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Glycogen is an α-glucan produced by rumen microbes from various feed carbohydrates. It may be digested ruminally or intestinally to provide nutrients. The physicochemical and enzymatic hydrolysis characteristics of microbial glycogen have not been described in detail, but do influence its conversion to absorbable nutrients in vivo, its nutritional comparability with plant starch sources, and its accurate analysis in vitro. The objectives of this study were to determine presence or absence of a gelatinization response and to describe enzymatic digestion characteristics of glycogen granules isolated from ruminal protozoa obtained from lactating dairy cows. Protozoal glycogen granules were determined to be 98.3% α-glucan. Granules displayed gelatinization, the breaking of hydrogen bonds between molecules or branches, at 65°C compared with purified wheat and corn starches, which initiated gelatinization at 50 and 65°C, respectively. Digestion of ungelatinized samples with amyloglucosidase for 2 h at 39°C showed approximately 3-fold greater hydrolysis to glucose for protozoal glycogen (25.2% of dry matter; DM) than for wheat (9.9% of DM) or corn (8.2% of DM) starches. Based on enzymatic digestion results, protozoal glycogen may be more readily digested than intact corn or wheat starches and should be gelatinized or the hydrogen bonds otherwise disrupted to allow more complete recovery in enzymatic analysis.

Our knowledge of the role of carbohydrates in dairy cattle nutrition has advanced substantially in the 100 years of the publication of the Journal of Dairy Science. In this review, we trace the history of scientific investigation and discovery from crude fiber, nitrogen-free extract, and "unidentified factors" to our present analytical schemes and understanding of ruminal and whole-animal utilization and effects of dietary carbohydrates. Historically, advances in research and new feeding standards occurred in parallel with and fostered by new methods of analysis. The 100 years of research reviewed here has bequeathed to us an impressive legacy of information, which we will continue to grow.

In response to drought-induced forage shortages along with increased corn and soy prices, this study was conducted to evaluate lactation responses of dairy cows to lower-forage diets supplemented with forage substitutes. By-product feeds were used to completely replace corn grain and soybean feeds. Forty-eight late-lactation cows were assigned to 1 of 4 diets using a randomized complete block design with a 2-wk covariate period followed by a 4-wk experimental period. The covariate diet contained corn grain, soybean meal, and 61% forage. Experimental diets contained chopped wheat straw (WS)/sugar beet pulp at 0/12, 3/9, 6/6, or 9/3 percentages of diet dry matter (DM). Corn silage (20%), alfalfa silage (20%), pelleted corn gluten feed (25.5%), distillers grains (8%), whole cottonseed (5%), cane molasses/whey blend (7%), and vitamin and mineral mix with monensin (2.5%) comprised the rest of diet DM. The WS/sugar beet pulp diets averaged 16.5% crude protein, 35% neutral detergent fiber, and 11% starch (DM basis). Cows consuming the experimental diets maintained a 3.5% fat- and protein-corrected milk production (35.2 kg; standard deviation=5.6 kg) that was numerically similar to that measured in the covariate period (35.3 kg; standard deviation=5.0 kg). Intakes of DM and crude protein declined linearly as WS increased, whereas neutral detergent fiber intake increased linearly. Linear increases in time spent ruminating (from 409 to 502 min/d) and eating (from 156 to 223 min/d) were noted as WS inclusion increased. Yields of milk fat and 3.5% fat-and protein-corrected milk did not change as WS increased, but those of protein and lactose declined linearly. Phosphorous intakes were in excess of recommended levels and decreased linearly with increasing WS inclusion. Nutritional model predictions for multiparous cows were closest to actual performance for the National Research Council 2001 model when a metabolizable protein basis was used; primiparous cow performance was better predicted by energy-based predictions made with the National Research Council or Cornell Net Carbohydrate and Protein System models. Model predictions of performance showed a quadratic diet effect with increasing WS. Lactating dairy cows maintained production on low-forage diets that included forage substitutes, and in which by-product feeds fully replaced corn grain and soybean. However, longer-term studies are needed to evaluate animal performance and to improve model predictions of performance on these nontraditional diets.

