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The duration of time that beef cattle are fed a high-grain diet affects the recovery from a bout of ruminal acidosis: Dry matter intake and ruminal fermentation

The duration of time that beef cattle are fed a high-grain diet affects the recovery from a bout... Abstract This study was conducted to determine if the duration of time cattle are fed a high-grain diet affects their susceptibility to and recovery from ruminal acidosis. Sixteen Angus heifers (BW ± SEM, 261 ± 6.1 kg) were assigned to 1 of 4 blocks and fed a backgrounding diet consisting of 60% barley silage, 30% barley grain, and 10% supplement (DM basis). Within block, cattle were randomly assigned to 1 of 2 treatments differing in the number of days they were fed the high-grain diet before an acidosis challenge: 34 d for long adapted (LA) and 8 d for short adapted (SA). All heifers were exposed to the same 20 d dietary transition to a high-grain diet containing 9% barley silage, 81% barley grain, and 10% supplement (DM basis). 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). Acidosis induction increased daily duration (531 to 1,020 min/d; P < 0.001) and area (176 to 595 (min × pH)/d; P < 0.001) that ruminal pH was <5.5 relative to BASE. Relative to BASE, inducing acidosis also increased the daily mean (0.3 to 11.4 mM; P = 0.013) and maximum (1.3 to 29.3 mM; P = 0.008) ruminal fluid lactate concentrations. There was no effect of dietary treatment on ruminal pH, lactate, or short-chain fatty acid (SCFA) concentrations (P > 0.050). However, during BASE and CHAL, SA heifers experienced greater linear (P = 0.031), quadratic (P = 0.016), and cubic (P = 0.008) coefficients for the duration of time that pH was <5.5. In addition, a treatment × day interaction for the duration that pH was <5.5 during REC1 suggested that LA cattle tended to recover from the challenge more rapidly than SA cattle (P = 0.085). Regression analysis confirmed that the LA heifers experienced a quicker linear (P = 0.019) recovery from induced acidosis over time. These results indicate adaptation of the ruminal epithelium continues with advancing time as evidenced by more stable ruminal pH both before and after an induced bout of acute ruminal acidosis but does not affect susceptibility of cattle to ruminal acidosis. INTRODUCTION Ruminal acidosis is thought to be the most prevalent digestive disorder in feedlot cattle (Nagaraja and Titgemeyer, 2007) and is associated with an increase in ruminal short-chain fatty acid (SCFA) production (Sutton et al., 2003; Loncke et al., 2009) and concentration (Penner et al., 2009a,c) and a decrease in ruminal pH (Penner et al., 2007). These effects have been associated with decreased energy intake (Fulton et al., 1979a,b; Brown et al., 2000) and growth and increased incidence of liver abscesses (Brent, 1976; Nagaraja and Chengappa, 1998). Additionally, exposure of the ruminal epithelium to low ruminal pH (<5.5) has been shown to decrease SCFA absorption (Gaebel et al., 1989; Wilson et al., 2012) and barrier function (Aschenbach and Gäbel, 2000; Penner et al., 2010; Wilson et al., 2012). Gradually transitioning cattle to high-grain diets may reduce the risk for ruminal acidosis (Bevans et al., 2005) by providing sufficient time for behavioral, microbial, and epithelial adaptation. Adaptations that increase SCFA absorption should correspond to lower risk for ruminal acidosis (Gäbel et al., 2002; Penner et al., 2009a); however, few studies have evaluated adaptation of the ruminal epithelium in beef cattle. Reported timelines for ruminal epithelial adaptation range between 7 d for initial increases in functional activity (Etschmann et al., 2009) to 42 d for measurable increases in surface area (Dirksen et al., 1985). This range suggests that even with a gradual dietary transition, the ruminal adaptive process may not be complete and that ruminal adaptation may proceed with advancing days on feed. Therefore, it was hypothesized that increasing the amount of time cattle are fed a high-grain diet would decrease the susceptibility to and the time required to recover from a bout of ruminal acidosis. The objective of the study was to determine if the duration of time that cattle are fed a high-grain diet influences their susceptibility to and recovery from a bout of ruminal acidosis. MATERIALS AND METHODS This manuscript evaluates whether the duration of time cattle are fed high-grain diets improves the resistance to and recovery from an induced bout of ruminal acidosis with a focus on DMI, ruminal SCFA concentrations, and ruminal pH. A companion paper (Schwaiger et al., 2013) reports the results obtained for SCFA and lactate absorption, saliva production, and blood metabolites. The procedures and heifers used in this study were preapproved by the Animal Care Committee of the Agriculture and Agri-Food Canada Lethbridge Research Centre (Lethbridge, AB, Canada) and the study was conducted according to the guidelines of the Canadian Council on Animal Care (Ottawa, ON, Canada). Unless otherwise stated, all analyses were conducted in triplicate and reanalyzed when the CV was >3.0%. Animals, Diets, and Experimental Design Sixteen ruminally cannulated Angus heifers were used in this study. Each heifer was fitted with a 10-cm ruminal cannula (model 9C; Bar Diamond Inc., Parma, ID) at approximately 9 mo of age and provided at least 5 wk for recovery following the surgery before the start of the study. The surgical procedure for ruminal cannulation was conducted according to Bar Diamond Inc. (2011) and at the time of surgery, the mean BW ± SEM was 261 ± 6.1 kg. Heifers were housed in tie-stalls and were provided exercise daily. Before the start of the study, heifers were transitioned from a high-forage diet [forage-to-concentrate ratio (F:C) of 95:5] to a diet with a F:C of 60:40, hereafter designated as the backgrounding diet (Table 1). The dietary transition from the high-forage diet to the backgrounding diet occurred over 7 d using 3 intermediate diets that all contained 10% supplement (DM basis). During the first 2 d heifers were fed 30% barley silage, 50% grass hay, 10% barley grain, and 10% supplement; for the following 3 d heifers were fed 40% barley silage, 30% grass hay, 20% barley grain, and 10% supplement; and during the last 2 d heifers were fed 55% barley silage, 10% grass hay, 25% barley grain, and 10% supplement (DM basis). Table 1. Dietary transition protocol, ingredient inclusion rates, and the chemical composition of diets fed to heifers provided a long adaptation (LA) or short adaption (SA) to the high-grain diet before the acidosis induction protocol   Experimental diet  Item  BG1  Step 1  Step 2  Step 3  Step 4  Step 5  HG2  Time on each diet, d      LA  7  4  4  4  4  4  34      SA  33  4  4  4  4  4  8  Ingredients, g/kg DM      Barley silage  600  500  400  300  200  130  90      Barley grain3  300  400  500  600  700  770  810      Supplement4  100  100  100  100  100  100  100  Chemical composition,5 g/kg ± SEM      n  8  4  4  4  4  4  16      DM  468 ± 7.4  486 ± 11.7  538 ± 11.5  597 ± 8.4  660 ± 7.9  732 ± 4.3  777 ± 4.3      CP  138 ± 1.0  134 ± 1.0  133 ± 1.5  135 ± 2.8  130 ± 2.8  131 ± 3.1  134 ± 1.8      NDF  377 ± 8.3  363 ± 7.4  330 ± 3.5  301 ± 3.5  273 ± 9.1  251 ± 7.9  248 ± 8.6      ADF  208 ± 4.3  192 ± 4.6  166 ± 2.5  140 ± 2.4  116 ± 1.4  107 ± 3.3  91 ± 1.0      Starch  320 ± 9.0  357 ± 3.0  389 ± 8.6  437 ± 12.5  478 ± 5.2  501 ± 13.3  510 ± 5.2    Experimental diet  Item  BG1  Step 1  Step 2  Step 3  Step 4  Step 5  HG2  Time on each diet, d      LA  7  4  4  4  4  4  34      SA  33  4  4  4  4  4  8  Ingredients, g/kg DM      Barley silage  600  500  400  300  200  130  90      Barley grain3  300  400  500  600  700  770  810      Supplement4  100  100  100  100  100  100  100  Chemical composition,5 g/kg ± SEM      n  8  4  4  4  4  4  16      DM  468 ± 7.4  486 ± 11.7  538 ± 11.5  597 ± 8.4  660 ± 7.9  732 ± 4.3  777 ± 4.3      CP  138 ± 1.0  134 ± 1.0  133 ± 1.5  135 ± 2.8  130 ± 2.8  131 ± 3.1  134 ± 1.8      NDF  377 ± 8.3  363 ± 7.4  330 ± 3.5  301 ± 3.5  273 ± 9.1  251 ± 7.9  248 ± 8.6      ADF  208 ± 4.3  192 ± 4.6  166 ± 2.5  140 ± 2.4  116 ± 1.4  107 ± 3.3  91 ± 1.0      Starch  320 ± 9.0  357 ± 3.0  389 ± 8.6  437 ± 12.5  478 ± 5.2  501 ± 13.3  510 ± 5.2  1BG = backgrounding diet. 2HG = high-grain finishing diet. 3Barley grain processing index (volume weight after processing expressed as a percentage of volume weight before processing, DM basis) was 82.8 ± 0.25%. 4Supplement prepared as a blend and contained (DM basis): 50.0% beet pulp, 33.0% canola meal, 12.0% calcium carbonate, 2.5% urea, 1.6% salt, 0.5% vitamin and mineral mix, 0.3% melengestrol acetate (200 mg/kg), and 0.1% molasses. The concentration of minerals and vitamins in the supplement for the high-grain diet (DM basis) were zinc sulfate monohydrate (55.7 mg/kg), copper sulfate pentahydrate (14.2 mg/kg), manganese sulfate monohydrate (25.6 mg/kg), Ethylene diamine dihydriodide (0.6 mg/kg), selenium (0.3 mg/kg), vitamin A (9,281 IU/kg), vitamin D3 (464 IU/kg), and vitamin E (13 IU/kg). 5All analysis except for DM are reported on a DM basis. View Large Table 1. Dietary transition protocol, ingredient inclusion rates, and the chemical composition of diets fed to heifers provided a long adaptation (LA) or short adaption (SA) to the high-grain diet before the acidosis induction protocol   Experimental diet  Item  BG1  Step 1  Step 2  Step 3  Step 4  Step 5  HG2  Time on each diet, d      LA  7  4  4  4  4  4  34      SA  33  4  4  4  4  4  8  Ingredients, g/kg DM      Barley silage  600  500  400  300  200  130  90      Barley grain3  300  400  500  600  700  770  810      Supplement4  100  100  100  100  100  100  100  Chemical composition,5 g/kg ± SEM      n  8  4  4  4  4  4  16      DM  468 ± 7.4  486 ± 11.7  538 ± 11.5  597 ± 8.4  660 ± 7.9  732 ± 4.3  777 ± 4.3      CP  138 ± 1.0  134 ± 1.0  133 ± 1.5  135 ± 2.8  130 ± 2.8  131 ± 3.1  134 ± 1.8      NDF  377 ± 8.3  363 ± 7.4  330 ± 3.5  301 ± 3.5  273 ± 9.1  251 ± 7.9  248 ± 8.6      ADF  208 ± 4.3  192 ± 4.6  166 ± 2.5  140 ± 2.4  116 ± 1.4  107 ± 3.3  91 ± 1.0      Starch  320 ± 9.0  357 ± 3.0  389 ± 8.6  437 ± 12.5  478 ± 5.2  501 ± 13.3  510 ± 5.2    Experimental diet  Item  BG1  Step 1  Step 2  Step 3  Step 4  Step 5  HG2  Time on each diet, d      LA  7  4  4  4  4  4  34      SA  33  4  4  4  4  4  8  Ingredients, g/kg DM      Barley silage  600  500  400  300  200  130  90      Barley grain3  300  400  500  600  700  770  810      Supplement4  100  100  100  100  100  100  100  Chemical composition,5 g/kg ± SEM      n  8  4  4  4  4  4  16      DM  468 ± 7.4  486 ± 11.7  538 ± 11.5  597 ± 8.4  660 ± 7.9  732 ± 4.3  777 ± 4.3      CP  138 ± 1.0  134 ± 1.0  133 ± 1.5  135 ± 2.8  130 ± 2.8  131 ± 3.1  134 ± 1.8      NDF  377 ± 8.3  363 ± 7.4  330 ± 3.5  301 ± 3.5  273 ± 9.1  251 ± 7.9  248 ± 8.6      ADF  208 ± 4.3  192 ± 4.6  166 ± 2.5  140 ± 2.4  116 ± 1.4  107 ± 3.3  91 ± 1.0      Starch  320 ± 9.0  357 ± 3.0  389 ± 8.6  437 ± 12.5  478 ± 5.2  501 ± 13.3  510 ± 5.2  1BG = backgrounding diet. 2HG = high-grain finishing diet. 3Barley grain processing index (volume weight after processing expressed as a percentage of volume weight before processing, DM basis) was 82.8 ± 0.25%. 4Supplement prepared as a blend and contained (DM basis): 50.0% beet pulp, 33.0% canola meal, 12.0% calcium carbonate, 2.5% urea, 1.6% salt, 0.5% vitamin and mineral mix, 0.3% melengestrol acetate (200 mg/kg), and 0.1% molasses. The concentration of minerals and vitamins in the supplement for the high-grain diet (DM basis) were zinc sulfate monohydrate (55.7 mg/kg), copper sulfate pentahydrate (14.2 mg/kg), manganese sulfate monohydrate (25.6 mg/kg), Ethylene diamine dihydriodide (0.6 mg/kg), selenium (0.3 mg/kg), vitamin A (9,281 IU/kg), vitamin D3 (464 IU/kg), and vitamin E (13 IU/kg). 