation with SOY showed significantly greater propionate production (P<0.05). Denaturing gradient gel electrophoresis (DGGE) fingerprint analysis showed a distinct profile of fecal microbiota with different samples. The findings indicate that consuming different types of pectin substrates might affect fecal microbiota composition and enhance the production of SCFAs. Funding provided by Arkansas Soybean Promotion Board is greatly appreciated.

The management of the transition from hay- to pasture-based diets affects milk fatty acid kinetics

As fatty acids (FA) may become included among the quality parameters setting milk prices, their variation during diet changes needs to be investigated. The aim of this work was to study the kinetics of milk FA during rapid or progressive transition from hay- to pasture-based diets. Two farms in the southwestern Alps were selected. The milk of five Valdostana Red Pie cows from each farm was analysed on days 0, 1, 2, 3, 5 and 7 (at day 1, pasture was introduced into the diet), and thereafter twice a week. Changes in the milk FA became significant after days 3 or 5. The FA from C4:0 to C18:0, C18:2n-6, C18:3n-3 and C20:0 became stable after day 5 for both transition types. The cis9-C18:1, trans10 + trans11-C18:1 and cis9trans11-conjugated linoleic acid (CLA) became stable 3, 16 and 16 days after the maximum pasture intake, respectively. The C4:0 peaked at 3 days after pasture introduction or increase in the diet, probably reflecting high butyrate production in the rumen due to the high carbohydrate concentration in fresh herbage. The kinetics of all FAs fitted a log-normal cumulate distribution (except for C4:0), but model coefficients did not differ between the rapid and the progressive transition. After diet transition, only cis9-C18, cis9trans11-CLA, C18:3n-3, the mono-unsaturated and poly-unsaturated FA, and the sum of CLA isomers increased faster in the rapid transition than in the progressive transition diet (higher a coefficients), because of the higher initial pasture intake. Knowledge of the kinetics of milk FA when pasture is introduced into the diet could help farmers to improve milk nutritional quality.

Starch-entrapped microspheres show a beneficial fermentation profile and decrease in potentially harmful bacteria duringin vitrofermentation in faecal microbiota obtained from patients with inflammatory bowel disease

The purpose of this research was to test the hypothesis that starch-entrapped microspheres would produce favourable fermentation profiles and microbial shifts during in vitro fermentation with the faecal microbiota from patients with inflammatory bowel disease (IBD). In vitro fermentation was carried out using a validated, dynamic, computer-controlled model of the human colon (Toegepast Natuurwetenschappelijk Onderzoek gastro-intestinal model-2) after inoculation with pooled faeces from healthy individuals, patients with inactive IBD (Crohn's disease (CD)) or patients with active IBD (ulcerative colitis (UC)). Starch-entrapped microspheres fermented more slowly and produced more butyrate than fructo-oligosaccharides (FOS) when fermented with the faecal microbiota from patients with active UC. When fermented with the microbiota from patients with inactive CD, starch-entrapped microspheres also fermented more slowly but produced similar amounts of butyrate compared with FOS. Starch-entrapped microspheres showed a greater ability to maintain a low pH during simulated-distal colon conditions compared with FOS. After fermentation with the microbiota from inactive CD patients, starch-entrapped microspheres resulted in lower concentrations of some potentially harmful gut bacteria, included in Bacteroides, Enterococcus, Fusobacterium and Veillonella, compared with FOS. These findings suggest that slow fermenting starch-entrapped microspheres may induce a favourable colonic environment in patients with IBD through high butyrate production, maintenance of low pH in the distal colon and inhibition of the growth of potentially harmful bacteria.

PH and peptide supply can radically alter bacterial populations and short-chain fatty acid ratios within microbial communities from the human colon.

The effects of changes in the gut environment upon the human colonic microbiota are poorly understood. The response of human fecal microbial communities from two donors to alterations in pH (5.5 or 6.5) and peptides (0.6 or 0.1%) was studied here in anaerobic continuous cultures supplied with a mixed carbohydrate source. Final butyrate concentrations were markedly higher at pH 5.5 (0.6% peptide mean, 24.9 mM; 0.1% peptide mean, 13.8 mM) than at pH 6.5 (0.6% peptide mean, 5.3 mM; 0.1% peptide mean, 7.6 mM). At pH 5.5 and 0.6% peptide input, a high butyrate production coincided with decreasing acetate concentrations. The highest propionate concentrations (mean, 20.6 mM) occurred at pH 6.5 and 0.6% peptide input. In parallel, major bacterial groups were monitored by using fluorescence in situ hybridization with a panel of specific 16S rRNA probes. Bacteroides levels increased from ca. 20 to 75% of total eubacteria after a shift from pH 5.5 to 6.5, at 0.6% peptide, coinciding with high propionate formation. Conversely, populations of the butyrate-producing Roseburia group were highest (11 to 19%) at pH 5.5 but fell at pH 6.5, a finding that correlates with butyrate formation. When tested in batch culture, three Bacteroides species grew well at pH 6.7 but poorly at pH 5.5, which is consistent with the behavior observed for the mixed community. Two Roseburia isolates grew equally well at pH 6.7 and 5.5. These findings suggest that a lowering of pH resulting from substrate fermentation in the colon may boost butyrate production and populations of butyrate-producing bacteria, while at the same time curtailing the growth of Bacteroides spp.