Microbial fermentation of carbohy- drates in the hindgut of dairy cattle is responsible for 5 to 10% of total-tract carbohydrate digestion. When dietary, animal, or environmental factors contribute to abnormal, excessive flow of fermentable carbohydrates from the small intestine, hindgut acidosis can occur. Hindgut acidosis is characterized by increased rates of production of short-chain fatty acids including lactic acid, decreased digesta pH, and damage to gut epithe- lium as evidenced by the appearance of mucin casts in feces. Hindgut acidosis is more likely to occur in high-producing animals fed diets with relatively greater proportions of grains and lesser proportions of forage. In these animals, ruminal acidosis and poor selective retention of fermentable carbohydrates by the rumen will increase carbohydrate flow to the hindgut. In more severe situations, hindgut acidosis is characterized by an inflammatory response; the resulting breach of the barrier between animal and digesta may contribute to laminitis and other disorders. In a research setting, effects of increased hindgut fermentation have been evaluated using pulse-dose or continuous abomasal infusions of varying amounts of fermentable carbohy- drates. Continuous small-dose abomasal infusions of 1 kg/d of pectin or fructans into lactating cows resulted in decreased diet digestibility and decreased milk fat percentage without affecting fecal pH or VFA concen- trations. The decreased diet digestibility likely resulted from increased bulk in the digestive tract or from in- creased digesta passage rate, reducing exposure of the digesta to intestinal enzymes and epithelial absorptive surfaces. The same mechanism is proposed to explain the decreased milk fat percentage because only milk concentrations of long-chain fatty acids were decreased. Pulse-dose abomasal fructan infusions (1 g/kg of BW) into steers resulted in watery feces, decreased fecal pH, and increased fecal VFA concentrations, without caus- ing an inflammatory response. Daily 12-h abomasal in- fusions of a large dose of starch (~4 kg/d) have also induced hindgut acidosis as indicated by decreased fe- cal pH and watery feces. On the farm, watery or foamy feces or presence of mucin casts in feces may indicate hindgut acidosis. In summary, hindgut acidosis occurs because of relatively high rates of large intestinal fer- mentation, likely due to digestive dysfunction in other parts of the gut. A better understanding of the relation- ship of this disorder to other animal health disorders is needed.

The concept of improving animal performance by going beyond simply meeting requirements and synchronizing ruminal availability of protein and energy has been with us for at least 3 decades. Although theoretically appealing, research and field results have not supported this approach to diet formulation. Why? Essential to successful ruminal synchrony is the ability to predict available amounts and fates of diverse substrates. The substrates come from varied sources; their efficiencies of use and yields of products are affected by inherent properties, interactions, transformations, and passage. However, substrate quality and availability in the rumen are affected only in part by diet. For example, NPN, true protein, and peptides are contributed by diet and intraruminal recycling, with additional endogenous NPN contributions by the cow. Changes in factors that alter the rate or extent of substrate fermentation, such as the rate of passage or ruminal pH, can alter nutrient yield from the rumen and must be accounted for in order for synchrony to work. Our ability to estimate ruminally available substrate is also challenged by normal variation in feed composition and imprecision in component and digestibility analyses. Current in vitro assays may not be adequate to accurately describe the digestibility of feed components in vivo in mixed diets. There are some indications that the amount or pattern of supply of fermentable carbohydrate has a greater impact on microbial production and efficiency than does the pattern of protein supply. Animal responses to modifications in the supply of true protein from the rumen may be masked if additional protein is oxidized by tissues or if AA from endogenous sources cover deficiencies. Animal factors, such as response to immune challenge and sustained damage to tissues, will also affect partitioning of nutrients for production and may alter an animal's response to changes in nutrient supply. With the array of factors internal and external to the diet that must be considered, "synchrony" implies a greater deliberate precision in diet manipulation than may be currently possible to effect. Perhaps we should consider balance. Within the rumen and cow, can we generate conditions so that needed substrates or nutrients are available from the diet or accessible from endogenous resources to meet requirements and enhance productivity and efficiency? This approach involves the whole animal, rather than only the rumen and feed we offer to the cow.