5All analysis except for DM are reported on a DM basis. View Large Based on time from surgery and the reduction in swelling and time since replacement of the 4C ruminal cannula (7.6 cm; Bar Diamond Inc.) with the 9C cannula (9 cm; Bar Diamond Inc.), heifers were assigned to 1 of 4 blocks and, within block, assigned to 1 of 2 treatments designated as long adapted (LA) or short adapted (SA). Within block, average BW across treatments was balanced. Heifers on the LA treatment were fed the backgrounding diet for 7 d before being transitioned to a barley-based finishing diet whereas SA heifers were fed the backgrounding diet for 33 d before being exposed to the same dietary transition protocol (Table 1). Differences in the duration of the backgrounding period allowed a delay in the start of the dietary transition for the SA animals so that the LA and SA heifers were fed the barley-based finishing diet for 34 and 8 d, respectively, before the induction of ruminal acidosis. The transition from the backgrounding diet to the finishing diet was accomplished over a period of 20 d using 5 intermediate diets with each diet fed for 4 d. Throughout the study, feed was offered once daily at 0900 h allowing for ad libitum intake by targeting 5 to 10% refusals on an as is basis, except for the day of and the day preceding the acidosis challenge. The study design consisted of 4 distinct measurement periods including 8 d for baseline measurements (BASE), the day of the acidosis challenge (CHAL), and 2 consecutive 8-d recovery periods (REC1 and REC2). The ruminal acidosis induction protocol was similar to that of Dohme et al. (2008) with the following modifications. Within each block, the proportion of feed intake relative to BW over 31 consecutive d (d 3 to d 33 on the finishing diet) for the LA heifers was used to calculate the feed restriction and challenge dose. Body weight was measured once a week during this time, and a linear growth rate was used to estimate daily intake as a proportion of BW. The challenge severity was normalized across dietary treatments by applying the same challenge dose relative to BW for LA and SA heifers. On the day before the challenge, feed intake for both LA and SA was restricted to 50% of DMI as a proportion of BW. Data from the day of feed restriction was not used in the statistical analysis. On the day of the CHAL, heifers (excluding those in block 1) were provided with an intraruminal infusion of ground barley grain (ground to pass through a 4.5-mm sieve) at 0900 h equating to 10% of DMI as a proportion of BW measured before feed restriction. Heifers in block 1 were infused with the same lot of ground barley but at 20% of DMI as a proportion of BW; however, because of the severity of the resulting acidosis, the amount of grain infused was subsequently reduced for the cattle in the remaining blocks. Heifers were then given their full diet allocation 1 h after the intraruminal infusion (1000 h). Beginning at the time of challenge, the pH of strained ruminal fluid from the ventral sac was measured every 2 h for the first 12 h and then every 4 h for the next 12 h using a portable pH meter (Accumet 25; Fisher Scientific, Ottawa, ON). Spot sampling pH measurement was used to monitor pH during the challenge, as the continuous indwelling pH system used did not provide a visible real-time pH reading. When ruminal pH was ≤4.2 an additional pH measurement was made 1 h later. If ruminal pH remained ≤4.2, heifers were provided an intraruminal dose of 250 g of sodium bicarbonate. This intervention was necessary for 2 of the 4 heifers exposed to the ruminal acidosis induction protocol in block 1 (both LA), and for this reason, subsequent intraruminal barley infusions were decreased from 20 to 10% of DMI as a proportion of BW, as indicated previously. Data from these heifers were used in the data analysis. Interventions were not required for cattle offered the modified challenge dose. Data and Sample Collection The amount of feed offered and refused was recorded daily throughout the study. Once the cattle were fed the high-grain diet, feed offered and refused were analyzed daily for DM content to determine DMI. In addition, TMR samples of the backgrounding and finishing diets were collected twice weekly, and samples of each transition diet were collected and stored at –20°C until analysis. Before analysis, TMR samples were allowed to thaw and the following feeding periods were composited by block to yield 4 samples each: steps 1 through 5, extended high-grain feeding (LA only), BASE, CHAL and REC1, and REC2. This resulted in a total of 16 samples used for chemical analysis of the high-grain diet and 4 samples for each of the transition diets (Table 1). In addition, the backgrounding diet was composited by treatment and block to yield 8 samples. Ingredients were also collected for DM content and chemical analysis. Barley silage was also collected twice weekly, grass hay and barley grain were collected weekly, and samples of the supplement were collected monthly as the same production lot was used for the entire study. All samples were composited to yield a monthly sample and were stored at –20°C for subsequent analysis. Ingredient and TMR samples were dried at 55°C for 48 h and ground to pass through a 1-mm sieve (SM100; Retsch, Hann, Germany). The analytical DM content was determined by drying samples at 135°C for 2 h (AOAC Int., 1995) and was used to calculate nutrient composition on a DM basis. Crude protein was estimated from the N concentration (CP = N × 6.25), which was determined using flash combustion (Carlo Erba Instruments, Milan, Italy). Neutral detergent fiber and ADF were determined using an Ankom Fiber Analyzer (Ankom Technology Corporation; Fairport, NY) using separate runs. Heat-stable α-amylase and sodium sulfite were used in the NDF procedure (Van Soest et al., 1991). Starch content was determined using enzymatic hydrolysis of α-linked glucose polymers as described by Rode et al. (1999) with minor modifications. Briefly, 100 to 500 mg of sample was diluted in 25 mL 0.1 N sodium acetate buffer and 200 μL of α-amylase (Termamyl; Novo Nordisk, Bagsvaerd, Denmark) was added. Tubes were vortexed immediately and at 10, 20, and 30 min while incubating at 95°C in a constantly shaking water bath. Incubation at 95°C continued for an additional 0.5 h. Subsequently, the incubation temperature was lowered to 65°C and 200 μL amyloglucosidase (208-469; Boehringer Mannheim, Laval, QC, Canada) was added to the tubes. Tubes were vortexed immediately and after 30 and 60 min of incubation at 65°C. Incubation at 65°C continued for an additional 1 h followed by cooling for 5 min. Tubes were then centrifuged (29,000 × g for 15 min at 4°C) and diluted 1:20 using double distilled water. Fifty microliters of sample was added to a microplate and 300-μL glucose trinder reagent (315-100; Sigma-Aldrich, St. Louis, MO) was added to all wells. Following a 20-min incubation at 39°C, glucose was determined by reading the absorbance at 508 nm (Appliscan Multiplate Reader; Thermo Electron Company, Waltham, MA). Means from 4 glucose determinations were compared in duplicate and results were confirmed when the CV < 5.0%. Ruminal pH Measurement Ruminal pH data were recorded every 1 min using an indwelling pH measurement system (LRCpH Data Logger system; Dascor, Escondido, CA) positioned at the bottom of the cranial-ventral sac as described by Penner et al. (2006). Measurement started on d 1 of BASE and persisted through to the final day of experimentation resulting in 26 d of measurements. Ruminal pH systems were standardized before ruminal incubation and were restandardized in pH buffers 7 and 4 at 39°C before and after cleaning in Terg-A-Zyme (Alconox; White Plains, NY) solution for 10 min on weekly removal from the rumen. The resulting data were transformed from millivolt recordings to pH based on the pre- and postincubation standardizations with the precleaning measurement used for the postincubation standardization values. A linear drift correction over time was used to correct pH data (Penner et al., 2006). The daily minimum, mean, and maximum pH as well as duration (DUR; min/d) and area (AREA; min × pH) that pH was <5.5 were calculated. The day of the dietary restriction before CHAL was discarded from analysis as were pH data from days when the temporarily isolated and washed reticulorumen procedure was conducted (d 5 of BASE, d 2 of REC1, and d 1 of REC2; see Schwaiger et al., 2013). After these deletions, 22 d of pH data remained for each heifer (7 d for BASE, 1 d for CHAL, and 7 d each for REC1 and REC2). Additionally, pH was monitored during the first 7 d that the LA and SA cattle were fed the high-grain diet to test whether the duration of time on the backgrounding diet affected the response. Ruminal Fluid Sampling and Analyses Ruminal digesta was sampled on d 3 of BASE, on the day of CHAL, and on d 7 of REC1. Starting at the time of feeding (0900 h), samples were collected every 2 h for a total of 7 samples over 12 h. Due to an observed continued response to the challenge during block 1, the sampling protocol was extended to cover a 24 h duration on the challenge day for blocks 2 to 4. The extended sampling was used to collect 3 additional ruminal fluid samples with a 4-h interval between consecutive samples. Digesta was collected from 3 locations in the rumen (cranial, central-ventral, and caudal sacs) with an equal volume from each location used to prepare a composite (250 mL each for 750 mL total). Composited digesta was strained through PECAP polyester monofilament (pore size 355 µm; part 7-355/47; B & SH Thompson, Ville Mont-Royal, QC, Canada) and aliquots were stored at –20°C for determination of SCFA (1.5 mL), lactate (1.5 mL), and osmolality (2 mL). For SCFA and lactate determination, 1.5-mL aliquots were preserved with 0.3 mL frozen 25% (wt/vol) metaphosphoric acid. Ruminal fluid osmolality (mOsm/kg) was determined in duplicate by freezing-point depression. Samples were centrifuged at 16,100 × g for 30 min 4°C, and 250 μL of supernatant was loaded into the osmometer (Advanced Instruments 3250; Advanced Instruments, Norwood, MA). For quality control, double distilled H2O and standards (290 and 500 mOsm/kg) were analyzed at the beginning and end of each day. Ruminal fluid SCFA and lactate concentrations were determined by gas chromatography using a flame ionization detector. The gas chromatograph (Hewlett-Packard 5890; Hewlett-Packard, Santa Clara, CA) was fitted with a Zebron capillary column (ZB-FFAP; 30 m by 0.32 mm i.d. by 1.0 µm phase thickness; Phenomenex, Torrance, CA), and crotonic acid (trans-2-butenoic acid) and malonic acid (propanedioic acid) were used as internal standards for SCFA and lactate, respectively. In both cases, helium was used as carrier gas (28.5 cm/s). For SCFA determination, 1 μL was injected using a split ratio of 50:1. The injector temperature was set at 225°C and the column temperature was held at 150°C for 1 min followed by a 5°C/min increase in temperature until reaching 195°C, after which temperature was held for 5 min. The detector temperature was held constant at 250°C. For lactate determination, lactate methyl esters were prepared using BF3–MeOH as described by Supelco (1998) and a 1-μL splitless injection was used. The injector temperature was set at 225°C and the column temperature was held at 45°C for 1 min followed by a 30°C/min increase in temperature until reaching 160°C and then a 5°C/min increase in temperature until reaching 195°C, after which temperature was held for 5 min. The detector temperature was held constant at 250°C. Lactate concentrations below the lowest standard (0.21 mM) were assumed to be 0 mM. Statistical Analyses Data were analyzed as a randomized complete block design using the PROC MIXED procedure of SAS (version 9.2; SAS Inst. Inc., Cary, NC). Block was considered a fixed effect and was left in the model except when performing analysis of covariance as described below. Significance was declared when P ≤ 0.05 and tendencies are discussed when 0.05 < P ≤ 0.10. Mean separation was conducted for all variables using the Tukey's post hoc mean separation test and the SAS PDMIX800 macro (Saxton, 1998). The PROC UNIVARIATE procedure of SAS was used to determine if residual data were normally, identically, and independently distributed (NIID). If necessary (based on the normal probability plot and the Shapiro-Wilk test), outliers were removed in a stepwise fashion until the normal probability plot indicated that residual data were NIID. In total 3 observations were removed for serum lactate during BASE and 4 observations during CHAL. For serum L-lactate, 3 observations were removed for BASE, and for serum D-lactate, 3 and 7 observations were removed during BASE and CHAL, respectively. Three observations during both BASE and REC1 were removed for plasma insulin. Ruminal pH and DMI data were summarized by day and heifer. The fixed effect of treatment was investigated over the first 7 d on the high-grain diet. To compare daily means across periods with a different number of days, a split-plot design was used. In this manner the fixed effects of treatment, period, treatment × period, period × day, and treatment × period × day were investigated with the random effects of heifer × block and heifer × period × treatment × block. The effects of period × day and treatment × period × day were left in the model but not reported due to their lack of physiological importance. The effects of treatment and treatment × period were also not reported, as each sampling period was considered biologically distinct. The fixed effects of treatment, day, and treatment × day were then investigated within BASE using day as a repeated measure for the subject heifer × treatment × block. The fixed effect of treatment was then investigated within CHAL and regression analysis was conducted for each variable with respect to day using daily means from BASE and CHAL to determine if there were significant linear, quadratic, or cubic effects of day. To accomplish this, the fixed effects of treatment, day, treatment × day, day2, treatment × day2, day3, and treatment × day3 were tested while accounting for repeated measures with heifer × treatment as the subject. Starting with the highest order term, insignificant (P > 0.05) terms were removed from the model in a stepwise fashion until only significant (P ≤ 0.05) terms remained in the model. When an interaction was significant, the lower order term was removed. The same regression approach as listed above was used to evaluate the recovery response during REC1. The fixed effects of treatment, period, and treatment × period were investigated for ruminal fluid fermentation products by summarizing the data by heifer and period. Only the first 12 h of challenge sampling was used for ruminal fluid fermentation products (extended sampling on CHAL was ignored). Period was used as a repeated measure for the subject heifer × treatment × block. Then, within each sampling period the fixed effects of treatment, time, and treatment × time were investigated for ruminal fluid fermentation products by summarizing the data by heifer, period, and time. Time was used as a repeated measure for the subject heifer × treatment × block. One SA heifer was removed from the study before the acidosis induction due to low intake, frothy ruminal contents, and keratinized epithelia; however, baseline