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.

This study was conducted to evaluate the effects of the forage-to-concentrate ratio of the partial mixed ration (PMR) and the quantity of concentrate offered in an automated milking system (AMS), in a feed-first guided-flow barn, on the behavior and performance of dairy cows. Eight ruminally cannulated multiparous Holstein cows were used in a replicated 4 × 4 Latin square balanced for carry-over effects. Treatments were arranged in a 2 × 2 factorial consisting of a PMR that contained (dry matter basis) either a low (54:46; L-FOR) or a high (64:36; H-FOR) forage-to-concentrate ratio and AMS concentrate provision to achieve low (2 kg/d; L-AMS) or high (6 kg/d; H-AMS) intake. Each period consisted of 28 d with 6 d for dietary transition, 13 d for adaptation, and 9 d of collection. The first 4 d of data and sample collection were used to evaluate behavioral data (milking frequency, feeding behavior, and standing and lying behavior) and ruminal pH. Subsequently, a sampling device removal day was provided, and the last 4 d were used to evaluate ruminal fermentation and apparent total-tract digestibility. All 9 d were used for milk yield measurement, and the 8 d were used for dry matter intake measurement. Cows fed the H-AMS consumed 3.5 kg/d less PMR while consuming 4.2 kg/d more AMS concentrate, but total dry matter intake (PMR+AMS) was not affected by treatments averaging 27.3 kg/d. Although cows fed H-AMS had greater concentrate intake, they also had greater variability for AMS concentrate intake among days (0.85 vs. 0.25 kg/d, respectively). The number of PMR meals and PMR eating behavior were not affected by the PMR or AMS treatments. Feeding H-AMS did not affect milking frequency averaging 3.63 milkings/d, but tended to increase milk yield by 1.25 kg/d relative to L-AMS. Likewise, cows fed the L-FOR tended to have greater milk yield relative to H-FOR (39.3 vs 37.9 kg/d, respectively), but had greater holding area time. Minimum ruminal pH tended to be lower for cows fed L-FOR compared with cows fed H-FOR but was not affected by the AMS concentrate treatment. When fed the L-FOR, feeding the H-AMS increased total short-chain fatty acid concentration in the rumen relative to cows fed L-AMS, whereas the response for H-FOR was not affected by the AMS concentrate. These data suggest that feeding H-AMS may improve milk yield, but also increases the day-to-day variability in AMS concentrate consumption. Feeding a L-FOR PMR may increase milk yield without affecting variability in AMS concentrate consumption; however, it may reduce ruminal pH and increase the time spent in the holding area compared with cows fed a H-FOR PMR.

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.

The objective of this study was to characterise the regional variation in the barrier function of the gastrointestinal tract in Holstein calves using the flux rates of mannitol and inulin as permeability markers and tissue conductance (Gt) as an electrophysiological indicator of barrier function. Six Holstein steer calves (6 months of age) fed a common diet were used. Calves were killed by captive bolt stunning and pithing, and tissues were collected from the rumen, omasum, duodenum, jejunum, ileum, caecum, proximal colon, and distal colon. Tissues were carefully washed using a pre-heated (38.5°C) buffer solution (pH 7.4) saturated with oxygen and then transported to the laboratory. The mucosa was prepared by hand stripping and mounted between two halves of an Ussing chamber (n = 3/region with an exposed surface area of 3.14 cm2 for rumen and omasum and 1 cm2 for all other tissues). All tissues were incubated under short-circuit conditions and exposed to a similar buffer solution except for the energy source; rumen, omasum, caecum, and colon tissues were incubated with buffer containing short-chain fatty acids while tissues from the small intestine were bathed in buffer containing glucose. The Gt and the serosal-to-mucosal flux rates of 14C-inulin and 3H-mannitol were measured as indicators of barrier function. The serosal-to-mucosal flux rate of mannitol was greatest (P < 0.001) in the jejunum [104.8 nmol/(cm2 × h)] and least in the rumen and omasum [20.3 and 18.6 nmol/(cm2 × h), respectively]. In contrast, the serosal-to-mucosal flux rate of inulin was greatest (P < 0.001) in the omasum [158.6 nmol/(cm2 × h)] followed by the rumen [87.3 nmol/(cm2 × h)] with no differences among the other regions [18.7 – 62.0 nmol/(cm2 × h)]. The Gt was greatest (P < 0.001) in the jejunum (34.6 mS/cm2) and least for the rumen (3.67 mS/cm2) and omasum (3.23 mS/cm2). The Gt was correlated with both inulin and mannitol flux rates in duodenum, caecum and proximal colon (P < 0.05); whereas, no such correlations existed in jejunum, ileum and distal colon. The Gt was correlated with the mannitol flux rate but not the inulin flux rate in rumen and omasum. For all regions but the rumen and omasum there was a positive correlation between mannitol and inulin flux rates. These data indicate that the translocation of a large molecule (inulin) across the omasum and rumen is greatest despite having an apparently tight epithelium based on Gt and mannitol flux rate, while the jejunum appears to have greatest potential for paracellular permeability.

The objective of this study was to determine if the quantity of concentrate provided in an automated milking system (AMS) affects dry matter intake (DMI), attendance to the AMS, milk and milk component yield, feeding behavior, cow activity, and ruminal fermentation of lactating dairy cows fed isocaloric diets. Eight ruminally cannulated primiparous Holstein cows were used in a replicated 4 × 4 Latin square design with 28-d periods. Cows were housed in a freestall facility with a guided-traffic (feed-first) flow barn design. Treatments included 0.5, 2.0, 3.5, or 5.0 kg/d of dry matter of pellet in the AMS with an equivalent reduction of the same pellet in the partial mixed ration (PMR). Days 21 to 24 of each treatment period were used for DMI, milking performance (visits, yield, and composition), behavior, and ruminal pH determination, and d 25 to 28 were used for ruminal short-chain fatty acid and ammonia concentrations as well as total-tract digestibility. As imposed, consumption of AMS pellet linearly increased, equating to 0.50, 2.00, 3.49, and 4.93 kg of dry matter/d for the 0.5, 2.0, 3.5, and 5.0 kg/d treatments, respectively. Correspondingly, the standard deviation in AMS pellet intake among days linearly increased from 0.06 to 0.85 kg of dry matter/d as the quantity of concentrate in the AMS increased from 0.5 to 5.0 kg. The PMR DMI decreased linearly with increasing AMS concentrate allocation, but total DMI (PMR + AMS) was not affected (25.3 kg/d). As the AMS concentrate allocation increased, the selection against particles retained on an 18-mm sieve linearly increased and selection against particles retained on the pan decreased. Milking frequency (3.22 milkings/d), milk yield (37.5 kg/d), milk fat yield (1.43 kg/d), and milk protein yield (1.22 kg/d) were not affected; however, milk urea nitrogen concentration decreased linearly with increasing AMS concentrate. Ruminal pH averaged 6.18 and was not affected by AMS concentrate. Total ruminal short-chain fatty acid concentration was greatest when 3.5 kg of concentrate was allocated in the AMS and ruminal ammonia decreased linearly with increasing AMS concentrate. Time spent lying, the number of lying bouts, and average bout duration were not affected by treatment. These data indicate that increasing the quantity of concentrate in the AMS increases daily variability in AMS concentrate intake while decreasing PMR intake without affecting voluntary visits to the AMS and milk or milk component yield. As such, under isocaloric dietary settings, increasing the supply of pellet in the AMS is not likely to affect voluntary visits to the AMS, milk and milk component yields, or ruminal fermentation.