Two experiments were conducted to evaluate the effect of molasses or molasses-urea supplementation with or without soybean hulls on digestibility, intake, and animal performance in cattle fed bahiagrass (Paspalum notatum Flugge) hay. In Experiment 1, 6 Holstein steers (average BW 256 ± 15 kg) were fed 1 of 6 treatment diets: 1) bahiagrass hay only; 2) hay plus molasses; 3) hay plus molasses-urea; 4) hay plus soybean hulls; 5) hay plus soybean hulls and molasses; and 6) hay plus soybean hulls and molasses-urea. Molasses were fed at 0.59% ofBW (DM basis), soybean hulls at 0.75%, and urea at 4.5% of the weight of the molasses. Dry matter intake and apparent digestibility were measured in an incomplete Latin square design with a 28-d recovery period between each period. Digestibility of dietary DM, OM, and ADF was increased with soybean hulls (P 0.10) by soybean hulls. Urea added to molasses increased nonesterified fatty acids (P 0.10) by treatments. Blood glucose was increased (P

Effects of sucrose (Suc) concentration on fermentation kinetics and products were evaluated using 3 concentrations of Suc, with 1 concentration of isolated NDF from Bermudagrass fermented together in batch culture in vitro with rumen inoculum. Fixed amounts of medium and inoculum were the protein sources, so protein:Suc decreased with increasing Suc. Kinetics were calculated from gas production over 48 h in a randomized complete block design (n = 28), and product yield was evaluated with sampling every 4 h for 24 h in a split-split plot in time design (n = 84). Fermentation vial was the experimental unit. Increasing Suc increased the lag time of rapidly (P 1 indicate incorporation of C from the medium, likely from AA and peptides. The results support the premises that direct effects of Suc concentration and perhaps protein:Suc alter yields of fermentation products. That substrate concentration altered fermentation products and kinetics, possibly due to interactions with the run conditions, advises the clear definition of substrates and fermentation conditions to determine how the results integrate into our knowledge of ruminant nutrition.

Fructans are an important nonfiber carbohydrate in cool season grasses. Their fermentation by ruminal microbes is not well described, though such information is needed to understand their nutritional value to ruminants. Our objective was to compare kinetics and product formation of orchardgrass fructan (phlein; PHL) to other nonfiber carbohydrates when fermented in vitro with mixed or pure culture ruminal microbes. Studies were carried out as randomized complete block designs. All rates given are first-order rate constants. With mixed ruminal microbes, rate of substrate disappearance tended to be greater for glucose (GLC) than for PHL and chicory fructan (inulin; INU), which tended to differ from each other (0.74, 0.62, and 0.33 h(-1), respectively). Disappearance of GLC had almost no lag time (0.04 h), whereas the fructans had lags of 1.4h. The maximum microbial N accumulation, a proxy for cell growth, tended to be 20% greater for PHL and INU than for GLC. The N accumulation rate for GLC (1.31h(-1)) was greater than for PHL (0.75 h(-1)) and INU (0.26 h(-1)), which also differed. More microbial glycogen (+57%) was accumulated from GLC than from PHL, though accumulation rates did not differ (1.95 and 1.44 h(-1), respectively); little glycogen accumulated from INU. Rates of organic acid formation were 0.80, 0.28, and 0.80 h(-1) for GLC, INU, and PHL, respectively, with PHL tending to be greater than INU. Lactic acid production was more than 7-fold greater for GLC than for the fructans. The ratio of microbial cell carbon to organic acid carbon tended to be greater for PHL (0.90) and INU (0.86) than for GLC (0.69), indicating a greater yield of cell mass per amount of substrate fermented with fructans. Reduced microbial yield for GLC may relate to the greater glycogen production that requires ATP, and lactate production that yields less ATP; together, these processes could have reduced ATP available for cell growth. Acetate molar proportion was less for GLC than for fructans, and less for PHL than for INU. In studies with pure cultures, all microbes evaluated showed differences in specific growth rate constants (μ) for GLC, fructose, sucrose, maltose, and PHL. Selenomonas ruminantium and Streptococcus bovis showed the highest μ for PHL (0.55 and 0.67 h(-1), respectively), which were 50 to 60% of the μ achieved for GLC. The 10 other species tested had μ between 0.01 and 0.11h(-1) with PHL. Ruminal microbes use PHL differently than they do GLC or INU.