data from this heifer were used in statistical analysis. A failed pH electrode resulted in the loss of 6 d of pH data from 1 heifer (SA; BASE d 6 to d 8, CHAL, REC1 d 1 to d 2). Dry matter intake data were missing for 1 heifer (SA) on d 5 of REC2. RESULTS Confirmation of the Experimental Model The Duration of Time Fed the Backgrounding Diet did not Affect DMI or Ruminal Fermentation. The aim of this study was to evaluate whether duration of time that heifers were fed a high-grain diet affects the susceptibility to and recovery from an induced bout of ruminal acidosis. To determine if the duration of time on the backgrounding diet confounded the response, comparisons were made for BW, DMI, and ruminal pH during the first 7 d that heifers from each treatment were fed the high-grain diet (Table 2). The 26 additional d that the SA cattle were fed the backgrounding diet increased BW (P < 0.001) and tended to increase DMI (P = 0.094) during the first 7 d on the high-grain diet; however, there was no effect of treatment on DMI when reported as a percentage of BW (P = 0.70). There were also no differences between treatments for minimum and mean ruminal pH, nor were there differences for DUR. On the other hand, maximum pH was greater for LA than SA (6.68 vs. 6.49; P = 0.001). Overall, we interpret these data to suggest that the duration of time that heifers were fed the backgrounding diet did not have a significant impact on DMI or ruminal fermentation, and therefore responses observed for the subsequent tables can be attributed to the duration of time that heifers were fed the high-grain diet. Table 2. Mean daily BW, DMI, and ruminal pH for the first 7 d that long-adapted (LA) and short-adapted (SA) heifers were fed the high-grain diet1   Treatment      Item  LA  SA  SEM  P value  BW,2 kg  314  342  2.4  <0.001  DMI, kg  8.7  9.3  0.25  0.094  DMI, % BW  2.8  2.7  0.07  0.70  Minimum pH3  5.04  5.06  0.032  0.65  Mean pH  5.79  5.72  0.042  0.24  Maximum pH  6.68  6.49  0.037  0.001  pH < 5.5      Duration, min/d  475  522  50.3  0.52      Area, (min × pH)/d  153  166  22.5  0.67    Treatment      Item  LA  SA  SEM  P value  BW,2 kg  314  342  2.4  <0.001  DMI, kg  8.7  9.3  0.25  0.094  DMI, % BW  2.8  2.7  0.07  0.70  Minimum pH3  5.04  5.06  0.032  0.65  Mean pH  5.79  5.72  0.042  0.24  Maximum pH  6.68  6.49  0.037  0.001  pH < 5.5      Duration, min/d  475  522  50.3  0.52      Area, (min × pH)/d  153  166  22.5  0.67  1Forage:concentrate ratio = 9:91. 2Mean BW was estimated assuming linear growth between measurements separated by 20 d (LA) and 13 d (SA). 3Data for 7 d of indwelling ruminal pH measurement. View Large Table 2. Mean daily BW, DMI, and ruminal pH for the first 7 d that long-adapted (LA) and short-adapted (SA) heifers were fed the high-grain diet1   Treatment      Item  LA  SA  SEM  P value  BW,2 kg  314  342  2.4  <0.001  DMI, kg  8.7  9.3  0.25  0.094  DMI, % BW  2.8  2.7  0.07  0.70  Minimum pH3  5.04  5.06  0.032  0.65  Mean pH  5.79  5.72  0.042  0.24  Maximum pH  6.68  6.49  0.037  0.001  pH < 5.5      Duration, min/d  475  522  50.3  0.52      Area, (min × pH)/d  153  166  22.5  0.67    Treatment      Item  LA  SA  SEM  P value  BW,2 kg  314  342  2.4  <0.001  DMI, kg  8.7  9.3  0.25  0.094  DMI, % BW  2.8  2.7  0.07  0.70  Minimum pH3  5.04  5.06  0.032  0.65  Mean pH  5.79  5.72  0.042  0.24  Maximum pH  6.68  6.49  0.037  0.001  pH < 5.5      Duration, min/d  475  522  50.3  0.52      Area, (min × pH)/d  153  166  22.5  0.67  1Forage:concentrate ratio = 9:91. 2Mean BW was estimated assuming linear growth between measurements separated by 20 d (LA) and 13 d (SA). 3Data for 7 d of indwelling ruminal pH measurement. View Large Confirmation that Ruminal Acidosis was Induced. To illustrate the effects of the induced challenge on ruminal pH, mean pH (10-min intervals) was plotted for the 2 treatments over a 5-d duration surrounding the induced bout of ruminal acidosis (Fig. 1). From the rapid drop in pH and the extent to which pH dropped, it is clear that a bout of ruminal acidosis was successfully induced using the acidosis challenge protocol. Figure 1. View largeDownload slide Mean ruminal pH for the 5 d surrounding the induced challenge. Data were summarized by minute for each heifer, and means were calculated for every 10 min for long-adapted (LA) and short-adapted (SA) heifers during the last 2 d of baseline (BASE 7 and 8), during the dietary restriction (REST), during the day of the induced challenge (CHAL), and during the first day of recovery from the challenge (REC1). All (n = 8) LA heifers were used for all means shown while 7 (n = 7) SA heifers were used during BASE and 6 (n = 6) SA heifers were used during REST, CHAL, and REC1. One SA heifer was removed from the study after BASE, and another had a failed pH electrode. Figure 1. View largeDownload slide Mean ruminal pH for the 5 d surrounding the induced challenge. Data were summarized by minute for each heifer, and means were calculated for every 10 min for long-adapted (LA) and short-adapted (SA) heifers during the last 2 d of baseline (BASE 7 and 8), during the dietary restriction (REST), during the day of the induced challenge (CHAL), and during the first day of recovery from the challenge (REC1). All (n = 8) LA heifers were used for all means shown while 7 (n = 7) SA heifers were used during BASE and 6 (n = 6) SA heifers were used during REST, CHAL, and REC1. One SA heifer was removed from the study after BASE, and another had a failed pH electrode. Comparisons by period were conducted to evaluate whether the acidosis protocol affected DMI and ruminal pH. It is important to note that there were no interactions between treatment and period for DMI, ruminal pH, SCFA, lactate, or osmolality (P ≥ 0.12; data not shown). Dry matter intake tended (P = 0.072) to be affected by period, whereby DMI in CHAL tended to be greater than during the other periods. The acidosis challenge reduced minimum (P < 0.001) and mean pH (P < 0.001) relative to BASE with the effect reversed during REC1 and REC2, such that minimum and mean pH were greater during REC1 and REC2 than during BASE and CHAL. Maximum pH increased on the day of CHAL due to the elevated pH caused by the dietary restriction (P < 0.001; Fig. 1). This elevated pH persisted for approximately 1 h following the induced challenge. The DUR (P < 0.001) and AREA (P < 0.001) was greater during CHAL relative to BASE. Specifically, the CHAL nearly doubled the DUR relative to BASE (1,020 vs. 531 min/d; Table 3). The increases for DUR and AREA during CHAL were both reversible with reduced DUR and AREA observed during REC1 and REC2 than during BASE, and the AREA during REC1 and REC2 were not different than during BASE but less than CHAL. Table 3. Mean daily DMI, ruminal pH, short-chain fatty acids, lactate, and osmolality across measurement periods when heifers were fed the high-grain diet.1 There were no treatment and treatment × period interactions (P ≥ 0.12).2   Period2      Item  BASE  CHAL  REC1  REC2  SEM  P value  Number of days, d  7  1  7  7      DMI, kg  9.3  10.5  9.2  9.6  0.46  0.072  Minimum pH  5.03b  4.57c  5.20a  5.26a  0.03  <0.001  Mean pH  5.73b  5.27c  5.86a  5.93a  0.043  <0.001  Maximum pH  6.54b  6.96a  6.52b  6.60b  0.038  <0.001  pH < 5.5      Duration, min/d  531b  1,020a  294c  259c  55.7  <0.001      Area, (pH × min)/d  176b  595a  72b  58b  36  <0.001  Total SCFA,3 mM      Mean  128.2  133  122  –  4.32  0.14      Maximum  157.7ab  165.7a  143.2b  –  5  0.011  Acetate, mM      Mean  62.2  67.8  63.6  –  1.62  0.22      Maximum  75.5  80  73.6  –  1.98  0.08  Propionate, mM      Mean  50.4  50  42.8  –  3.88  0.12      Maximum  65.0a  61.7ab  51.0b  –  4.35  0.018  Butyrate, mM      Mean  15.2  17.9  15  –  1.55  0.23      Maximum  20.1  24.5  19.4  –  2.1  0.20  Lactate, mM      Mean  0.3b  11.4a  0.1b  –  1.04  0.013      Maximum  1.3b  29.3a  0.5b  –  2.55  0.008  Osmolality, mOsm/kg      Mean  369  369  369  –  5.3  0.99      Maximum  412ab  432a  407b  –  6.9  0.030    Period2      Item  BASE  CHAL  REC1  REC2  SEM  P value  Number of days, d  7  1  7  7      DMI, kg  9.3  10.5  9.2  9.6  0.46  0.072  Minimum pH  5.03b  4.57c  5.20a  5.26a  0.03  <0.001  Mean pH  5.73b  5.27c  5.86a  5.93a  0.043  <0.001  Maximum pH  6.54b  6.96a  6.52b  6.60b  0.038  <0.001  pH < 5.5      Duration, min/d  531b  1,020a  294c  259c  55.7  <0.001      Area, (pH × min)/d  176b  595a  72b  58b  36  <0.001  Total SCFA,3 mM      Mean  128.2  133  122  –  4.32  0.14      Maximum  157.7ab  165.7a  143.2b  –  5  0.011  Acetate, mM      Mean  62.2  67.8  63.6  –  1.62  0.22      Maximum  75.5  80  73.6  –  1.98  0.08  Propionate, mM      Mean  50.4  50  42.8  –  3.88  0.12      Maximum  65.0a  61.7ab  51.0b  –  4.35  0.018  Butyrate, mM      Mean  15.2  17.9  15  –  1.55  0.23      Maximum  20.1  24.5  19.4  –  2.1  0.20  Lactate, mM      Mean  0.3b  11.4a  0.1b  –  1.04  0.013      Maximum  1.3b  29.3a  0.5b  –  2.55  0.008  Osmolality, mOsm/kg      Mean  369  369  369  –  5.3  0.99      Maximum  412ab  432a  407b  –  6.9  0.030  a,b,cMeans within a dependent variable differ (P ≤ 0.05). The Tukey post hoc mean separation test was used for mean separation. 1Forage:concentrate ratio = 9:91. 2For ruminal pH BASE = baseline measurement period that consisted of 8 d on the final diet before dietary restriction (50% DMI/BW). CHAL = challenge day that occurred 1 d after dietary restriction and consisted of an intraruminal infusion of ground barley grain (10% DMI/BW) followed by full diet allocation. REC = recovery period that started 24 h after the challenge and is separated into 2 consecutive 8 d periods (REC1 and REC2). Data from days where the temporarily isolated and washed reticulorumen technique was conducted during BASE, REC1, and REC2 have been omitted. For ruminal fluid, BASE sampling occurred 7 d prior to CHAL, CHAL sampling occurred on d of induced challenge, REC1 sampling occurred 7 d after challenge. Daily mean and maximum values were determined from samples collected at the time of feeding and every 2 hours for 12 h after feeding. 3SCFA = short-chain fatty acid. View Large Table 3. Mean daily DMI, ruminal pH, short-chain fatty acids, lactate, and osmolality across measurement periods when heifers were fed the high-grain diet.1 There were no treatment and treatment × period interactions (P ≥ 0.12).2   Period2      Item  BASE  CHAL  REC1  REC2  SEM  P value  Number of days, d  7  1  7  7      DMI, kg  9.3  10.5  9.2  9.6  0.46  0.072  Minimum pH  5.03b  4.57c  5.20a  5.26a  0.03  <0.001  Mean pH  5.73b  5.27c  5.86a  5.93a  0.043  <0.001  Maximum pH  6.54b  6.96a  6.52b  6.60b  0.038  <0.001  pH < 5.5      Duration, min/d  531b  1,020a  294c  259c  55.7  <0.001      Area, (pH × min)/d  176b  595a  72b  58b  36  <0.001  Total SCFA,3 mM      Mean  128.2  133  122  –  4.32  0.14      Maximum  157.7ab  165.7a  143.2b  –  5  0.011  Acetate, mM      Mean  62.2  67.8  63.6  –  1.62  0.22      Maximum  75.5  80  73.6  –  1.98  0.08  Propionate, mM      Mean  50.4  50  42.8  –  3.88  0.12      Maximum  65.0a  61.7ab  51.0b  –  4.35  0.018  Butyrate, mM      Mean  15.2  17.9  15  –  1.55  0.23      Maximum  20.1  24.5  19.4  –  2.1  0.20  Lactate, mM      Mean  0.3b  11.4a  0.1b  –  1.04  0.013      Maximum  1.3b  29.3a  0.5b  –  2.55  0.008  Osmolality, mOsm/kg      Mean  369  369  369  –  5.3  0.99      Maximum  412ab  432a  407b  –  6.9  0.030    Period2      Item  BASE  CHAL  REC1  REC2  SEM  P value  Number of days, d  7  1  7  7      DMI, kg  9.3  10.5  9.2  9.6  0.46  0.072  Minimum pH  5.03b  4.57c  5.20a  5.26a  0.03  <0.001  Mean pH  5.73b  5.27c  5.86a  5.93a  0.043  <0.001  Maximum pH  6.54b  6.96a  6.52b  6.60b  0.038  <0.001  pH < 5.5      Duration, min/d  531b  1,020a  294c  259c  55.7  <0.001      Area, (pH × min)/d  176b  595a  72b  58b  36  <0.001  Total SCFA,3 mM      Mean  128.2  133  122  –  4.32  0.14      Maximum  157.7ab  165.7a  143.2b  –  5  0.011  Acetate, mM      Mean  62.2  67.8  63.6  –  1.62  0.22      Maximum  75.5  80  73.6  –  1.98  0.08  Propionate, mM      Mean  50.4  50  42.8  –  3.88  0.12      Maximum  65.0a  61.7ab  51.0b  –  4.35  0.018  Butyrate, mM      Mean  15.2  17.9  15  –  1.55  0.23      Maximum  20.1  24.5  19.4  –  2.1  0.20  Lactate, mM      Mean  0.3b  11.4a  0.1b  –  1.04  0.013      Maximum  1.3b  29.3a  0.5b  –  2.55  0.008  Osmolality, mOsm/kg      Mean  369  369  369  –  5.3  0.99      Maximum  412ab  432a  407b  –  6.9  0.030  a,b,cMeans within a dependent variable differ (P ≤ 0.05). The Tukey post hoc mean separation test was used for mean separation. 1Forage:concentrate ratio = 9:91. 2For ruminal pH BASE = baseline measurement period that consisted of 8 d on the final diet before dietary restriction (50% DMI/BW). CHAL = challenge day that occurred 1 d after dietary restriction and consisted of an intraruminal infusion of ground barley grain (10% DMI/BW) followed by full diet allocation. REC = recovery period that started 24 h after the challenge and is separated into 2 consecutive 8 d periods (REC1 and REC2). Data from days where the temporarily isolated and washed reticulorumen technique was conducted during BASE, REC1, and REC2 have been omitted. For ruminal fluid, BASE sampling occurred 7 d prior to CHAL, CHAL sampling occurred on d of induced challenge, REC1 sampling occurred 7 d after challenge. Daily mean and maximum values were determined from samples collected at the time of feeding and every 2 hours for 12 h after feeding. 