The objectives of this study were to determine the effect of an increase in diet fermentability on 1) the rate and extent to which short-chain fatty acid (SCFA) absorption pathways adapt relative to changes in Na(+) transport, 2) the epithelial surface area (SA), and 3) the barrier function of the bovine ruminal epithelium. Twenty-five Holstein steer calves were assigned to either the control diet (CON; 91.5% hay and 8.5% supplement) or a moderately fermentable diet (50% hay; 41.5% barley grain (G), and 8.5% supplement) fed for 3 (G3), 7 (G7), 14 (G14), or 21 days (G21). All calves were fed at 2.25% body weight at 0800. Calves were killed (at 1000), and ruminal tissue was collected to determine the rate and pathway of SCFA transport, Na(+) transport and barrier function in Ussing chambers. Tissue was also collected for SA measurement and gene expression. Mean reticular pH decreased from 6.90 for CON to 6.59 for G7 and then increased (quadratic P < 0.001). While effective SA of the ruminal epithelium was not affected (P > 0.10) by dietary treatment, the net Na(+) flux increased by 125% within 7 days (quadratic P = 0.016). Total acetate and butyrate flux increased from CON to G21, where passive diffusion was the primary SCFA absorption pathway affected. Increased mannitol flux, tissue conductance, and tendencies for increased expression of IL-1β and TLR2 indicated reduced rumen epithelium barrier function. This study indicates that an increase in diet fermentability acutely increases Na(+) and SCFA absorption in the absence of increased SA, but reduces barrier function.

Urea transport (UT-B) proteins are known to facilitate urea movement across the ruminal epithelium; however, other mechanisms may be involved as well because inhibiting UT-B does not completely abolish urea transport. Of the aquaporins (AQP), which are a family of membrane-spanning proteins that are predominantly involved in the movement of water, AQP-3, AQP-7, and AQP-10 are also permeable to urea, but it is not clear if they contribute to urea transport across the ruminal epithelium. The objectives of this study were to determine (1) the functional roles of AQP and UT-B in the serosal-to-mucosal urea flux (Jsm-urea) across rumen epithelium; and (2) whether functional adaptation occurs in response to increased diet fermentability. Twenty-five Holstein steer calves (n=5) were assigned to a control diet (CON; 91.5% hay and 8.5% vitamin and mineral supplement) or a medium grain diet (MGD; 41.5% barley grain, 50% hay, and 8.5% vitamin and mineral) that was fed for 3, 7, 14, or 21 d. Calves were killed and ruminal epithelium was collected for mounting in Ussing chambers under short-circuit conditions and for analysis of mRNA abundance of UT-B and AQP-3, AQP-7, and AQP-10. To mimic physiologic conditions, the mucosal buffer (pH 6.2) contained no urea, whereas the serosal buffer (pH 7.4) contained 1 mM urea. The fluxes of (14)C-urea (Jsm-urea; 26 kBq/10 mL) and (3)H-mannitol (Jsm-mannitol; 37 kBq/10 mL) were measured, with Jsm-mannitol being used as an indicator of paracellular or hydrophilic movement. Serosal addition of phloretin (1 mM) was used to inhibit UT-B-mediated urea transport, whereas NiCl2 (1 mM) was used to inhibit AQP-mediated urea transport. Across treatments, the addition of phloretin or NiCl2 reduced the Jsm-urea from 116.5 to 54.0 and 89.5 nmol/(cm(2) × h), respectively. When both inhibitors were added simultaneously, Jsm-urea was further reduced to 36.8 nmol/(cm(2) × h). Phloretin-sensitive and NiCl2-sensitive Jsm-urea were not affected by diet. The Jsm-urea tended to increase linearly as the duration of adaptation to MGD increased, with the lowest Jsm-urea being observed in animals fed CON [107.7 nmol/(cm(2) × h)] and the highest for those fed the MGD for 21 d [144.2 nmol/(cm(2) × h)]. Phloretin-insensitive Jsm-urea tended to increase linearly as the duration of adaptation to MGD increased, whereas NiCl2-insensitive Jsm-urea tended to be affected by diet. Gene transcript abundance for AQP-3 and UT-B in ruminal epithelium increased linearly as the duration of MGD adaptation increased. For AQP-7 and AQP-10, gene transcript abundance in animals that were fed the MGD was greater compared with that of CON animals. These results demonstrate that both AQP and UT-B play significant functional roles in urea transport, and they may play a role in urea transport during dietary adaptation to fermentable carbohydrates.