Evaluation of physical, chemical, and enzymatic hydrolysis characteristics of protozoal glycogen is best performed on a pure substrate to avoid interference from other cell components. A method for isolating protozoal glycogen granules without use of detergents or other potentially contaminating chemicals was developed. Rumen inoculum was incubated anerobically in vitro with glucose. Glycogen-laden protozoa produced in the fermentation, primarily isotrichids, were allowed to sediment in a separatory funnel and were dispensed. The protozoa were processed through repeated centrifugations and sonication to isolate glycogen granules largely free of feed and cellular debris. The final water-insoluble lyophilized product analyzed as 98.3% α-glucan with very rare starch granules and 1.9% protein. Observed losses of glycogen granules during the clean-up process indicate that this procedure should not be used for quantitative assessment of protozoal glycogen from fermentations. Further optimization of this procedure to enhance the amount of glycogen obtained per fermentation may be possible.

Microbial glycogen measurement is used to account for fates of carbohydrate substrates. It is commonly applied to washed cells or pure cultures which can be accurately subsampled, allowing the use of smaller sample sizes. However, the nonhomogeneous fermentation pellets produced with strained rumen inoculum cannot be accurately subsampled, requiring analysis of the entire pellet. In this study, two microbial glycogen methods were compared for analysis of such fermentation pellets: boiling samples for 3h in 30% KOH (KOH) or for 15min in 0.2M NaOH (NaOH), followed by enzymatic hydrolysis with α-amylase and amyloglucosidase, and detection of released glucose. Total α-glucan was calculated as glucose×0.9. KOH and NaOH did not differ in the α-glucan detected in fermentation pellets (29.9 and 29.6mg, respectively; P=0.61). Recovery of different control α-glucans was also tested using KOH, NaOH, and a method employing 45min of bead beating (BB). For purified beef liver glycogen (water-soluble) recovery, BB (95.0%)KOH (91.4%)NaOH (87.4%; P0.05), and for wheat starch (water-insoluble granules) recovery, NaOH (96.9%)BB (93.8%)KOH (91.0%; P0.05). Recovery of isolated protozoal glycogen (water-insoluble granules) did not differ among KOH (87.0%), NaOH (87.6%), and BB (86.0%; P=0.81), but recoveries for all were below 90%. Differences among substrates in the need for gelatinization and susceptibility to destruction by alkali likely affected the results. In conclusion, KOH and NaOH glycogen methods provided comparable determinations of fermentation pellet α-glucan. The tests on purified α-glucans indicated that assessment of recovery in glycogen methods can differ by the control α-glucan selected.