3SCFA = short-chain fatty acid. View Large No differences were detected for mean SCFA, mean acetate, mean propionate, mean and maximum butyrate concentrations, and mean osmolality when compared across periods (Table 3). Maximum acetate concentration tended (P = 0.08) to increase during CHAL and decrease during REC1. Maximum propionate concentration was greatest during BASE and least during REC1 (P = 0.018) while both maximum total SCFA (165.7 vs. 143.2 mM; P = 0.011) and maximum ruminal fluid osmolality (432 vs. 407 mOsm/kg; P = 0.03) were greatest during the CHAL and least during REC1. Mean (P = 0.013) and maximum (P = 0.008) ruminal fluid lactate concentration increased during the CHAL (11.4 and 29.3 mM) relative to BASE (0.3 and 1.3 mM) but BASE and REC1 (0.1 and 0.5 mM; Table 3) were not different. Ruminal lactate concentration >5.0 mM was observed in 12 of the 15 heifers during CHAL. In contrast, none of the heifers experienced a maximal daily ruminal lactate concentration ≥5.0 mM during either BASE or REC1 (data is not shown). Although there was significant between-heifer variability in response to the challenge, we were able to successfully induce an average bout of acute ruminal acidosis when data from all heifers were considered (Table 3). By implementing our challenge model, we observed a significant increase in ruminal fluid lactate concentration (Table 3) with a peak mean lactate concentration of 19.0 mM observed 8 h after providing the challenge dose. The daily mean lactate concentration of 11.4 mM, and daily maximum lactate concentration of 29.3 mM are both above the suggested threshold of 5.0 mM to indicate acute ruminal lactic acidosis (Aschenbach et al., 2011). Dry Matter Intake and Ruminal Fermentation during the Baseline Measurement Period To evaluate the effect of treatment and day on DMI and pH and the effect of treatment and time on ruminal SCFA and osmolality before CHAL, the data were investigated within BASE (Tables 4 and 5). Differences observed during BASE are deemed to be part of the adaptation response. There was no effect of treatment on DMI or pH variables during BASE (P ≥ 0.088). Dry matter intake was greatest on the first day of BASE (P = 0.038); however, minimum (P = 0.01), mean (P = 0.03), and maximum (P = 0.027) pH all reached the lowest values on d 6 of BASE, during which time both DUR (P = 0.064) and AREA (pH × min/d; P = 0.075) tended to be greatest. A tendency for a treatment × day interaction for maximum pH (P = 0.063) indicated that the LA heifers tended to experience an increase in maximum pH between d 1 and 4 during BASE while the SA heifers tended to experience a decrease in maximum pH during this time (data not shown). Table 4. Mean daily DMI and pH variables for the 8 d before the induced acidosis challenge (BASE1). Long-adapted (LA) heifers were fed the high-grain diet2 for 26 additional d before this measurement period; short-adapted (SA) heifers received the high-grain diet on d 1 of the BASE period.   Treatment    Day of baseline period (BASE)    P value3  Item  LA  SA  SEM  1  2  3  4  6  7  8  SEM  T  D  T × D  DMI, kg  9.3  9.3  0.64  10.5  9.5  9.6  8.3  8.8  8.5  9.6  0.58  1.00  0.038  0.97  Minimum pH  4.99  5.06  0.037  5.11ab  5.02ab  4.93ab  5.04ab  4.88b  5.12a  5.09ab  0.058  0.20  0.010  0.69  Mean pH  5.73  5.72  0.056  5.78  5.66  5.63  5.82  5.58  5.82  5.78  0.078  0.93  0.030  0.56  Maximum pH  6.59  6.48  0.043  6.65a  6.56ab  6.52ab  6.55ab  6.45b  6.46ab  6.56ab  0.060  0.088  0.027  0.063  Ruminal pH < 5.5      Duration, min/d  547  516  72.6  474  581  639  485  680  397  465  94.3  0.77  0.064  0.29      Area, (min × pH)/d  190  167  31.2  138  210  223  147  261  127  141  44.2  0.60  0.075  0.53    Treatment    Day of baseline period (BASE)    P value3  Item  LA  SA  SEM  1  2  3  4  6  7  8  SEM  T  D  T × D  DMI, kg  9.3  9.3  0.64  10.5  9.5  9.6  8.3  8.8  8.5  9.6  0.58  1.00  0.038  0.97  Minimum pH  4.99  5.06  0.037  5.11ab  5.02ab  4.93ab  5.04ab  4.88b  5.12a  5.09ab  0.058  0.20  0.010  0.69  Mean pH  5.73  5.72  0.056  5.78  5.66  5.63  5.82  5.58  5.82  5.78  0.078  0.93  0.030  0.56  Maximum pH  6.59  6.48  0.043  6.65a  6.56ab  6.52ab  6.55ab  6.45b  6.46ab  6.56ab  0.060  0.088  0.027  0.063  Ruminal pH < 5.5      Duration, min/d  547  516  72.6  474  581  639  485  680  397  465  94.3  0.77  0.064  0.29      Area, (min × pH)/d  190  167  31.2  138  210  223  147  261  127  141  44.2  0.60  0.075  0.53  a,bMeans within a dependent variable differ (P ≤ 0.050). The Tukey post hoc mean separation test was used for mean separation. For DMI and mean pH, mean separation was not achieved despite a significant effect of day. 1BASE = the baseline measurement period consisted of 8 d on the final diet before dietary restriction and the acidosis challenge. Day 5 was the day of the temporarily isolated and washed reticulorumen technique. Due to manipulation of the rumen, DMI and pH data were not included. 2Forage:concentrate ratio = 9:91. 3T = treatment; D = day of baseline period. View Large Table 4. Mean daily DMI and pH variables for the 8 d before the induced acidosis challenge (BASE1). Long-adapted (LA) heifers were fed the high-grain diet2 for 26 additional d before this measurement period; short-adapted (SA) heifers received the high-grain diet on d 1 of the BASE period.   Treatment    Day of baseline period (BASE)    P value3  Item  LA  SA  SEM  1  2  3  4  6  7  8  SEM  T  D  T × D  DMI, kg  9.3  9.3  0.64  10.5  9.5  9.6  8.3  8.8  8.5  9.6  0.58  1.00  0.038  0.97  Minimum pH  4.99  5.06  0.037  5.11ab  5.02ab  4.93ab  5.04ab  4.88b  5.12a  5.09ab  0.058  0.20  0.010  0.69  Mean pH  5.73  5.72  0.056  5.78  5.66  5.63  5.82  5.58  5.82  5.78  0.078  0.93  0.030  0.56  Maximum pH  6.59  6.48  0.043  6.65a  6.56ab  6.52ab  6.55ab  6.45b  6.46ab  6.56ab  0.060  0.088  0.027  0.063  Ruminal pH < 5.5      Duration, min/d  547  516  72.6  474  581  639  485  680  397  465  94.3  0.77  0.064  0.29      Area, (min × pH)/d  190  167  31.2  138  210  223  147  261  127  141  44.2  0.60  0.075  0.53    Treatment    Day of baseline period (BASE)    P value3  Item  LA  SA  SEM  1  2  3  4  6  7  8  SEM  T  D  T × D  DMI, kg  9.3  9.3  0.64  10.5  9.5  9.6  8.3  8.8  8.5  9.6  0.58  1.00  0.038  0.97  Minimum pH  4.99  5.06  0.037  5.11ab  5.02ab  4.93ab  5.04ab  4.88b  5.12a  5.09ab  0.058  0.20  0.010  0.69  Mean pH  5.73  5.72  0.056  5.78  5.66  5.63  5.82  5.58  5.82  5.78  0.078  0.93  0.030  0.56  Maximum pH  6.59  6.48  0.043  6.65a  6.56ab  6.52ab  6.55ab  6.45b  6.46ab  6.56ab  0.060  0.088  0.027  0.063  Ruminal pH < 5.5      Duration, min/d  547  516  72.6  474  581  639  485  680  397  465  94.3  0.77  0.064  0.29      Area, (min × pH)/d  190  167  31.2  138  210  223  147  261  127  141  44.2  0.60  0.075  0.53  a,bMeans within a dependent variable differ (P ≤ 0.050). The Tukey post hoc mean separation test was used for mean separation. For DMI and mean pH, mean separation was not achieved despite a significant effect of day. 1BASE = the baseline measurement period consisted of 8 d on the final diet before dietary restriction and the acidosis challenge. Day 5 was the day of the temporarily isolated and washed reticulorumen technique. Due to manipulation of the rumen, DMI and pH data were not included. 2Forage:concentrate ratio = 9:91. 3T = treatment; D = day of baseline period. View Large Table 5. Ruminal short-chain fatty acid (SCFA) concentration and osmolality before the induced acidosis challenge (BASE1). Long-adapted (LA) heifers were fed the high-grain diet2 for 26 additional d before this measurement period; short-adapted (SA) heifers received the high-grain diet on d 1 of the BASE2 period.   Treatment    Hour relative to feeding (h)    P value2  LA  SA  SEM  0  2  4  6  8  10  12  SEM  T  h  T × h  Total SCFA, mM  133.5  122.1  4.12  94.7c  109.1bc  125.8ab  139.9a  144.8a  133.7a  146.8a  4.12  0.065  <0.001  0.59  Acetate, mM  63.7  60.8  1.58  47.6c  56.5b  62.5ab  68.0a  68.9a  63.4ab  68.9a  2.31  0.23  <0.001  0.70  Propionate, mM  54.1  46.6  2.93  35.2c  39.7c  48.1b  55.5ab  58.6a  54.6ab  60.9a  3.08  0.093  <0.001  0.52  Butyrate, mM  15.8  14.7  1.89  11.9ab  13.0ab  15.2b  16.5ab  17.4a  15.8ab  17.1ab  1.62  0.71  0.015  0.53  Osmolality, mOsm/kg  380a  358b  6.6  321c  350b  369ab  384a  390a  376ab  391a  7.3  0.038  <0.001  0.42    Treatment    Hour relative to feeding (h)    P value2  LA  SA  SEM  0  2  4  6  8  10  12  SEM  T  h  T × h  Total SCFA, mM  133.5  122.1  4.12  94.7c  109.1bc  125.8ab  139.9a  144.8a  133.7a  146.8a  4.12  0.065  <0.001  0.59  Acetate, mM  63.7  60.8  1.58  47.6c  56.5b  62.5ab  68.0a  68.9a  63.4ab  68.9a  2.31  0.23  <0.001  0.70  Propionate, mM  54.1  46.6  2.93  35.2c  39.7c  48.1b  55.5ab  58.6a  54.6ab  60.9a  3.08  0.093  <0.001  0.52  Butyrate, mM  15.8  14.7  1.89  11.9ab  13.0ab  15.2b  16.5ab  17.4a  15.8ab  17.1ab  1.62  0.71  0.015  0.53  Osmolality, mOsm/kg  380a  358b  6.6  321c  350b  369ab  384a  390a  376ab  391a  7.3  0.038  <0.001  0.42  a,b,cMeans within a dependent variable differ (P ≤ 0.05). The Tukey post hoc mean separation test was used for mean separation. 1BASE = the baseline measurement period consisted of 8 d on the final diet before dietary restriction and the acidosis challenge. Day 5 was the day of the temporarily isolated and washed reticulorumen technique. Due to manipulation of the rumen, DMI and pH data were not included. 2Forage:concentrate ratio = 9:91. 3T = treatment. View Large Table 5. Ruminal short-chain fatty acid (SCFA) concentration and osmolality before the induced acidosis challenge (BASE1). Long-adapted (LA) heifers were fed the high-grain diet2 for 26 additional d before this measurement period; short-adapted (SA) heifers received the high-grain diet on d 1 of the BASE2 period.   Treatment    Hour relative to feeding (h)    P value2  LA  SA  SEM  0  2  4  6  8  10  12  SEM  T  h  T × h  Total SCFA, mM  133.5  122.1  4.12  94.7c  109.1bc  125.8ab  139.9a  144.8a  133.7a  146.8a  4.12  0.065  <0.001  0.59  Acetate, mM  63.7  60.8  1.58  47.6c  56.5b  62.5ab  68.0a  68.9a  63.4ab  68.9a  2.31  0.23  <0.001  0.70  Propionate, mM  54.1  46.6  2.93  35.2c  39.7c  48.1b  55.5ab  58.6a  54.6ab  60.9a  3.08  0.093  <0.001  0.52  Butyrate, mM  15.8  14.7  1.89  11.9ab  13.0ab  15.2b  16.5ab  17.4a  15.8ab  17.1ab  1.62  0.71  0.015  0.53  Osmolality, mOsm/kg  380a  358b  6.6  321c  350b  369ab  384a  390a  376ab  391a  7.3  0.038  <0.001  0.42    Treatment    Hour relative to feeding (h)    P value2  LA  SA  SEM  0  2  4  6  8  10  12  SEM  T  h  T × h  Total SCFA, mM  133.5  122.1  4.12  94.7c  109.1bc  125.8ab  139.9a  144.8a  133.7a  146.8a  4.12  0.065  <0.001  0.59  Acetate, mM  63.7  60.8  1.58  47.6c  56.5b  62.5ab  68.0a  68.9a  63.4ab  68.9a  2.31  0.23  <0.001  0.70  Propionate, mM  54.1  46.6  2.93  35.2c  39.7c  48.1b  55.5ab  58.6a  54.6ab  60.9a  3.08  0.093  <0.001  0.52  Butyrate, mM  15.8  14.7  1.89  11.9ab  13.0ab  15.2b  16.5ab  17.4a  15.8ab  17.1ab  1.62  0.71  0.015  0.53  Osmolality, mOsm/kg  380a  358b  6.6  321c  350b  369ab  384a  390a  376ab  391a  7.3  0.038  <0.001  0.42  a,b,cMeans within a dependent variable differ (P ≤ 0.05). The Tukey post hoc mean separation test was used for mean separation. 1BASE = the baseline measurement period consisted of 8 d on the final diet before dietary restriction and the acidosis challenge. Day 5 was the day of the temporarily isolated and washed reticulorumen technique. Due to manipulation of the rumen, DMI and pH data were not included. 2Forage:concentrate ratio = 9:91. 3T = treatment. View Large No interactions between treatment and hour of sampling were observed for ruminal SCFA concentration or osmolality during BASE (Table 5). The concentration of SCFA and ruminal fluid osmolality were all affected by time such that the greatest concentrations and osmolality were generally observed 8 to 12 h postfeeding. Dry Matter Intake and Ruminal Fermentation during an Induced Bout of Ruminal Acidosis The impact of the duration of dietary adaptation on the susceptibility to an induced bout of acidosis was investigated solely using data within CHAL (Table 6). As with BASE, there was no effect of treatment on DMI, pH variables, SCFA concentrations, lactate concentration, or osmolality during CHAL (P ≥ 0.44; Table 6). Table 6. Mean DMI, ruminal pH, short-chain fatty acid (SCFA) concentration, and osmolality variables on the challenge day. Long-adapted (LA) heifers were fed the high-grain diet1 for 34 d compared to 8 d for the short-adapted (SA) heifers.   