G. B. Penner

RUMINANT NUTRITION SYMPOSIUM: Role of fermentation acid absorption in the regulation of ruminal pH12

Highly fermentable diets are rapidly converted to organic acids [i.e., short-chain fatty acids (SCFA) and lactic acid] within the rumen. The resulting release of protons can constitute a challenge to the ruminal ecosystem and animal health. Health disturbances, resulting from acidogenic diets, are classified as subacute and acute acidosis based on the degree of ruminal pH depression. Although increased acid production is a nutritionally desired effect of increased concentrate feeding, the accumulation of protons in the rumen is not. Consequently, mechanisms of proton removal and their quantitative importance are of major interest. Saliva buffers (i.e., bicarbonate, phosphate) have long been identified as important mechanisms for ruminal proton removal. An even larger proportion of protons appears to be removed from the rumen by SCFA absorption across the ruminal epithelium, making efficiency of SCFA absorption a key determinant for the individual susceptibility to subacute ruminal acidosis. Proceeding initially from a model of exclusively diffusional absorption of fermentation acids, several protein-dependent mechanisms have been discovered over the last 2 decades. Although the molecular identity of these proteins is mostly uncertain, apical acetate absorption is mediated, to a major degree, via acetate-bicarbonate exchange in addition to another nitrate-sensitive, bicarbonate-independent transport mechanism and lipophilic diffusion. Propionate and butyrate also show partially bicarbonate-dependent transport modes. Basolateral efflux of SCFA and their metabolites has to be mediated primarily by proteins and probably involves the monocarboxylate transporter (MCT1) and anion channels. Although the ruminal epithelium removes a large fraction of protons from the rumen, it also recycles protons to the rumen via apical sodium-proton exchanger, NHE. The latter is stimulated by ruminal SCFA absorption and salivary Na(+) secretion and protects epithelial integrity. Finally, SCFA absorption also accelerates urea transport into the rumen, which via ammonium recycling, may remove protons from rumen to the blood. Ammonium absorption into the blood is also stimulated by luminal SCFA. It is suggested that the interacting transport processes for SCFA, urea, and ammonia represent evolutionary adaptations of ruminants to actively coordinate energy fermentation, protein assimilation, and pH regulation in the rumen.

Gluconeogenesis in dairy cows: the secret of making sweet milk from sour dough.

Gluconeogenesis is a crucial process to support glucose homeostasis when nutritional supply with glucose is insufficient. Because ingested carbohydrates are efficiently fermented to short‐chain fatty acids in the rumen, ruminants are required to meet the largest part of their glucose demand by de novo genesis after weaning. The qualitative difference to nonruminant species is that propionate originating from ruminal metabolism is the major substrate for gluconeogenesis. Disposal of propionate into gluconeogenesis via propionyl‐CoA carboxylase, methylmalonyl‐CoA mutase, and the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK) has a high metabolic priority and continues even if glucose is exogenously supplied. Gluconeogenesis is regulated at the transcriptional and several posttranscriptional levels and is under hormonal control (primarily insulin, glucagon, and growth hormone). Transcriptional regulation is relevant for regulating precursor entry into gluconeogenesis (propionate, alanine and other amino acids, lactate, and glycerol). Promoters of the bovine pyruvate carboxylase (PC) and PEPCK genes are directly controlled by metabolic products. The final steps decisive for glucose release (fructose 1,6‐bisphosphatase and glucose 6‐phosphatase) appear to be highly dependent on posttranscriptional regulation according to actual glucose status. Glucogenic precursor entry, together with hepatic glycogen dynamics, is mostly sufficient to meet the needs for hepatic glucose output except in high‐producing dairy cows during the transition from the dry period to peak lactation. Lactating cows adapt to the increased glucose requirement for lactose production by mobilization of endogenous glucogenic substrates and increased hepatic PC expression. If these adaptations fail, lipid metabolism may be altered leading to fatty liver and ketosis. Increasing feed intake and provision of glucogenic precursors from the diet are important to ameliorate these disturbances. An improved understanding of the complex mechanisms underlying gluconeogenesis may further improve our options to enhance the postpartum health status of dairy cows.