Effects of duration of grain feeding on the concentration of endotoxic lipopolysaccharide (LPS) in digesta throughout the digestive tract and on acute phase proteins and LPS in peripheral blood were determined in Holstein yearling calves. Twenty-five Holstein yearling steer calves received either a forage-based diet containing 92% hay and 8% of a mineral and vitamin pellet on a dry matter basis (CON) or a moderate-grain diet, obtained by replacing 41.5% of the hay in the forage-based diet with barley grain, for 3 (MG3), 7 (MG7), 14 (MG14), or 21 d (MG21) before slaughter. Immediately before slaughter, blood samples were collected from the jugular vein. Immediately after slaughter, digesta samples were collected from the rumen, jejunum, ileum, cecum, colon, and rectum. Rumen liquid digesta, digesta from the intestines, and peripheral blood plasma were analyzed for LPS. Peripheral blood plasma and serum were analyzed for the acute phase proteins amyloid A, haptoglobin, and LPS-binding protein. Feeding the grain diet increased the LPS concentration in rumen fluid linearly from 15,488 endotoxin units (EU)/mL for CON to 70,146 EU/mL for MG7. Concentrations of LPS in rumen fluid in MG14 and MG21 were 61,944 and 56,234 EU/mL, respectively, and did not differ. The LPS concentrations in jejunal digesta were much lower than that in digesta elsewhere in the digestive tract, which suggests that ruminal LPS is broken down in the abomasum or proximal jejunum. The concentration of digesta LPS in the ileum was higher than that of digesta elsewhere in the intestines and similar to that in rumen fluid. The duration of grain feeding increased the LPS concentration in digesta in the ileum and cecum and tended to increase that in the colon cubically. Concentrations of LPS in this part of the digestive tract were highest in the MG3 and MG21 groups. The highest concentrations of LPS in digesta in the cecum, colon, and rectum were 3.7, 3.8, and 5.6 times higher than that in CON, respectively. Grain feeding and the increase in LPS in digesta were not accompanied by an acute phase response or a detectable concentration of LPS in peripheral blood. The absence of LPS in peripheral blood and the lack of increase in acute phase proteins indicated that the grain feeding protocol used in the current study and the accompanying changes in LPS concentrations of the digesta did not result in systemic inflammation.

The aim of this study was to determine the time course for adaptation of the reticulo-rumen, omasum, abomasum, and small intestine in response to an abrupt increase in the proportion of grain in the diet. Adaptive responses include tissue and digesta mass, small intestinal length, and brush border enzyme activity in the duodenum, proximal jejunum, and ileum. Twenty-five Holstein steers (213 ± 23 kg; 5 to 7 mo of age) were blocked by body weight, and within block were randomly assigned to 1 of 5 treatments: the control diet (CTRL; 92% chopped grass hay and 8% mineral and vitamin supplement on a dry matter basis) or a moderate grain diet (MGD; 50% chopped grass hay, 42% rolled barley grain, and 8% mineral and vitamin supplement) that was fed for 3 (MGD3), 7 (MGD7), 14 (MGD14), or 21 d (MGD21). Dry matter intake was limited to 2.25% of body weight to ensure that changes in dry matter intake did not confound the results. On the last day of the dietary exposure, steers were slaughtered 2 h after feeding. Reticulo-rumen tissue mass and ruminal epithelium mass in the ventral sac of the rumen were not affected by the MGD. Wet reticulo-ruminal digesta mass decreased from CTRL to MGD7 and then increased, but reticulo-ruminal digesta dry matter mass did not differ between treatments. Omasal mass, omasal tissue mass, and omasum digesta mass decreased linearly with the number of days fed MGD, but abomasal tissue mass tended to increase linearly. Duodenal tissue mass tended to increase linearly, and ileal length increased linearly with the number of days fed MGD. Lactase activity in the proximal jejunum increased linearly and maltase activity in duodenum tended to increase linearly with days fed MGD. Aminopeptidase N activity in the proximal jejunum increased cubically with days fed MGD, and dipeptidylpeptidase IV activity in ileum tended to decrease from CTRL to MGD14 and then tended to increase. Adaptation to a diet with a greater proportion of concentrate involves changes in the mass and length of regions of the gastrointestinal tract and brush border enzyme activity. These changes take place gradually over at least 3 wk.

Effects of the duration of moderate grain feeding on the taxonomic composition of gastrointestinal microbiota were determined in 15 Holstein yearling steers. Treatments included feeding a diet of 92% dry matter (DM) hay (D0), and feeding a 41.5% barley grain diet for 7 (D7) or 21 d (D21) before slaughter. At slaughter, digesta samples were collected from six regions, i.e., the rumen, jejunum, ileum, cecum, colon, and rectum. Extracted DNA from these samples was analyzed using MiSeq Illumina sequencing of the V4 region of the 16S rRNA gene. Three distinct PCoA clusters existed, i.e., the rumen, the jejunum/ileum, and the cecum/colon/rectum. Feeding the grain diet for 7 d reduced microbial diversity in all regions, except the ileum. Extending the duration of grain feeding from 7 to 21 d did not affect this diversity further. Across regions, treatment changed the relative abundances of 89 genera. Most of the changes between D0 and D7 and between D7 and D21 were opposite, demonstrating the resilience of gastrointestinal microbiota to a moderate increase in grain feeding. Results show that the duration of a moderate increase in grain feeding affects how gastrointestinal microbiota respond to this increase.