Volatile fatty acid concentrations ([VFA], mM) have long been used to assess the effect of dietary treatments on ruminal fermentation in vivo. However, discrepancies in statistical results between [VFA] and VFA pool size (VFAmol) possibly related to ruminal digesta liquid amount (LIQ, kg) indicate potential issues with the use of [VFA]. We investigated relationships among [VFA], VFAmol, and LIQ measured 2 h postfeeding using individual lactating cow data (n=175) from 7 separate feeding studies. Regression analyses were performed using mixed models with "study" as a discrete random variable. The mean across studies and average range of values within studies, respectively, were 151 and 75 for [VFA], 11.2 and 9.8 for VFAmol, 73.3 and 41.0 for LIQ, and 289 and 83 mmol/kg for rumen fluid osmolality. Liquid amount changed with VFAmol (3.76 VFAmol+31.2; average within-study R2=0.69), but the relationship was weak between [VFA] and LIQ (0.524 LIQ+112.8; average within-study R2=0.12). The relationship between LIQ and VFAmol was likely a function of the osmotic gradient between rumen liquid and blood. The VFA are a major ruminal solute; VFAmol amounts can affect water flux in the rumen as similar tonicities of rumen fluid and blood are maintained. This also has a damping effect on ruminal solute concentration, creating the weak relationship between [VFA] and LIQ. Within studies, similar [VFA] were found in LIQ differing by 30 kg or more. The difference between minimum and maximum LIQ within cow within study was 12.7 kg (standard deviation=7.1), so inclusion of "cow" in analyses did not correct for the variation in LIQ. To allow valid comparisons of experimental treatments, responses must be on an equivalent basis; concentrations in different LIQ are not on an equivalent basis and so are not valid to use for comparing treatment effects. The [VFA] changed with VFAmol (5.80 VFAmol+86.3; average within-study R2=0.56). However, the ratio of [VFA] to VFAmol ranged from 9.0 to 24.1 as a function of 1,000/LIQ; this reflects the inherent calculated relationship among the variables. The varying relationship of [VFA] to VFAmol further indicates that [VFA] is not an appropriate measure to evaluate the progress or effect of treatments on ruminal fermentation. Predictions of LIQ and VFAmol using cow and ruminal measures were insufficiently precise to be used in research. Previously drawn conclusions based on [VFA] need to be reevaluated, and alternate evaluations for in vivo ruminal fermentation are needed.

An important objective is to identify nutrients or dietary factors that are most critical for advancing our knowledge of, and improving our ability to predict, milk protein production. The Dairy NRC (2001) model is sensitive to prediction of microbial protein synthesis, which is among the most important component of models integrating requirement and corresponding supply of metabolizable protein or amino acids. There are a variety of important considerations when assessing appropriate use of microbial marker methodology. Statistical formulas and examples are included to document and explain limitations in using a calibration equation from a source publication to predict duodenal flow of purine bases from measured urinary purine derivatives in a future study, and an improved approach was derived. Sources of specific carbohydrate rumen-degraded protein components probably explain microbial interactions and differences among studies. Changes in microbial populations might explain the variation in ruminal outflow of biohydrogenation intermediates that modify milk fat secretion. Finally, microbial protein synthesis can be better integrated with the production of volatile fatty acids, which do not necessarily reflect volatile fatty acid molar proportions in the rumen. The gut and splanchnic tissues metabolize varying amounts of volatile fatty acids, and propionate has important hormonal responses influencing milk protein percentage. Integration of ruminal metabolism with that in the mammary and peripheral tissues can be improved to increase the efficiency of conversion of dietary nutrients into milk components for more efficient milk production with decreased environmental impact.

Though ≥22 species of Primates consume fungi, most do so at low rates, comprising

Achieving optimal rumen function requires an understanding of feeds and systems of nutritional evaluation. Key influences on optimal function include achieving good dry matter intake. The function of feeds in the rumen depends on other factors including chemical composition, rate of passage, degradation rate of the feed, availability of other substrates and cofactors, and individual animal variation. This article discusses carbohydrate, protein, and fat metabolism in the rumen, and provides practical means of evaluation of rations in the field. Conditions under which rumen function is suboptimal (ie, acidosis and bloat) are discussed, and methods for control examined.

A new method for determining neutral detergent-soluble fiber (NDSF) estimates the non-starch polysaccharide present in 90% ethanol-insoluble residue (EIR) but absent from neutral detergent residue (NDF). Unextracted feeds, EIR and NDF of feeds high in pectic substances were fermented in vitro with mixed ruminal microbes and gas production was measured. Soybean hulls were fermented for 48 h, and all other feeds for 24 h. Fermentation kinetics of NDSF were determined from gas curves produced by point by point subtraction of the gas production curves of NDF from those of EIR. Single-pool logistic fermentation rates and lag times for NDSF and NDF were determined from the gas production data. The single-pool logistic rates for NDSF for dried citrus pulp, dried beet pulp, soybean hulls, mature alfalfa stems and leaves and immature alfalfa stems and leaves were 0.13, 0.16, 0.05, 0.11, 0.14, 0.18 and 0.13 h−1, respectively. Fermentation rates tended to be faster and lag times shorter for NDSF than for NDF. Acetate to propionate ratios were numerically higher for EIR fermentations than for unextracted feeds or NDF. The fermentation characteristics of NDSF for the feeds in this study are consistent with reports of pectic substance fermentations indicating that NDSF does describe the soluble fiber fraction. The NDSF and gas production measurement methods offer nutritionally relevant ways to estimate soluble fiber content and its digestion characteristics in feedstuffs.