Microbial butyrate and its role for barrier function in the gastrointestinal tract

Butyrate production in the large intestine and ruminant forestomach depends on bacterial butyryl‐CoA/acetate‐CoA transferase activity and is highest when fermentable fiber and nonstructural carbohydrates are balanced. Gastrointestinal epithelia seem to use butyrate and butyrate‐induced endocrine signals to adapt proliferation, apoptosis, and differentiation to the growth of the bacterial community. Butyrate has a potential clinical application in the treatment of inflammatory bowel disease (IBD; ulcerative colitis). Via inhibited release of tumor necrosis factor α and interleukin 13 and inhibition of histone deacetylase, butyrate may contribute to the restoration of the tight junction barrier in IBD by affecting the expression of claudin‐2, occludin, cingulin, and zonula occludens poteins (ZO‐1, ZO‐2). Further evaluation of the molecular events that link butyrate to an improved tight junction structure will allow for the elucidation of the cofactors affecting the reliability of butyrate as a clinical treatment tool.

Ruminant Nutrition Symposium: Molecular adaptation of ruminal epithelia to highly fermentable diets.

Feeding highly fermentable diets to ruminants is one strategy to increase energy intake. The increase in short-chain fatty acid (SCFA) production and reduced ruminal pH associated with highly fermentable diets imposes a challenge to the metabolism and the regulation of intracellular pH homeostasis of ruminal epithelia. The ruminal epithelia respond to these challenges in a coordinated manner. Whereas the enlargement of absorptive surface area is well documented, emerging evidence at the mRNA and transporter and enzyme activity levels indicate that changes in epithelial cell function may be the initial response. It is not surprising that gene expression analysis has identified pathways involved in fatty acid metabolism, ion transport, and intracellular homeostasis to be the pathways dominantly affected during adaptation and after adaptation to a highly fermentable diet. These findings are important because the intraepithelial metabolism of SCFA, particularly butyrate, helps to maintain the concentration gradient between the cytosol and lumen, thereby facilitating absorption. Butyrate metabolism also controls the intracellular availability of butyrate, which is widely regarded as a signaling molecule. Current data indicate that for butyrate metabolism, 3-hydroxy-3-methylglutaryl-CoA synthase and acetyl-CoA acetyltransferase are potential regulatory points with transient up- and downregulation during diet adaptation. In addition to nutrient transport and utilization, genes involved in the maintenance of cellular tight junction integrity and induction of inflammation have been identified as differentially expressed genes during adaptation to highly fermentable diets. This may have important implications on ruminal epithelial barrier function and the inflammatory response often associated with subacute ruminal acidosis. The objective of this review is to summarize ruminal epithelial adaptation to highly fermentable diets focusing on the changes at the enzyme and transporter activity levels, as well as the underlying molecular changes at the mRNA and protein expression levels.

Short-term feed restriction impairs the absorptive function of the reticulo-rumen and total tract barrier function in beef cattle1

The objective of this study was to evaluate whether different severities of short-term feed restriction (FR) affect the absorptive function of the reticulo-rumen and total tract barrier function in beef cattle. Eighteen ruminally cannulated and ovariectomized Angus × Hereford heifers were blocked by BW into 3 blocks, with blocks conducted sequentially. Treatments were imposed during the 5-d FR period by restricting heifers to 75 (FR75), 50 (FR50) or 25% (FR25) of the ad libitum feed intake measured during a 5-d baseline period (BASE) occurring immediately before FR. Throughout the study, heifers were housed in individual pens (9 m(2)) and were fed the same diet (60% forage:40% concentrate) with free access to water. Dry matter intake was measured daily and ruminal pH was measured every 2 min throughout the study. Ruminal fluid and blood samples were collected on d 3 of the BASE and FR periods, and the temporarily isolated and washed reticulo-rumen technique was used to evaluate short-chain fatty acid (SCFA) absorption on d 5 of the BASE and FR periods. Total tract barrier function was evaluated starting on d 2 of the BASE and FR periods using a pulse dose of Cr-EDTA followed by 48 h of total urine collection. Data were analyzed using the Proc Mixed procedure of SAS with the fixed effects of block, treatment, period, and the treatment × period interaction, the random effect of cow nested in block with period included as a repeated measure. Dry matter intake did not differ among treatments during BASE but, as imposed by the experimental model, DMI during FR relative to BASE equated to 70, 49, and 25%, which was close to the targeted values of 75, 50, and 25% (treatment × period, P < 0.001). A treatment × period interaction (P < 0.001) was also detected for ruminal SCFA concentration with the concentration decreasing as the severity of FR increased, whereas there were no differences during BASE. Mean ruminal pH increased during FR with increasing severity of FR, but was not different during BASE (treatment × period, P < 0.001). Absorption of SCFA across the reticulo-rumen tended to decrease with increasing severity of FR (P = 0.08). For individual SCFA, acetate absorption (mmol/h) tended (P = 0.057) to be less for FR25 and FR50 when compared with FR75 and decreased (P = 0.05) by almost 70 mmol/h at FR25 and FR50 relative to BASE (322mmol/h). Heifers restricted to 25% (FR25) feed had greater urinary Cr recovery during FR than BASE, whereas no changes were detected for FR75 and FR50. This study indicates that moderate severities of short-term FR decrease the absorptive function of the reticulo-rumen, but more severe FR is required to compromise total tract barrier function in beef cattle.