Effect of pH on metabolic pathway shift in fermentation of xylose by Clostridium tyrobutyricum

The effect of pH (between 5.0 and 6.3) on butyric acid fermentation of xylose by Clostridium tyrobutyricum was studied. At pH 6.3, the fermentation gave a high butyrate production of 57.9 g l −1 with a yield of 0.38–0.59 g g −1 xylose and a reactor productivity up to 3.19 g l −1 h −1. However, at low pHs (<5.7), the fermentation produced more acetate and lactate as the main products, with only a small amount of butyric acid. The metabolic shift from butyrate formation to lactate and acetate formation in the fermentation was found to be associated with changes in the activities of several key enzymes. The activities of phosphotransbutyrylase (PTB), which is the key enzyme controlling butyrate formation, and NAD-independent lactate dehydrogenase (iLDH), which catalyzes the conversion of lactate to pyruvate, were higher in cells producing mainly butyrate at pH 6.3. In contrast, cells at pH 5.0 had higher activities of phosphotransacetylase (PTA), which is the key enzyme controlling acetate formation, and lactate dehydrogenase (LDH), which catalyzes the conversion of pyruvate to lactate. Also, PTA was very sensitive to the inhibition by butyric acid. Difference in the specific metabolic rate of xylose at different pHs suggests that the balance in NADH is a key in controlling the metabolic pathway used by the cells in the fermentation.

Stimulation of Butyrate Production by Gluconic Acid in Batch Culture of Pig Cecal Digesta and Identification of Butyrate-Producing Bacteria

Gluconic acid reaches the large intestine to stimulate lactic acid bacteria. However, the fermentation pattern of gluconic acid has yet to be elucidated. Accordingly, we examined the fermentation properties induced by gluconic acid in the pig cecal digesta in vitro. We also tested sorbitol and glucose, substrates for which the fermentation rate and patterns are known. The gluconic acid-utilizing bacteria were further isolated from pig cecal digesta and identified to examine the effect of gluconic acid on hind gut fermentation. Gluconic acid was fermented more slowly than were the other two substrates. Gluconic acid stimulated butyrate production; the butyrate molar percentage reached 26%, which is considered a high butyrate production. The majority of gluconic acid fermenters were identified as lactic acid bacteria, such as Lactobacillus reuteri and L. mucosae, and acid-utilizing bacteria, such as Megasphaera elsdenii and Mitsuokella multiacida. The gluconic acid fermented by lactic acid bacteria, and the lactate and acetate that were produced were used to form butyrate by acid-utilizing bacteria, such as M. elsdenii. Gluconic acid may be useful as a prebiotic to stimulate butyrate production in the large intestine.

Fecal high butyrate production of clinical relevance with plantago ovata seeds (POS)

Fecal high butyrate production of clinical relevance with plantago ovata seeds (POS)

Cornstarch fermentation by the colonic microbial community yields more butyrate than does cabbage fiber fermentation; cornstarch fermentation rates correlate negatively with methanogenesis

Fermentations of cornstarch and a cabbage-fiber preparation by human fecal suspensions were studied. The molar percent of butyrate of total short-chain fatty acid products was significantly higher when cornstarch was the substrate. Higher molar percents of butyrate were also produced from cornstarch as compared with endogenous substrate when rat fecal suspensions were used. A range of cornstarch fermentation rates was found with suspensions from 20 human subjects. Rapid fermentaion was associated with the absence of methane production. Methane-negative rat fecal suspensions also fermented cornstarch more rapidly than did methane-positive suspensions. High butyrate production may be important because butyrate provides energy to colonocytes and it regulates differentiation of cultured cells.

Contribution of Propionate to Glucose Synthesis in the Lactating and Nonlactating Cow

Ruminal volatile fatty acid production rates and blood glucose entry rate were measured in two cows, nonlactating and again lactating, with a constant infusion isotope dilution technique. Incorporation of ruminal acetate, propionate, and buty rate carbon into blood glucose was determined, and the contribution of propionate to glucose synthesis calculated.When the cows were nonlactating and consuming 9.5kg dry matter of a high grain ration, acetate and butyrate production averaged 42 and 8 moles per day. Because of an infusion error, propionate production could not be calculated, but was estimated to be 12 moles per day. When cows were lactating and consuming 17.0kg dry matter of the same ration, acetate and butyrate production averaged 77 and 6 moles per day. Propionate production was estimated at 25 moles per day. Total ruminal volatile acid production per kilogram ration dry matter, including the estimated propionate production, was 6.4 and 6.7 moles during the lactating and nonlactating phases. Average blood glucose entry rates during the lactating and nonlactating phases were 14.0 and 7.6 moles per day (∼0.81 and 0.44g/hr/kg body weight3/4).Propionate contributed to a minimum of 32% of the blood glucose entry rate in the nonlactating phase and 45% in the lactating phase. These estimates are minimum values, based upon the transfer of propionate-2-14C carbon to glucose. A maximum estimate, based upon the extent of carbon cycling in the citric acid cycle, suggested that propionate contributed to not more than 60% of the glucose formed during the lactating phase. The amount of cycling in the citric acid cycle was calculated from the distribution of radio-activity in glucose derived from propionate-2-14C.