Treatment    Hour relative to feeding    P value2  Item  LA  SA  SEM  0  2  4  6  8  10  12  16  20  24  SEM  T  h  T × h  DMI, kg  11  10.1  0.82  –  –  –  –  –  –  –  –  –  –    0.47  –  –  Minimum pH  4.6  4.58  0.134  –  –  –  –  –  –  –  –  –  –    0.9  –  –  Mean pH  5.3  5.31  0.095  –  –  –  –  –  –  –  –  –  –    0.94  –  –  Maximum pH  6.95  6.96  0.066  –  –  –  –  –  –  –  –  –  –    0.93  –  –  Ruminal pH < 5.5      Duration, min/d  951  1,013  58.6  –  –  –  –  –  –  –  –  –  –    0.51  –  –      Area, (min × pH)/d  583  545  123.1  –  –  –  –  –  –  –  –  –  –    0.85  –  –      Total SCFA, mM  121.6  122.7  6.71  47.8e  88.3d  123.5c  149.9ab  156.3a  136.6bc  136.5abc  135.7abc  130.4abc  116.2bcd  7.43  0.91  <0.001  0.34      Acetate, mM  61.6  63.1  2.66  28.4g  51.7f  66.4cde  74.8ab  74.8ac  66.0bde  67.9abcd  69.0abcd  66.0abcdf  59.1df  3.24  0.7  <0.001  0.39      Propionate, mM  45.7  43.4  4.77  12.8e  25.5d  42.3c  54.1ab  59.1a  51.7bc  51.1abc  52.1abc  51.4abc  45.3abc  3.95  0.74  <0.001  0.17      Butyrate, mM  14.2  16.3  1.82  6.4d  11.1c  14.8bc  20.8ab  22.3a  18.8ab  17.6abc  14.8bc  13.6bcd  12.2bc  1.75  0.44  <0.001  0.41      Lactate, mM  12.7  11.9  5.22  ND4  5.3bc  3.6bc  10.0b  19.0a  20.5abc  21.4abc  22.4abc  8.2abc  0.7c  4.29  0.92  0.027  0.53      Osmolality, mOsm/kg  376  376  6.0  285d  325c  367b  404a  414a  386ab  388ab  416a  398ab  375ab  9.8  0.98  <0.001  0.075    Treatment    Hour relative to feeding    P value2  Item  LA  SA  SEM  0  2  4  6  8  10  12  16  20  24  SEM  T  h  T × h  DMI, kg  11  10.1  0.82  –  –  –  –  –  –  –  –  –  –    0.47  –  –  Minimum pH  4.6  4.58  0.134  –  –  –  –  –  –  –  –  –  –    0.9  –  –  Mean pH  5.3  5.31  0.095  –  –  –  –  –  –  –  –  –  –    0.94  –  –  Maximum pH  6.95  6.96  0.066  –  –  –  –  –  –  –  –  –  –    0.93  –  –  Ruminal pH < 5.5      Duration, min/d  951  1,013  58.6  –  –  –  –  –  –  –  –  –  –    0.51  –  –      Area, (min × pH)/d  583  545  123.1  –  –  –  –  –  –  –  –  –  –    0.85  –  –      Total SCFA, mM  121.6  122.7  6.71  47.8e  88.3d  123.5c  149.9ab  156.3a  136.6bc  136.5abc  135.7abc  130.4abc  116.2bcd  7.43  0.91  <0.001  0.34      Acetate, mM  61.6  63.1  2.66  28.4g  51.7f  66.4cde  74.8ab  74.8ac  66.0bde  67.9abcd  69.0abcd  66.0abcdf  59.1df  3.24  0.7  <0.001  0.39      Propionate, mM  45.7  43.4  4.77  12.8e  25.5d  42.3c  54.1ab  59.1a  51.7bc  51.1abc  52.1abc  51.4abc  45.3abc  3.95  0.74  <0.001  0.17      Butyrate, mM  14.2  16.3  1.82  6.4d  11.1c  14.8bc  20.8ab  22.3a  18.8ab  17.6abc  14.8bc  13.6bcd  12.2bc  1.75  0.44  <0.001  0.41      Lactate, mM  12.7  11.9  5.22  ND4  5.3bc  3.6bc  10.0b  19.0a  20.5abc  21.4abc  22.4abc  8.2abc  0.7c  4.29  0.92  0.027  0.53      Osmolality, mOsm/kg  376  376  6.0  285d  325c  367b  404a  414a  386ab  388ab  416a  398ab  375ab  9.8  0.98  <0.001  0.075  a,b,c,d,e,fMeans within a dependent variable differ (P ≤ 0.05). The Tukey post hoc mean separation test was used for mean separation. 1Forage:concentrate ratio = 9:91. 2T = treatment. View Large Table 6. Mean DMI, ruminal pH, short-chain fatty acid (SCFA) concentration, and osmolality variables on the challenge day. Long-adapted (LA) heifers were fed the high-grain diet1 for 34 d compared to 8 d for the short-adapted (SA) heifers.   Treatment    Hour relative to feeding    P value2  Item  LA  SA  SEM  0  2  4  6  8  10  12  16  20  24  SEM  T  h  T × h  DMI, kg  11  10.1  0.82  –  –  –  –  –  –  –  –  –  –    0.47  –  –  Minimum pH  4.6  4.58  0.134  –  –  –  –  –  –  –  –  –  –    0.9  –  –  Mean pH  5.3  5.31  0.095  –  –  –  –  –  –  –  –  –  –    0.94  –  –  Maximum pH  6.95  6.96  0.066  –  –  –  –  –  –  –  –  –  –    0.93  –  –  Ruminal pH < 5.5      Duration, min/d  951  1,013  58.6  –  –  –  –  –  –  –  –  –  –    0.51  –  –      Area, (min × pH)/d  583  545  123.1  –  –  –  –  –  –  –  –  –  –    0.85  –  –      Total SCFA, mM  121.6  122.7  6.71  47.8e  88.3d  123.5c  149.9ab  156.3a  136.6bc  136.5abc  135.7abc  130.4abc  116.2bcd  7.43  0.91  <0.001  0.34      Acetate, mM  61.6  63.1  2.66  28.4g  51.7f  66.4cde  74.8ab  74.8ac  66.0bde  67.9abcd  69.0abcd  66.0abcdf  59.1df  3.24  0.7  <0.001  0.39      Propionate, mM  45.7  43.4  4.77  12.8e  25.5d  42.3c  54.1ab  59.1a  51.7bc  51.1abc  52.1abc  51.4abc  45.3abc  3.95  0.74  <0.001  0.17      Butyrate, mM  14.2  16.3  1.82  6.4d  11.1c  14.8bc  20.8ab  22.3a  18.8ab  17.6abc  14.8bc  13.6bcd  12.2bc  1.75  0.44  <0.001  0.41      Lactate, mM  12.7  11.9  5.22  ND4  5.3bc  3.6bc  10.0b  19.0a  20.5abc  21.4abc  22.4abc  8.2abc  0.7c  4.29  0.92  0.027  0.53      Osmolality, mOsm/kg  376  376  6.0  285d  325c  367b  404a  414a  386ab  388ab  416a  398ab  375ab  9.8  0.98  <0.001  0.075    Treatment    Hour relative to feeding    P value2  Item  LA  SA  SEM  0  2  4  6  8  10  12  16  20  24  SEM  T  h  T × h  DMI, kg  11  10.1  0.82  –  –  –  –  –  –  –  –  –  –    0.47  –  –  Minimum pH  4.6  4.58  0.134  –  –  –  –  –  –  –  –  –  –    0.9  –  –  Mean pH  5.3  5.31  0.095  –  –  –  –  –  –  –  –  –  –    0.94  –  –  Maximum pH  6.95  6.96  0.066  –  –  –  –  –  –  –  –  –  –    0.93  –  –  Ruminal pH < 5.5      Duration, min/d  951  1,013  58.6  –  –  –  –  –  –  –  –  –  –    0.51  –  –      Area, (min × pH)/d  583  545  123.1  –  –  –  –  –  –  –  –  –  –    0.85  –  –      Total SCFA, mM  121.6  122.7  6.71  47.8e  88.3d  123.5c  149.9ab  156.3a  136.6bc  136.5abc  135.7abc  130.4abc  116.2bcd  7.43  0.91  <0.001  0.34      Acetate, mM  61.6  63.1  2.66  28.4g  51.7f  66.4cde  74.8ab  74.8ac  66.0bde  67.9abcd  69.0abcd  66.0abcdf  59.1df  3.24  0.7  <0.001  0.39      Propionate, mM  45.7  43.4  4.77  12.8e  25.5d  42.3c  54.1ab  59.1a  51.7bc  51.1abc  52.1abc  51.4abc  45.3abc  3.95  0.74  <0.001  0.17      Butyrate, mM  14.2  16.3  1.82  6.4d  11.1c  14.8bc  20.8ab  22.3a  18.8ab  17.6abc  14.8bc  13.6bcd  12.2bc  1.75  0.44  <0.001  0.41      Lactate, mM  12.7  11.9  5.22  ND4  5.3bc  3.6bc  10.0b  19.0a  20.5abc  21.4abc  22.4abc  8.2abc  0.7c  4.29  0.92  0.027  0.53      Osmolality, mOsm/kg  376  376  6.0  285d  325c  367b  404a  414a  386ab  388ab  416a  398ab  375ab  9.8  0.98  <0.001  0.075  a,b,c,d,e,fMeans within a dependent variable differ (P ≤ 0.05). The Tukey post hoc mean separation test was used for mean separation. 1Forage:concentrate ratio = 9:91. 2T = treatment. View Large However, SCFA concentrations increased from the time of feeding achieving peak concentrations between 6 and 8 h postchallenge (P < 0.001). Ruminal acetate concentrations remained elevated between 6 and 20 h after challenge induction (Table 6; P < 0.001). Ruminal propionate (P < 0.001), butyrate (P < 0.001), total SCFA (P < 0.001), and lactate concentrations (P = 0.027) all peaked at 8 h after challenge induction. Ruminal fluid osmolality reached a maximum at h 6, 8, and 16 following the barley infusion (P < 0.001). The mean and maximum daily ruminal lactate concentrations ranged from 0.3 to 30.5 mM and 1.0 to 76.4 mM, respectively. Dry Matter Intake and Ruminal Fermentation during the First and Second Recovery Periods The effect of treatment and day on DMI and pH after the CHAL was investigated within REC1 (Table 7) to determine whether the duration of time fed a high-concentrate diet influenced the recovery response from an induced bout of ruminal acidosis. There was no effect of treatment on DMI or pH variables during REC1 (P > 0.10). Table 7. Mean daily DMI and ruminal pH during recovery (REC1) after an induced acidosis challenge. Long-adapted (LA) heifers were fed the high-grain diet1 for 34 d compared to 8 d for the short-adapted (SA) heifers.   Treatment    Day of recovery (REC1)2    P value3  Item  LA  SA  SEM  1  3  4  5  6  7  8  SEM  T  D  T × D  DMI, kg  9.1  9.5  0.56  8.0cd  8.6d  8.8bcd  9.9abc  9.5abcd  10.0ab  10.2a  0.48  0.69  0.001  0.38  Minimum pH  5.16  5.25  0.043  5.07  5.26  5.18  5.26  5.28  5.2  5.19  0.064  0.17  0.21  0.088  Mean pH  5.87  5.84  0.039  5.75  5.86  5.78  5.94  5.94  5.91  5.81  0.058  0.64  0.012  0.31  Maximum pH  6.56  6.49  0.053  6.44  6.51  6.52  6.60  6.56  6.53  6.54  0.051  0.39  0.54  1.00  pH < 5.5      Duration, min/d  297  274  56.0  458  281  407  192  167  167  326  75.8  0.78  0.019  0.085      Area, (min × pH)/d  83  71  23.2  178ab  61ab  106a  42b  32ab  44ab  76ab  28.6  0.70  0.018  0.37    Treatment    Day of recovery (REC1)2    P value3  Item  LA  SA  SEM  1  3  4  5  6  7  8  SEM  T  D  T × D  DMI, kg  9.1  9.5  0.56  8.0cd  8.6d  8.8bcd  9.9abc  9.5abcd  10.0ab  10.2a  0.48  0.69  0.001  0.38  Minimum pH  5.16  5.25  0.043  5.07  5.26  5.18  5.26  5.28  5.2  5.19  0.064  0.17  0.21  0.088  Mean pH  5.87  5.84  0.039  5.75  5.86  5.78  5.94  5.94  5.91  5.81  0.058  0.64  0.012  0.31  Maximum pH  6.56  6.49  0.053  6.44  6.51  6.52  6.60  6.56  6.53  6.54  0.051  0.39  0.54  1.00  pH < 5.5      Duration, min/d  297  274  56.0  458  281  407  192  167  167  326  75.8  0.78  0.019  0.085      Area, (min × pH)/d  83  71  23.2  178ab  61ab  106a  42b  32ab  44ab  76ab  28.6  0.70  0.018  0.37  a,b,c,dMeans within a dependent variable differ (P ≤ 0.050). Tukey was used for mean separation. For mean pH and duration of pH < 5.5, mean separation was not achieved despite a significant effect of day. 1Forage:concentrate ratio = 9:91 2REC1 = recovery 1 measurement period that started 24 h after the acidosis challenge and consisted of 8 d. Day 2 was the day of the temporarily isolated and washed reticulorumen technique. Due to manipulation of the rumen, DMI and pH data were not included. 3T = treatment; D = day of recovery. View Large Table 7. Mean daily DMI and ruminal pH during recovery (REC1) after an induced acidosis challenge. Long-adapted (LA) heifers were fed the high-grain diet1 for 34 d compared to 8 d for the short-adapted (SA) heifers.   Treatment    Day of recovery (REC1)2    P value3  Item  LA  SA  SEM  1  3  4  5  6  7  8  SEM  T  D  T × D  DMI, kg  9.1  9.5  0.56  8.0cd  8.6d  8.8bcd  9.9abc  9.5abcd  10.0ab  10.2a  0.48  0.69  0.001  0.38  Minimum pH  5.16  5.25  0.043  5.07  5.26  5.18  5.26  5.28  5.2  5.19  0.064  0.17  0.21  0.088  Mean pH  5.87  5.84  0.039  5.75  5.86  5.78  5.94  5.94  5.91  5.81  0.058  0.64  0.012  0.31  Maximum pH  6.56  6.49  0.053  6.44  6.51  6.52  6.60  6.56  6.53  6.54  0.051  0.39  0.54  1.00  pH < 5.5      Duration, min/d  297  274  56.0  458  281  407  192  167  167  326  75.8  0.78  0.019  0.085      Area, (min × pH)/d  83  71  23.2  178ab  61ab  106a  42b  32ab  44ab  76ab  28.6  0.70  0.018  0.37    Treatment    Day of recovery (REC1)2    P value3  Item  LA  SA  SEM  1  3  4  5  6  7  8  SEM  T  D  T × D  DMI, kg  9.1  9.5  0.56  8.0cd  8.6d  8.8bcd  9.9abc  9.5abcd  10.0ab  10.2a  0.48  0.69  0.001  0.38  Minimum pH  5.16  5.25  0.043  5.07  5.26  5.18  5.26  5.28  5.2  5.19  0.064  0.17  0.21  0.088  Mean pH  5.87  5.84  0.039  5.75  5.86  5.78  5.94  5.94  5.91  5.81  0.058  0.64  0.012  0.31  Maximum pH  6.56  6.49  0.053  6.44  6.51  6.52  6.60  6.56  6.53  6.54  0.051  0.39  0.54  1.00  pH < 5.5      Duration, min/d  297  274  56.0  458  281  407  192  167  167  326  75.8  0.78  0.019  0.085      Area, (min × pH)/d  83  71  23.2  178ab  61ab  106a  42b  32ab  44ab  76ab  28.6  0.70  0.018  0.37  a,b,c,dMeans within a dependent variable differ (P ≤ 0.050). Tukey was used for mean separation. For mean pH and duration of pH < 5.5, mean separation was not achieved despite a significant effect of day. 1Forage:concentrate ratio = 9:91 2REC1 = recovery 1 measurement period that started 24 h after the acidosis challenge and consisted of 8 d. Day 2 was the day of the temporarily isolated and washed reticulorumen technique. Due to manipulation of the rumen, DMI and pH data were not included. 3T = treatment; D = day of recovery. View Large Dry matter intake increased linearly (P < 0.001; data not shown) from d 1 to d 8, culminating in the greatest value on d 8 (P = 0.001; Table 7). A tendency for a treatment × day interaction for minimum pH (P = 0.088) indicated that minimum pH tended to increase to a greater extent between d 1 and d 3 for LA cattle but reached a greater value for SA cattle on d 6 and 7 (data not shown). Although there was no effect of day for maximum pH, mean pH reached its greatest values on d 5 and 6 (P = 0.012) while minimum values for DUR (P = 0.019) and AREA (P = 0.018) were observed on d 7 and 5, respectively. A tendency for a treatment × day interaction for DUR (P = 0.085) indicated that DUR tended to decrease between d 1 and d 3 for LA cattle and instead increased from d 1 to d 4 for SA cattle (data not shown). Ruminal SCFA concentrations were not affected by treatment or the treatment × hour interaction during REC1 (Table 8). However, SCFA concentration and osmolality increased over time after feeding with peak concentrations observed between 6 and 12 h postfeeding. Ruminal osmolality reached a peak at 6 and 12 h postfeeding. Table 8. Ruminal short-chain fatty acid (SCFA) and osmolality during recovery (REC1) after an induced acidosis challenge. Long-adapted (LA) heifers were fed the high-grain diet1 for 34 d compared to 8 d for the short-adapted (SA) heifers.   Treatment    Hour relative to feeding    P value2  Item  LA  SA  SEM  0  2  4  6  8  10  12  SEM  T  h  T × h  Total SCFA, mM  120.8  124.6  3.15  104.2c  117.7b  126.0ab  130.5ab  122.6b  120.6b  137.2a  3.80  0.41  <0.001  0.63  Acetate, mM  64  63  1.3  54.7c  62.7ab  65.5ab  67.3ab  62.6ab  62.2bc  69.3a  1.77  0.58  <0.001  0.65  Propionate, mM  42.4  45.9  5.17  37.5d  41.2bde  45.7ac  47.0abc  44.8abcd  42.9cde  49.9ab  3.95  0.64  <0.001  0.35  Butyrate, mM  14.4  15.9  2.21  12.0bd  13.9abc  14.9abc  16.3ac  15.4abc  15.6cd  18.0ab  1.77  0.64  <0.001  0.97  Osmolality, mOsm/kg  368  372  7.3  344b  368ab  373ab  383a  367ab  369ab  387a  8.4  0.73  0.002  0.68    Treatment    Hour relative to feeding    P value2  Item  LA  SA  SEM  0  2  4  6  8  10  12  SEM  T  h  T × h  Total SCFA, mM  120.8  124.6  3.15  104.2c  117.7b  126.0ab  130.5ab  122.6b  120.6b  137.2a  3.80  0.41  <0.001  0.63  Acetate, mM  64  63  1.3  54.7c  62.7ab  65.5ab  67.3ab  62.6ab  62.2bc  69.3a  1.77  0.58  <0.001  0.65  Propionate, mM  42.4  45.9  5.17  37.5d  41.2bde  45.7ac  47.0abc  44.8abcd  42.9cde  49.9ab  3.95  0.64  <0.001  0.35  Butyrate, mM  14.4  15.9  2.21  12.0bd  13.9abc  14.9abc  16.3ac  15.4abc  15.6cd  18.0ab  1.77  0.64  <0.001  0.97  Osmolality, mOsm/kg  368  372  7.3  344b  368ab  373ab  383a  367ab  369ab  387a  8.4  0.73  0.002  0.68  a,b,c,dMeans within a dependent variable differ (P ≤ 0.050). The Tukey post hoc test was used for mean separation. 1Forage:concentrate ratio = 9:91. 2T = treatment. View Large Table 8. Ruminal short-chain fatty acid (SCFA) and osmolality during recovery (REC1) after an induced acidosis challenge. Long-adapted (LA) heifers were fed the high-grain diet1 for 34 d compared to 8 d for the short-adapted (SA) heifers.   