Individual animal variability in ruminal bacterial communities and ruminal acidosis in primiparous Holstein cows during the periparturient period

The purpose of this study was to investigate variability among individual cows in their severity of ruminal acidosis (RA) pre- and postpartum, and determine whether this variability was related to differences in their ruminal bacterial community composition (BCC). Variability in the severity of RA among individual cows was characterized based on ruminal fermentation variables. Effects of prepartum dietary treatment on the severity of RA were also examined. Fourteen Holstein heifers paired by expected calving date and BCS were allotted to 1 of 2 prepartum dietary treatments: low-concentrate or high-concentrate diets. All cows received the same lactation diet postpartum. Microbial DNA extracted from 58 ruminal digesta samples in total collected prepartum (d -50, -31, and -14; 27 samples) and postpartum (d +14 and +52; 31 samples) and amplified by PCR were subjected to automated ribosomal intergenic spacer analysis. Changes in ruminal variables over time [pH, volatile fatty acids (VFA), and acidosis indicators, including duration and area under the rumen pH curve below 5.8, 5.5, and 5.2, measured on d -54, -35, -14, -3, +3, +17, +37, and +58] were analyzed using principal components analysis. Based on the shift (defined as the distance of the mean loadings) between the prepartum and postpartum period for each cow, the 14 cows were classified into 3 groups: least acidotic (n=5), most acidotic (n=5), and intermediate (n=4). Cows in the most acidotic group had greater severity of RA (measured as duration of total RA, mild RA, moderate RA, and acute RA; area under the pH curve for total RA, mild RA, and moderate RA) postpartum than prepartum, and this difference between periods was greater than for the least acidotic cows. Similarly, the RA index (total area of pH <5.8 normalized to intake) showed an interaction between severity of RA and period. The variation in the severity of RA was independent of intake, total VFA concentration, and individual VFA proportions. Production variables (milk yield, fat percentage, fat yield, fat-corrected milk, and efficiency of milk production) were not influenced by the severity of RA. Ruminal BCC was not influenced by dietary treatment or period. However, some cows experienced greater shift in BCC than other cows across the periods. Based on the magnitude of the shift in BCC (distance between mean ordination values across the periods for each cow), cows were grouped into 3 BCC profile categories: stable (5 cows with lesser shift), unstable (5 cows with greater shift), and intermediate (4 cows with average shift). Cows demonstrating a greater shift in BCC were not necessarily those in the most acidotic group and vice versa. The shift in ruminal fermentation variables (principal components analysis rankings) and the shift in BCC (automated ribosomal intergenic spacer analysis rankings) between pre- and postpartum were not related (n=14; R(2)=0.00). It was concluded that not all cows are equally susceptible to RA and postpartum shifts in BCC appear to be independent of the differences in the severity of RA postpartum.

Supplemental butyrate does not enhance the absorptive or barrier functions of the isolated ovine ruminal epithelia1