The goal of this in vitro study was to determine the influence of isotrichid protozoa (IP) on the conversion of glucose (Glc) to glycogen (Glyc) and transformation of Glc into fermentation products. Treatments were ruminal inoculum mechanically processed (blended) to destroy IP (B+, verified microscopically) or not mechanically processed (B-). Accumulated microbial Glyc was measured at 3h of fermentation with (L+; protozoa+bacteria) or without (L- predominantly protozoa) lysis of bacterial cells in the fermentation solids with 0.2 N NaOH. Two 3-h in vitro fermentations were performed using Goering-Van Soest medium in batch culture vessels supplemented with 78.75 mg of Glc/vessel in a 26.5-mL liquid volume. Rumen inoculum from 2 cannulated cows was filtered through cheesecloth, combined, and maintained under CO(2) for all procedures. At 3h, 0.63 and 0.38 mg of Glc remained in B- and B+. Net microbial Glyc accumulation (and Glc in Glyc as % of added Glc) detected at 3h of fermentation were 3.32 (4.69%), -1.42 (-2.01%), 6.45 (9.10%), and 3.65 (5.15%) mg for B-L-, B+L-, B-L+ and B+L+, respectively. Treatments B+ and L+ gave lower Glyc values than B- and L-, respectively. Treatment B+L- demonstrated net utilization of α-glucan contributed by inoculum with no net Glyc production. With destruction of IP, total Glyc accumulation declined by 44%, but estimated bacterial Glyc increased. Microbial accumulation of N increased 17.7% and calculated CH(4) production decreased 24.7% in B+ compared with B-, but accumulation of C in microbes, production of organic acids or C in organic acids, calculated CO(2), and carbohydrates in cell-free medium did not differ between B+ and B-. Given the short 3-h timeframe, increased N accumulation in B+ was attributed to decreased Glyc sequestration by IP rather than decreased predation on bacteria. After correction for estimates of C from AA and peptides utilized by microbes, 15% of substrate Glc C could not be accounted for in measured products in B+ or B-. Approximately 30% of substrate Glc was consumed by energetic costs associated with Glc transport and Glyc synthesis. The substantial accumulation of Glyc and changes in microbial N and Glyc accumulation related to presence of IP suggest that these factors should be considered in predicting profiles and amounts of microbial products and yield of nutrients to the cow as related to utilization of glucose. Determination of applicability of these findings to other soluble carbohydrates could be useful.

Soyhulls (original form), neutral detergent and acid detergent fiber extracted from soyhulls were characterized by rates of dry matter disappearance by in situ and in vitro enzyme procedures. Enzyme combinations were cellulase, pectinase, cellulase – hemicellulase, cellulase – pectinase, and cellulase – hemicellulase – pectinase. Incubation times for both in situ and in vitro procedures were 0, 1, 2, 6, 8, 12, and 24 h. Two additional independent time sequences of soyhulls used in situ and cellulase procedures for extended times of 36, 48 h and 36, 48, 60, and 72 h. In situ rates of dry matter disappearance were similar to in vitro enzyme systems with cellulase or cellulase – hemicellulase for original hulls at 24 h. In situ neutral detergent fiber disappearance was similar to cellulase, and acid detergent in situ disappearance similar to cellulase – pectinase. Addition of pectinase to cellulase or cellulase – hemicellulase increased dry matter disappearance for original and neutral detergent hulls but not acid detergent hulls. In situ rates of dry matter disappearance for original soyhulls increased with incubation time at 72 h compared to 24 and 48 h, emphasizing the importance of extended incubation for this technique. In vitro rates for cellulase decreased with incubation beyond 24 h, suggesting caution in interpretation of rate for in vitro enzymatic systems using extended intervals.