Treatment    Hour relative to feeding    P value2  Item  LA  SA  SEM  0  2  4  6  8  10  12  SEM  T  h  T × h  Total SCFA, mM  120.8  124.6  3.15  104.2c  117.7b  126.0ab  130.5ab  122.6b  120.6b  137.2a  3.80  0.41  <0.001  0.63  Acetate, mM  64  63  1.3  54.7c  62.7ab  65.5ab  67.3ab  62.6ab  62.2bc  69.3a  1.77  0.58  <0.001  0.65  Propionate, mM  42.4  45.9  5.17  37.5d  41.2bde  45.7ac  47.0abc  44.8abcd  42.9cde  49.9ab  3.95  0.64  <0.001  0.35  Butyrate, mM  14.4  15.9  2.21  12.0bd  13.9abc  14.9abc  16.3ac  15.4abc  15.6cd  18.0ab  1.77  0.64  <0.001  0.97  Osmolality, mOsm/kg  368  372  7.3  344b  368ab  373ab  383a  367ab  369ab  387a  8.4  0.73  0.002  0.68    Treatment    Hour relative to feeding    P value2  Item  LA  SA  SEM  0  2  4  6  8  10  12  SEM  T  h  T × h  Total SCFA, mM  120.8  124.6  3.15  104.2c  117.7b  126.0ab  130.5ab  122.6b  120.6b  137.2a  3.80  0.41  <0.001  0.63  Acetate, mM  64  63  1.3  54.7c  62.7ab  65.5ab  67.3ab  62.6ab  62.2bc  69.3a  1.77  0.58  <0.001  0.65  Propionate, mM  42.4  45.9  5.17  37.5d  41.2bde  45.7ac  47.0abc  44.8abcd  42.9cde  49.9ab  3.95  0.64  <0.001  0.35  Butyrate, mM  14.4  15.9  2.21  12.0bd  13.9abc  14.9abc  16.3ac  15.4abc  15.6cd  18.0ab  1.77  0.64  <0.001  0.97  Osmolality, mOsm/kg  368  372  7.3  344b  368ab  373ab  383a  367ab  369ab  387a  8.4  0.73  0.002  0.68  a,b,c,dMeans within a dependent variable differ (P ≤ 0.050). The Tukey post hoc test was used for mean separation. 1Forage:concentrate ratio = 9:91. 2T = treatment. View Large Data for REC2 is not shown as there were no treatment × day or treatment effects and mean values for REC2 did not differ from REC1 (Table 3). Regression Analysis Detects Differences in the Recovery Response after Ruminal Acidosis but not for the Susceptibility to Ruminal Acidosis Although treatment differences were not detected within BASE or CHAL, regression analysis was used to comprehensively evaluate the effect of treatment on the slope of the responses for the DUR before and leading into the CHAL and during REC1. Regression analysis, accounting for repeated measures, is a more appropriate method of detecting a change in a variable over time than mean separation. From Fig. 2, it is evident that over the 9 d BASE and CHAL, DUR was more variable in SA than LA heifers among days. The fitted data indicated significant treatment differences between the y-intercepts (P = 0.047), linear slopes (P = 0.007), quadratic coefficients (P = 0.003), and cubic coefficients (P = 0.001) with trend line equations of y = 567.4 + 48.7 d – 23.1 d2 + 2.2 d3 for LA and y = –173.6 + 653.1 d – 150.5 d2 + 9.8 d3 for SA. In support of this observed increase in between-day variability in pH, SA heifers also tended to have a greater SD in the duration of time that pH was <5.5 over the 8 d considered in this analysis when compared to LA heifers (data not shown; P = 0.096). The negative intercept observed for SA heifers is likely a response arising from being fed the high-grain diet for fewer days (i.e., d 1 was the first day SA heifers were fed the high-grain diet) and greater pH when fed the previous diet. Figure 2. View largeDownload slide Change in the duration of time that pH < 5.5 over the days leading up to and including the induced challenge. Long-adapted (LA) heifers were fed the high-grain diet for 26 d before this measurement period; short-adapted (SA) heifers received the high-grain diet on d 1 of the baseline period. Trend lines were constructed using analysis of covariance. Daily means were used from the 7 d during baseline (BASE; d 5 = day where the temporarily isolated and washed reticulo-rumen technique was performed and as a result pH data were removed.) and the challenge day depicted as C in the figure above.. Data from the day of restriction (R) was not included in the analysis. Significant treatment differences were found between y-intercepts (P = 0.047), linear slopes (P = 0.007), quadratic coefficients (P = 0.003), and cubic coefficients (P = 0.001). The resulting equations, therefore, account for a significant amount of variation in the dependant variable and were used in constructing the trend lines: y = 567.4 + 48.7 d – 23.1 d2 + 2.2 d3 (LA) and y = –173.6 + 653.1 d – 150.5 d2 + 9.8 d3 (SA). Figure 2. View largeDownload slide Change in the duration of time that pH < 5.5 over the days leading up to and including the induced challenge. Long-adapted (LA) heifers were fed the high-grain diet for 26 d before this measurement period; short-adapted (SA) heifers received the high-grain diet on d 1 of the baseline period. Trend lines were constructed using analysis of covariance. Daily means were used from the 7 d during baseline (BASE; d 5 = day where the temporarily isolated and washed reticulo-rumen technique was performed and as a result pH data were removed.) and the challenge day depicted as C in the figure above.. Data from the day of restriction (R) was not included in the analysis. Significant treatment differences were found between y-intercepts (P = 0.047), linear slopes (P = 0.007), quadratic coefficients (P = 0.003), and cubic coefficients (P = 0.001). The resulting equations, therefore, account for a significant amount of variation in the dependant variable and were used in constructing the trend lines: y = 567.4 + 48.7 d – 23.1 d2 + 2.2 d3 (LA) and y = –173.6 + 653.1 d – 150.5 d2 + 9.8 d3 (SA). The tendencies observed for the treatment × day interaction for minimum pH (P = 0.088) and DUR (P = 0.085) during REC1 can both be interpreted to suggest that the LA heifers recovered more quickly from the CHAL. During REC1 (Fig. 3) the LA cattle experienced a greater linear reduction in the duration of time that pH was <5.5 over the first few days after the challenge (Fig. 3; –588 vs. 369 min/d; P = 0.019). Figure 3. View largeDownload slide Change in the duration of time that pH < 5.5 over the days following the induced challenge. Long-adapted (LA) heifers were fed the high-grain diet for 34 d compared to 8 d for the short-adapted (SA) heifers. Trend lines were constructed using analysis of covariance. Daily means were used from the first 7 d following the induced challenge (REC1; d 2 = the day that the temporarily isolated and washed reticulo-rumen technique was performed and therefore pH data were removed.). Significant treatment differences were found between y-intercepts (P = 0.048), linear slopes (P = 0.019), quadratic coefficients (P = 0.026), and cubic coefficients (P = 0.037). The resulting equations, therefore, account for a significant amount of variation in the dependant variable and were used in constructing the trend lines: y = 1,176.4 – 587.8 d + 100.9d2 – 5.06 d3 (LA) and y = 162.8 + 369.4 d – 123.7 d2 + 10.0 d3 (SA). Figure 3. View largeDownload slide Change in the duration of time that pH < 5.5 over the days following the induced challenge. Long-adapted (LA) heifers were fed the high-grain diet for 34 d compared to 8 d for the short-adapted (SA) heifers. Trend lines were constructed using analysis of covariance. Daily means were used from the first 7 d following the induced challenge (REC1; d 2 = the day that the temporarily isolated and washed reticulo-rumen technique was performed and therefore pH data were removed.). Significant treatment differences were found between y-intercepts (P = 0.048), linear slopes (P = 0.019), quadratic coefficients (P = 0.026), and cubic coefficients (P = 0.037). The resulting equations, therefore, account for a significant amount of variation in the dependant variable and were used in constructing the trend lines: y = 1,176.4 – 587.8 d + 100.9d2 – 5.06 d3 (LA) and y = 162.8 + 369.4 d – 123.7 d2 + 10.0 d3 (SA). DISCUSSION In North American feedlots, beef cattle are typically fed diets with a high proportion of grain to maximize energy intake and improve performance and feed efficiency (Owens et al., 1997). However, feeding highly fermentable diets increases the risk for ruminal acidosis (Aschenbach et al., 2011) as rates of acid production in the rumen may exceed rates of acid removal (Penner et al., 2009c). While prevalence rates for ruminal acidosis in feedlot cattle are not currently available, several studies using continuous ruminal pH measurement in feedlot cattle indicate that based on a threshold pH value of 5.5, the risk for ruminal acidosis and likelihood of cattle experiencing ruminal acidosis is high (Wierenga et al., 2010; Li et al., 2011; Moya et al., 2011). In addition, acute ruminal acidosis is primarily characterized by an increase in ruminal lactate concentration (Nocek, 1997; Nagaraja and Titgemeyer, 2007; Aschenbach et al., 2011). For these reasons, we used the DUR and AREA that pH was <5.5 to detect ruminal acidosis (both subacute and acute) and ruminal fluid lactate concentrations to detect acute ruminal acidosis. A number of studies have investigated changes in ruminal pH, SCFA, and lactate concentrations that occur in beef cattle subjected to an induced bout of ruminal acidosis (Hibbard et al., 1995; Goad et al., 1998; Brown et al., 2000). However, none of those studies have examined the DUR and AREA of ruminal pH depression in beef cattle following feed restriction and a grain challenge using a continuous indwelling ruminal pH measurement system (Dado and Allen, 1993; Penner et al., 2006, 2009b). Continuous measurement of pH has been widely used in dairy cattle (Krause and Oetzel, 2005; Dohme et al., 2008; Khafipour et al., 2009) and has facilitated the growing knowledge gap between the characterization of ruminal acidosis in dairy cattle (Nocek, 1997; Krause and Oetzel, 2006; Kleen and Cannizzo, 2012) and beef cattle (Huber, 1976; Owens et al., 1998; Nagaraja and Titgemeyer, 2007). As the dietary inclusion rates of cereal grains are much higher in finishing diets for beef cattle relative to diets fed to lactating dairy cattle, it is important that comprehensive measurement approaches are used to improve our understanding of ruminal acidosis in general and to improve our knowledge regarding the severity and prevalence of ruminal acidosis in beef cattle. Acute Ruminal Acidosis was Effectively Induced Our challenge model was successful in decreasing mean and minimum ruminal pH and increasing both the duration and area that pH was <5.5. However, the extent of ruminal pH depression and lactate accumulation was more severe than most previous studies implementing a grain challenge to induce ruminal acidosis in dairy (Krause and Oetzel, 2005; Dohme et al., 2008; Khafipour et al., 2009) and beef (Burrin and Britton, 1986; Goad et al., 1998) cattle. In fact, the challenge in the current study was more in line with a challenge model using ruminal glucose infusion (Harmon et al., 1985) or consecutive challenges (Nagaraja et al., 1985; Coe et al., 1999). The severe challenge induced in the current study was likely the result of low ruminal pH for heifers when fed the basal diet. In fact, the mean duration pH was <5.5 was 531 min/d during BASE. This prolonged duration of pH depression during BASE was greater than the severity of subacute ruminal acidosis induced in dairy cattle by Keunen et al. (2002), Osborne et al. (2004), and Gozho et al. (2007) and was similar in severity to the grain challenge imposed on hay-adapted and grain-adapted steers by Goad et al. (1998) as well as the most severe challenge induced by Dohme et al. (2008) in lactating dairy cattle (9.5 h/d that pH was <5.5). Therefore, it is clear that the heifers in our study were already coping with a substantial acidotic challenge before the induced bout of acidosis. The severity of ruminal acidosis observed during BASE was caused primarily by the high fermentability of the high-grain diet and was similar to the severity of acidosis observed in other studies feeding similar high-grain barley-based finishing diets to beef cattle (Bevans et al., 2005; Wierenga et al., 2010; Moya et al., 2011). This severely depressed pH can greatly increase the risk of an acute bout of ruminal acidosis, as is evident by the severity of our challenge and studies that have investigated multiple challenges as part of an acidosis induction protocol in cattle (Nagaraja et al., 1985; Coe et al., 1999; Dohme et al., 2008). The current findings in combination with past studies underline the importance of continued work with indwelling pH measurement systems and beef cattle fed finishing feedlot diets. It is evident that we induced acute acidosis. The nearly doubling of the DUR during CHAL relative to that during BASE and the observed minimum pH of 4.57 are supported by the high ruminal fluid lactate concentrations. Because the observed minimum pH was below the pKa of SCFA (4.8 to 4.9; Aschenbach et al., 2011), the SCFA would have actually been acting as bases by stabilizing pH to approximately 4.8. However, the observed concomitant increase in lactate concentration (pKa = 3.9; Aschenbach et al., 2011) ensured that pH continued to plummet after maximum SCFA and lactate concentrations were observed. When both SCFA and lactate concentrations peaked at 8 h after the challenge, SCFA concentrations were eightfold greater than lactate concentrations. Over the next 8 h, a shift in organic acid accumulation and presumably production resulted in a decrease in SCFA concentrations while lactate concentration remained elevated. However, the concentration of SCFA in the ruminal fluid remained sixfold greater than lactate at 16 h after the challenge. While for subacute ruminal acidosis, high production rates and dissociation of SCFA is clearly the driving factor for low ruminal pH, the observed response is an example of when ruminal lactate becomes a more effective organic acid at reducing ruminal pH than