Our objective was to determine if increasing the ruminal butyrate concentration would improve the selective permeability of ruminal epithelia. Suffolk wether lambs (n = 18) with an initial BW of 47.4 ±1.4 kg were housed in individual pens (1.5 × 1.5 m) with rubber mats on the floor. Lambs were blocked by initial BW into 6 blocks and, within block, were randomly assigned to either the control (CON) or 1 of 2 butyrate supplementation amounts (i.e., 1.25% or 2.50% butyrate as a proportion of DMI). With the exception of butyrate supplementation, all lambs were fed a common diet (90% concentrate and 10% barley silage). After a 14-d feeding period, lambs were killed, and ruminal epithelia from the ventral sac were mounted in Ussing chambers. To facilitate the Ussing chamber measurements, only 1 lamb was killed on an individual day. Thus, the starting date was staggered so that all lambs were exposed to the same experimental protocol. In Ussing chambers, epithelia were incubated using separate mucosal (pH 6.2) and serosal (pH 7.4) bathing solutions. Then 1-14C-butyrate (74 kBq/10 mL) was added to the mucosal side and was used to measure the mucosal-to-serosal flux (J(ms-butyrate)) in 2 consecutive 60-min flux periods with simultaneous measurement of transepithelial conductance (G(t)). During the first (challenge) flux period, the mucosal buffer solution was either acidified to pH 5.2 (ACID) or used as a control (pH 6.2; SHAM). Buffer solutions bathing the epithelia were replaced before the second flux period (recovery). Total ruminal short-chain fatty acid and butyrate concentrations were greater (P = 0.001) in lambs fed 2.50% compared with those fed 0% or 1.25% butyrate. The J(ms-butyrate) was less for lambs fed 1.25% and 2.50% butyrate [3.00 and 3.12 μmol/(cm2·h), respectively] than for CON [3.91 μmol/(cm2· h)]. However, no difference (P = 0.13)was observed for G(t). An ex vivo treatment × flux period interaction was detected (P = 0.003) for J(ms-butyrate), where no differences were present between ACID and SHAM during the challenge period, but the Jms-butyrate was less for ACID than for SHAM during recovery. These results indicate that large increases in the ruminal butyrate concentration decrease the selective permeability of the isolated ruminal epithelia.

Duration of time that beef cattle are fed a high-grain diet affects the recovery from a bout of ruminal acidosis: Short-chain fatty acid and lactate absorption, saliva production, and blood metabolites1

This study was conducted to determine if the duration of time that beef cattle are fed a high-grain diet affects short-chain fatty acid (SCFA) absorption, saliva production, and blood metabolites before, during, and following an induced bout of ruminal acidosis. Sixteen Angus heifers were assigned to 1 of 4 blocks and within block to 1 of 2 treatments designated as long adapted (LA) or short adapted (SA). Long adapted and SA heifers were fed a backgrounding diet [forage:concentrate (F:C) = 60:40] for 33 and 7 d, respectively, and then transitioned over 20 d to a high-grain diet (F:C = 9:91) with the timing of dietary transition staggered such that the LA and SA heifers were fed the high-grain diet for 34 and 8 d, respectively, before inducing ruminal acidosis. Ruminal acidosis was induced by restricting feed to 50% of DMI:BW for 24 h followed by an intraruminal infusion of ground barley at 10% DMI:BW. Heifers were then given their regular diet allocation 1 h after the intraruminal infusion. Data were collected during an 8 d baseline period (BASE), on the day of the acidosis challenge (CHAL), and during 2 consecutive 8 d recovery periods (REC1 and REC2). When pooled across periods, the fractional rates of propionate (42 vs. 34%/h; P = 0.045) and butyrate (45 vs. 36%/h; P = 0.019) absorption, measured using the isolated and washed reticulorumen technique, were greater for LA than SA heifers. Moreover, overall, LA heifers tended to have greater absolute rates of butyrate absorption (94 vs. 79 mmol/h; P = 0.087) and fractional rates of total SCFA absorption (37 vs. 32%/h; P = 0.100). Treatment × period interactions for lactate absorption (P = 0.024) and serum D-lactate concentration (P = 0.003) were detected with LA heifers having greater D-lactate concentrations during CHAL and greater fractional rates of lactate absorption during REC1 than SA. The absolute and fractional absorption of acetate, propionate, and butyrate increased between REC1 and REC2, with intermediate values for BASE (P ≤ 0.05). Although fractional rates of SCFA absorption were low during REC1, saliva production (P = 0.018) increased between BASE and REC1, with intermediate values for REC2. These results suggest that the duration of time that animals are fed a high-grain diet may increase propionate, butyrate, and lactate absorption, and that cattle may decrease SCFA absorption and increase saliva production shortly after an acute bout of ruminal acidosis.

Effect of maturity at harvest on yield, chemical composition, and in situ degradability for annual cereals used for swath grazing

The objective of this study was to determine how harvest maturity of whole-crop cereals commonly used in swath grazing systems in western Canada affects yield, chemical composition, and in situ digestibility. We hypothesized that the increase in yield with advancing maturity would not offset the decline in digestibility and, thus, the yield of effectively degradable DM (EDDM) would decline with advanced stages of maturity. Four replicate plots of barley (Hordeum vulgare L.; cv. CDC Cowboy), millet (Panicum milliaceum; cv. Red Proso), oat (Avena sativa L., spp.; CDC Weaver), and wheat (Triticum aestivum L.; cv. 07FOR21) were grown, with a subsection in each replicate harvested at 4 different maturities: head elongation, late milk, hard dough, and fully mature. At each stage of maturity, the wet and DM yields, and chemical composition (DM, OM, NDF, crude fat, and nonfiber carbohydrates; NFC) were determined. Whole-crop samples were ground (2-mm screen) and weighed into nylon bags (pore size of 53 ± 10 µm), and duplicate incubation runs were conducted by crop type. For each incubation run, nylon bags were randomly allocated (randomized by field replication, stage of maturity, and incubation time) to 1 of 7 heifers (32 bags/heifer during each run). Degradation rates were determined using a first-order kinetic model and data were analyzed with stage of maturity as a fixed effect and plot as a random effect. The DM, OM, and NFC yields increased linearly for barley and oat (P < 0.001), and increased quadratically for millet and wheat (P ≤ 0.025). Neutral detergent fiber yield increased linearly for barley (P = 0.005) and quadratically for millet, oat, and wheat (P = 0.044). There were no changes in CP yield observed for barley, millet, or oat with advancing maturity, but there was a linear increase observed for wheat (P = 0.002). The NFC concentration increased linearly for barley, millet, and oat (P < 0.001), and quadratically for wheat (P < 0.001), whereas the EDDM concentration decreased quadratically for millet, oat, and wheat (P = 0.003). The degradation rate of NDF decreased linearly with advancing maturity (P ≤ 0.014) for millet, oat, and wheat, but was not affected for barley (P = 0.13). The yield EDDM increased linearly for barley and oat (P < 0.001), and increased quadratically for millet and wheat (P ≤ 0.025). These findings suggest that harvesting whole-crop annual cereals at the hard dough and mature stages may maximize the yield of EDDM.

Expression and activity of key hepatic gluconeogenesis enzymes in response to increasing intravenous infusions of glucose in dairy cows

The present study aimed at investigating whether increasing concentrations of glucose supply have a depressive effect on the mRNA abundance and activity of key gluconeogenic enzymes in dairy cows. Twelve Holstein-Friesian dairy cows in mid-lactation were intravenously infused with saline (SI; n = 6) or a 40% glucose solution (GI; n = 6). For GI cows, the infusion dose increased by 1.25%/d relative to the initial NE(l) requirement until a maximum dose equating to surplus 30% NE(l) was reached on d 24. Cows receiving SI received an equivalent volume of 0.9% saline solution. Blood samples were taken every 2 d, and liver biopsies were collected every 8 d. A treatment x quadratic dose interaction (P < 0.01) was observed for the concentration of plasma glucose and serum insulin. The interactions were due to positive quadratic responses of the concentrations of glucose and insulin for GI cows, whereas the concentrations of glucose and insulin did not change over time for SI cows. The concentration of beta-hydroxybutyric acid (BHBA) and serum urea nitrogen (BUN) responded in a treatment x quadratic dose manner, such that greater decreases (P < 0.01) in BHBA and BUN concentrations were observed for cows receiving GI than SI as the dosage increased. Serum NEFA concentration tended to follow a similar pattern as serum BHBA and BUN; however, the interaction was not significant (P = 0.07). The mRNA abundance of gluconeogenesis enzymes followed a linear treatment x dose interaction (P < 0.05) for only pyruvate carboxylase (PC), which was paralleled by a trend for a linear treatment x dose interaction (P = 0.13) for PC enzyme activity. The least PC expression and activity were observed at the largest glucose dosage. The activity, but not mRNA abundance, of fructose 1,6-bisphosphatase (FBPase) showed treatment x quadratic dose interactions (P < 0.05) with decreasing activity at increasing glucose dose. Activities and expression of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase were not affected (P > 0.25) by treatment. In conclusion, hepatic gluconeogenic enzymes are only moderately affected by slowly increasing glucose supply, including a translational or posttranslational downregulation of FBPase activity and a decrease in the mRNA abundance of PC with possible consequences for PC enzyme activity.