the more abundant SCFA. Duration of Time Cattle are Fed the High-Grain Diet Influences Variability in Ruminal pH One major challenge with feeding cattle high-grain diets is the variation in the ruminal pH response among cattle, even when fed the same diet (Brown et al., 2000; Bevans et al., 2005; Nagaraja and Titgemeyer, 2007). Leading up to and including the challenge, we observed that SA heifers had greater variability for DUR across days within BASE (based on the quadratic and cubic regression coefficients), suggesting that the short duration on the high-grain diet may have increased the between-day variability in ruminal pH buffering, SCFA production, or eating behavior. Greater day-to-day variation in ruminal pH for the SA cattle was observed despite a lack of a treatment effect on the between-day rate of change for DMI. Bevans et al. (2005) reported that dramatic differences in the variation in ruminal pH occurred in feedlot cattle during a dietary transition protocol to a high-grain diet. They also demonstrated that accelerating the rate of dietary transition to a high-grain finishing diet increased the variability in the ruminal pH response without affecting daily DMI. The decreased pH variability for LA relative to SA heifers observed in our study and that of Bevans et al. (2005) for cattle that were provided a gradual dietary adaptation suggests that extending the time on feed may improve the ability to resist dramatic changes in ruminal pH that are inherent when consuming a high-grain diet. However, the day-to-day variation in pH does not appear to be linked to similar variation in DMI indicating that ruminal acid production, buffering strategies, or meal patterns and amounts may differ for cattle exposed to SA and LA. This finding also presents a new opportunity to assess the between day variability in ruminal pH as an appropriate indicator for cattle experiencing ruminal acidosis. Increasing the Duration of Time Fed a High Grain Diet does not Affect Risk for Ruminal Acidosis but Hastens the Recovery Response While numerous studies have investigated ruminal acidosis (Dohme et al., 2008; Khafipour et al., 2009; Penner et al., 2009a), few have evaluated risk for ruminal acidosis as affected by dietary adaptation or the recovery response following an episode of ruminal acidosis (Gaebel and Martens, 1988; Krehbiel et al., 1995; Zhang et al., 2013). Our results clearly show that while variation in DUR was greater for SA than LA, the severity of the induced acidosis did not differ by treatment. This indicates that the duration of time for adaptation to the high-grain diet does not influence the risk for ruminal acidosis. Based on the recovery pattern following the induced bout of acidosis, LA cattle experienced their lowest daily DUR after approximately 4 d while it took the SA cattle approximately 6 d to experience their lowest daily DUR. The mean daily DUR was 294 min/d when averaged over both treatment groups; however, it took the LA heifers 3 d to reduce the DUR below 294 min/d while SA heifers required 5 d. The reduced amount of time required for the LA heifers to reach mean and minimum values for DUR during the first week of recovery from the induced challenge can be appropriately quantified by their greater linear decrease for DUR during this time (Fig. 3). This is the first study, known to the authors, to suggest that time on high-grain feed may decrease the time necessary for beef cattle to recover from an induced bout of acute ruminal acidosis. Epithelial adaptation to high-grain diets (Thorlacius and Lodge, 1973; Gäbel et al., 1991; Sehested et al., 2000) has been suggested to increase the buffering capacity of the rumen. Therefore, increased rates of SCFA absorption with advancing days on the high-grain diet offers one potential explanation for the observed reduced recovery time in LA cattle, and is addressed in our companion study (Schwaiger et al., 2013). In addition, it is also possible that advancing days on high-grain feed may have stabilized the microbial communities (Mackie et al., 1978; Mackie and Gilchrist, 1979; Counotte et al., 1981) or altered eating behavior. For example, the populations of lactate-utilizing bacteria may have increased to help prevent lactate accumulation during the days following CHAL (Goad et al., 1998); however, we did not evaluate whether microbial composition differed among treatments and over time and therefore cannot confirm or refute this mechanism. It was recently reported by Zhang et al. (2013) that the severity of feed restriction influences the rate of recovery for DMI and ruminal pH when beef cattle resume ad libitum consumption of a diet containing 40% concentrate. In that study, heifers that were subjected to an imposed feed restriction experienced elevated DUR and AREA when ad libitum feeding resumed. The recovery, with respect to DMI and ruminal pH, from the feed restriction challenge imposed by Zhang et al. (2013) was complete by the second week of ad libitum feeding and was greatest for those cattle who were subjected to the greatest severity of imposed feed restriction. The challenge induced in the current study was much more severe than that of Zhang et al. (2013), as was the time necessary for pH to stabilize. Taken together with the results of the current study, it can be hypothesized that the severity of the ruminal acidosis bout may positively correlate to the number of days required for recovery of ruminal pH and that cattle exposed to more time for adaptation before acute ruminal acidosis may have a greater ability to recover following a bout of ruminal acidosis. Conclusions Although the severity of acidosis was equivalent across treatments, heifers that spent more time on a high-concentrate diet before the bout of imposed ruminal acidosis exhibited decreased between-day variation in ruminal pH (based on the quadratic and cubic regression coefficients) without differences in DMI. However, the observed increased stability in ruminal pH leading up to the challenge did not influence the susceptibility to a bout of induced ruminal acidosis. 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Google Scholar CrossRef Search ADS PubMed  Footnotes 1 Funding for the project was provided through the Alberta Livestock and Meat Agency and Agriculture and Agri-Food Canada. American Society of Animal Science http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Animal Science Oxford University Press

The duration of time that beef cattle are fed a high-grain diet affects the recovery from a bout of ruminal acidosis: Dry matter intake and ruminal fermentation

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0021-8812
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1525-3163
DOI
10.2527/jas.2013-6471
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24158369
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Abstract

Abstract This study was conducted to determine if the duration of time cattle are fed a high-grain diet affects their susceptibility to and recovery from ruminal acidosis. Sixteen Angus heifers (BW ± SEM, 261 ± 6.1 kg) were assigned to 1 of 4 blocks and fed a backgrounding diet consisting of 60% barley silage, 30% barley grain, and 10% supplement (DM basis). Within block, cattle were randomly assigned to 1 of 2 treatments differing in the number of days they were fed the high-grain diet before an acidosis challenge: 34 d for long adapted (LA) and 8 d for short adapted (SA). All heifers were exposed to the same 20 d dietary transition to a high-grain diet containing 9% barley silage, 81% barley grain, and 10% supplement (DM basis). 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). Acidosis induction increased daily duration (531 to 1,020 min/d; P < 0.001) and area (176 to 595 (min × pH)/d; P < 0.001) that ruminal pH was <5.5 relative to BASE. Relative to BASE, inducing acidosis also increased the daily mean (0.3 to 11.4 mM; P = 0.013) and maximum (1.3 to 29.3 mM; P = 0.008) ruminal fluid lactate concentrations. There was no effect of dietary treatment on ruminal pH, lactate, or short-chain fatty acid (SCFA) concentrations (P > 0.050). However, during BASE and CHAL, SA heifers experienced greater linear (P = 0.031), quadratic (P = 0.016), and cubic (P = 0.008) coefficients for the duration of time that pH was <5.5. In addition, a treatment × day interaction for the duration that pH was <5.5 during REC1 suggested that LA cattle tended to recover from the challenge more rapidly than SA cattle (P = 0.085). Regression analysis confirmed that the LA heifers experienced a quicker linear (P = 0.019) recovery from induced acidosis over time. These results indicate adaptation of the ruminal epithelium continues with advancing time as evidenced by more stable ruminal pH both before and after an induced bout of acute ruminal acidosis but does not affect susceptibility of cattle to ruminal acidosis. INTRODUCTION Ruminal acidosis is thought to be the most prevalent digestive disorder in feedlot cattle (Nagaraja and Titgemeyer, 2007) and is associated with an increase in ruminal short-chain fatty acid (SCFA) production (Sutton et al., 2003; Loncke et al., 2009) and concentration (Penner et al., 2009a,c) and a decrease in ruminal pH (Penner et al., 2007). These effects have been associated with decreased energy intake (Fulton et al., 1979a,b; Brown et al., 2000) and growth and increased incidence of liver abscesses (Brent, 1976; Nagaraja and Chengappa, 1998). Additionally, exposure of the ruminal epithelium to low ruminal pH (<5.5) has been shown to decrease SCFA absorption (Gaebel et al., 1989; Wilson et al., 2012) and barrier function (Aschenbach and Gäbel, 2000; Penner et al., 2010; Wilson et al., 2012). Gradually transitioning cattle to high-grain diets may reduce the risk for ruminal acidosis (Bevans et al., 2005) by providing sufficient time for behavioral, microbial, and epithelial adaptation. Adaptations that increase SCFA absorption should correspond to lower risk for ruminal acidosis (Gäbel et al., 2002; Penner et al., 2009a); however, few studies have evaluated adaptation of the ruminal epithelium in beef cattle. Reported timelines for ruminal epithelial adaptation range between 7 d for initial increases in functional activity (Etschmann et al., 2009) to 42 d for measurable increases in surface area (Dirksen et al., 1985). This range suggests that even with a gradual dietary transition, the ruminal adaptive process may not be complete and that ruminal adaptation may proceed with advancing days on feed. Therefore, it was hypothesized that increasing the amount of time cattle are fed a high-grain diet would decrease the susceptibility to and the time required to recover from a bout of ruminal acidosis. The objective of the study was to determine if the duration of time that cattle are fed a high-grain diet influences their susceptibility to and recovery from a bout of ruminal acidosis. MATERIALS AND METHODS This manuscript evaluates whether the duration of time cattle are fed high-grain diets improves the resistance to and recovery from an induced bout of ruminal acidosis with a focus on DMI, ruminal SCFA concentrations, and ruminal pH. A companion paper (Schwaiger et al., 2013) reports the results obtained for SCFA and lactate absorption, saliva production, and blood metabolites. The procedures and heifers used in this study were preapproved by the Animal Care Committee of the Agriculture and Agri-Food Canada Lethbridge Research Centre (Lethbridge, AB, Canada) and the study was conducted according to the guidelines of the Canadian Council on Animal Care (Ottawa, ON, Canada). Unless otherwise stated, all analyses were conducted in triplicate and reanalyzed when the CV was >3.0%. Animals, Diets, and Experimental Design Sixteen ruminally cannulated Angus heifers were used in this study. Each heifer was fitted with a 10-cm ruminal cannula (model 9C; Bar Diamond Inc., Parma, ID) at approximately 9 mo of age and provided at least 5 wk for recovery following the surgery before the start of the study. The surgical procedure for ruminal cannulation was conducted according to Bar Diamond Inc. (2011) and at the time of surgery, the mean BW ± SEM was 261 ± 6.1 kg. Heifers were housed in tie-stalls and were provided exercise daily. Before the start of the study, heifers were transitioned from a high-forage diet [forage-to-concentrate ratio (F:C) of 95:5] to a diet with a F:C of 60:40, hereafter designated as the backgrounding diet (Table 1). The dietary transition from the high-forage diet to the backgrounding diet occurred over 7 d using 3 intermediate diets that all contained 10% supplement (DM basis). During the first 2 d heifers were fed 30% barley silage, 50% grass hay, 10% barley grain, and 10% supplement; for the following 3 d heifers were fed 40% barley silage, 30% grass hay, 20% barley grain, and 10% supplement; and during the last 2 d heifers were fed 55% barley silage, 10% grass hay, 25% barley grain, and 10% supplement (DM basis). Table 1. Dietary transition protocol, ingredient inclusion rates, and the chemical composition of diets fed to heifers provided a long adaptation (LA) or short adaption (SA) to the high-grain diet before the acidosis induction protocol   Experimental diet  Item  BG1  Step 1  Step 2  Step 3  Step 4  Step 5  HG2  Time on each diet, d      LA  7  4  4  4  4  4  34      SA  33  4  4  4  4  4  8  Ingredients, g/kg DM      Barley silage  600  500  400  300  200  130  90      Barley grain3  300  400  500  600  700  770  810      Supplement4  100  100  100  100  100  100  100  Chemical composition,5 g/kg ± SEM      n  8  4  4  4  4  4  16      DM  468 ± 7.4  486 ± 11.7  538 ± 11.5  597 ± 8.4  660 ± 7.9  732 ± 4.3  777 ± 4.3      CP  138 ± 1.0  134 ± 1.0  133 ± 1.5  135 ± 2.8  130 ± 2.8  131 ± 3.1  134 ± 1.8      NDF  377 ± 8.3  363 ± 7.4  330 ± 3.5  301 ± 3.5  273 ± 9.1  251 ± 7.9  248 ± 8.6      ADF  208 ± 4.3  192 ± 4.6  166 ± 2.5  140 ± 2.4  116 ± 1.4  107 ± 3.3  91 ± 1.0      Starch  320 ± 9.0  357 ± 3.0  389 ± 8.6  437 ± 12.5  478 ± 5.2  501 ± 13.3  510 ± 5.2    Experimental diet  Item  BG1  Step 1  Step 2  Step 3  Step 4  Step 5  HG2  Time on each diet, d      LA  7  4  4  4  4  4  34      SA  33  4  4  4  4  4  8  Ingredients, g/kg DM      Barley silage  600  500  400  300  200  130  90      Barley grain3  300  400  500  600  700  770  810      Supplement4  100  100  100  100  100  100  100  Chemical composition,5 g/kg ± SEM      n  8  4  4  4  4  4  16      DM  468 ± 7.4  486 ± 11.7  538 ± 11.5  597 ± 8.4  660 ± 7.9  732 ± 4.3  777 ± 4.3      CP  138 ± 1.0  134 ± 1.0  133 ± 1.5  135 ± 2.8  130 ± 2.8  131 ± 3.1  134 ± 1.8      NDF  377 ± 8.3  363 ± 7.4  330 ± 3.5  301 ± 3.5  273 ± 9.1  251 ± 7.9  248 ± 8.6      ADF  208 ± 4.3  192 ± 4.6  166 ± 2.5  140 ± 2.4  116 ± 1.4  107 ± 3.3  91 ± 1.0      Starch  320 ± 9.0  357 ± 3.0  389 ± 8.6  437 ± 12.5  478 ± 5.2  501 ± 13.3  510 ± 5.2  1BG = backgrounding diet. 2HG = high-grain finishing diet. 3Barley grain processing index (volume weight after processing expressed as a percentage of volume weight before processing, DM basis) was 82.8 ± 0.25%. 4Supplement prepared as a blend and contained (DM basis): 50.0% beet pulp, 33.0% canola meal, 12.0% calcium carbonate, 2.5% urea, 1.6% salt, 0.5% vitamin and mineral mix, 0.3% melengestrol acetate (200 mg/kg), and 0.1% molasses. The concentration of minerals and vitamins in the supplement for the high-grain diet (DM basis) were zinc sulfate monohydrate (55.7 mg/kg), copper sulfate pentahydrate (14.2 mg/kg), manganese sulfate monohydrate (25.6 mg/kg), Ethylene diamine dihydriodide (0.6 mg/kg), selenium (0.3 mg/kg), vitamin A (9,281 IU/kg), vitamin D3 (464 IU/kg), and vitamin E (13 IU/kg). 5All analysis except for DM are reported on a DM basis. View Large Table 1. Dietary transition protocol, ingredient inclusion rates, and the chemical composition of diets fed to heifers provided a long adaptation (LA) or short adaption (SA) to the high-grain diet before the acidosis induction protocol   Experimental diet  Item  BG1  Step 1  Step 2  Step 3  Step 4  Step 5  HG2  Time on each diet, d      LA  7  4  4  4  4  4  34      SA  33  4  4  4  4  4  8  Ingredients, g/kg DM      Barley silage  600  500  400  300  200  130  90      Barley grain3  300  400  500  600  700  770  810      Supplement4  100  100  100  100  100  100  100  Chemical composition,5 g/kg ± SEM      n  8  4  4  4  4  4  16      DM  468 ± 7.4  486 ± 11.7  538 ± 11.5  597 ± 8.4  660 ± 7.9  732 ± 4.3  777 ± 4.3      CP  138 ± 1.0  134 ± 1.0  133 ± 1.5  135 ± 2.8  130 ± 2.8  131 ± 3.1  134 ± 1.8      NDF  377 ± 8.3  363 ± 7.4  330 ± 3.5  301 ± 3.5  273 ± 9.1  251 ± 7.9  248 ± 8.6      ADF  208 ± 4.3  192 ± 4.6  166 ± 2.5  140 ± 2.4  116 ± 1.4  107 ± 3.3  91 ± 1.0      Starch  320 ± 9.0  357 ± 3.0  389 ± 8.6  437 ± 12.5  478 ± 5.2  501 ± 13.3  510 ± 5.2    Experimental diet  Item  BG1  Step 1  Step 2  Step 3  Step 4  Step 5  HG2  Time on each diet, d      LA  7  4  4  4  4  4  34      SA  33  4  4  4  4  4  8  Ingredients, g/kg DM      Barley silage  600  500  400  300  200  130  90      Barley grain3  300  400  500  600  700  770  810      Supplement4  100  100  100  100  100  100  100  Chemical composition,5 g/kg ± SEM      n  8  4  4  4  4  4  16      DM  468 ± 7.4  486 ± 11.7  538 ± 11.5  597 ± 8.4  660 ± 7.9  732 ± 4.3  777 ± 4.3      CP  138 ± 1.0  134 ± 1.0  133 ± 1.5  135 ± 2.8  130 ± 2.8  131 ± 3.1  134 ± 1.8      NDF  377 ± 8.3  363 ± 7.4  330 ± 3.5  301 ± 3.5  273 ± 9.1  251 ± 7.9  248 ± 8.6      ADF  208 ± 4.3  192 ± 4.6  166 ± 2.5  140 ± 2.4  116 ± 1.4  107 ± 3.3  91 ± 1.0      Starch  320 ± 9.0  357 ± 3.0  389 ± 8.6  437 ± 12.5  478 ± 5.2  501 ± 13.3  510 ± 5.2  1BG = backgrounding diet. 2HG = high-grain finishing diet. 3Barley grain processing index (volume weight after processing expressed as a percentage of volume weight before processing, DM basis) was 82.8 ± 0.25%. 4Supplement prepared as a blend and contained (DM basis): 50.0% beet pulp, 33.0% canola meal, 12.0% calcium carbonate, 2.5% urea, 1.6% salt, 0.5% vitamin and mineral mix, 0.3% melengestrol acetate (200 mg/kg), and 0.1% molasses. The concentration of minerals and vitamins in the supplement for the high-grain diet (DM basis) were zinc sulfate monohydrate (55.7 mg/kg), copper sulfate pentahydrate (14.2 mg/kg), manganese sulfate monohydrate (25.6 mg/kg), Ethylene diamine dihydriodide (0.6 mg/kg), selenium (0.3 mg/kg), vitamin A (9,281 IU/kg), vitamin D3 (464 IU/kg), and vitamin E (13 IU/kg). 5All analysis except for DM are reported on a DM basis. View Large Based on time from surgery and the reduction in swelling and time since replacement of the 4C ruminal cannula (7.6 cm; Bar Diamond Inc.) with the 9C cannula (9 cm; Bar Diamond Inc.), heifers were assigned to 1 of 4 blocks and, within block, assigned to 1 of 2 treatments designated as long adapted (LA) or short adapted (SA). Within block, average BW across treatments was balanced. Heifers on the LA treatment were fed the backgrounding diet for 7 d before being transitioned to a barley-based finishing diet whereas SA heifers were fed the backgrounding diet for 33 d before being exposed to the same dietary transition protocol (Table 1). Differences in the duration of the backgrounding period allowed a delay in the start of the dietary transition for the SA animals so that the LA and SA heifers were fed the barley-based finishing diet for 34 and 8 d, respectively, before the induction of ruminal acidosis. The transition from the backgrounding diet to the finishing diet was accomplished over a period of 20 d using 5 intermediate diets with each diet fed for 4 d. Throughout the study, feed was offered once daily at 0900 h allowing for ad libitum intake by targeting 5 to 10% refusals on an as is basis, except for the day of and the day preceding the acidosis challenge. The study design consisted of 4 distinct measurement periods including 8 d for baseline measurements (BASE), the day of the acidosis challenge (CHAL), and 2 consecutive 8-d recovery periods (REC1 and REC2). The ruminal acidosis induction protocol was similar to that of Dohme et al. (2008) with the following modifications. Within each block, the proportion of feed intake relative to BW over 31 consecutive d (d 3 to d 33 on the finishing diet) for the LA heifers was used to calculate the feed restriction and challenge dose. Body weight was measured once a week during this time, and a linear growth rate was used to estimate daily intake as a proportion of BW. The challenge severity was normalized across dietary treatments by applying the same challenge dose relative to BW for LA and SA heifers. On the day before the challenge, feed intake for both LA and SA was restricted to 50% of DMI as a proportion of BW. Data from the day of feed restriction was not used in the statistical analysis. On the day of the CHAL, heifers (excluding those in block 1) were provided with an intraruminal infusion of ground barley grain (ground to pass through a 4.5-mm sieve) at 0900 h equating to 10% of DMI as a proportion of BW measured before feed restriction. Heifers in block 1 were infused with the same lot of ground barley but at 20% of DMI as a proportion of BW; however, because of the severity of the resulting acidosis, the amount of grain infused was subsequently reduced for the cattle in the remaining blocks. Heifers were then given their full diet allocation 1 h after the intraruminal infusion (1000 h). Beginning at the time of challenge, the pH of strained ruminal fluid from the ventral sac was measured every 2 h for the first 12 h and then every 4 h for the next 12 h using a portable pH meter (Accumet 25; Fisher Scientific, Ottawa, ON). Spot sampling pH measurement was used to monitor pH during the challenge, as the continuous indwelling pH system used did not provide a visible real-time pH reading. When ruminal pH was ≤4.2 an additional pH measurement was made 1 h later. If ruminal pH remained ≤4.2, heifers were provided an intraruminal dose of 250 g of sodium bicarbonate. This intervention was necessary for 2 of the 4 heifers exposed to the ruminal acidosis induction protocol in block 1 (both LA), and for this reason, subsequent intraruminal barley infusions were decreased from 20 to 10% of DMI as a proportion of BW, as indicated previously. Data from these heifers were used in the data analysis. Interventions were not required for cattle offered the modified challenge dose. Data and Sample Collection The amount of feed offered and refused was recorded daily throughout the study. Once the cattle were fed the high-grain diet, feed offered and refused were analyzed daily for DM content to determine DMI. In addition, TMR samples of the backgrounding and finishing diets were collected twice weekly, and samples of each transition diet were collected and stored at –20°C until analysis. Before analysis, TMR samples were allowed to thaw and the following feeding periods were composited by block to yield 4 samples each: steps 1 through 5, extended high-grain feeding (LA only), BASE, CHAL and REC1, and REC2. This resulted in a total of 16 samples used for chemical analysis of the high-grain diet and 4 samples for each of the transition diets (Table 1). In addition, the backgrounding diet was composited by treatment and block to yield 8 samples. Ingredients were also collected for DM content and chemical analysis. Barley silage was also collected twice weekly, grass hay and barley grain were collected weekly, and samples of the supplement were collected monthly as the same production lot was used for the entire study. All samples were composited to yield a monthly sample and were stored at –20°C for subsequent analysis. Ingredient and TMR samples were dried at 55°C for 48 h and ground to pass through a 1-mm sieve (SM100; Retsch, Hann, Germany). The analytical DM content was determined by drying samples at 135°C for 2 h (AOAC Int., 1995) and was used to calculate nutrient composition on a DM basis. Crude protein was estimated from the N concentration (CP = N × 6.25), which was determined using flash combustion (Carlo Erba Instruments, Milan, Italy). Neutral detergent fiber and ADF were determined using an Ankom Fiber Analyzer (Ankom Technology Corporation; Fairport, NY) using separate runs. Heat-stable α-amylase and sodium sulfite were used in the NDF procedure (Van Soest et al., 1991). Starch content was determined using enzymatic hydrolysis of α-linked glucose polymers as described by Rode et al. (1999) with minor modifications. Briefly, 100 to 500 mg of sample was diluted in 25 mL 0.1 N sodium acetate buffer and 200 μL of α-amylase (Termamyl; Novo Nordisk, Bagsvaerd, Denmark) was added. Tubes were vortexed immediately and at 10, 20, and 30 min while incubating at 95°C in a constantly shaking water bath. Incubation at 95°C continued for an additional 0.5 h. Subsequently, the incubation temperature was lowered to 65°C and 200 μL amyloglucosidase (208-469; Boehringer Mannheim, Laval, QC, Canada) was added to the tubes. Tubes were vortexed immediately and after 30 and 60 min of incubation at 65°C. Incubation at 65°C continued for an additional 1 h followed by cooling for 5 min. Tubes were then centrifuged (29,000 × g for 15 min at 4°C) and diluted 1:20 using double distilled water. Fifty microliters of sample was added to a microplate and 300-μL glucose trinder reagent (315-100; Sigma-Aldrich, St. Louis, MO) was added to all wells. Following a 20-min incubation at 39°C, glucose was determined by reading the absorbance at 508 nm (Appliscan Multiplate Reader; Thermo Electron Company, Waltham, MA). Means from 4 glucose determinations were compared in duplicate and results were confirmed when the CV < 5.0%. Ruminal pH Measurement Ruminal pH data were recorded every 1 min using an indwelling pH measurement system (LRCpH Data Logger system; Dascor, Escondido, CA) positioned at the bottom of the cranial-ventral sac as described by Penner et al. (2006). Measurement started on d 1 of BASE and persisted through to the final day of experimentation resulting in 26 d of measurements. Ruminal pH systems were standardized before ruminal incubation and were restandardized in pH buffers 7 and 4 at 39°C before and after cleaning in Terg-A-Zyme (Alconox; White Plains, NY) solution for 10 min on weekly removal from the rumen. The resulting data were transformed from millivolt recordings to pH based on the pre- and postincubation standardizations with the precleaning measurement used for the postincubation standardization values. A linear drift correction over time was used to correct pH data (Penner et al., 2006). The daily minimum, mean, and maximum pH as well as duration (DUR; min/d) and area (AREA; min × pH) that pH was <5.5 were calculated. The day of the dietary restriction before CHAL was discarded from analysis as were pH data from days when the temporarily isolated and washed reticulorumen procedure was conducted (d 5 of BASE, d 2 of REC1, and d 1 of REC2; see Schwaiger et al., 2013). After these deletions, 22 d of pH data remained for each heifer (7 d for BASE, 1 d for CHAL, and 7 d each for REC1 and REC2). Additionally, pH was monitored during the first 7 d that the LA and SA cattle were fed the high-grain diet to test whether the duration of time on the backgrounding diet affected the response. Ruminal Fluid Sampling and Analyses Ruminal digesta was sampled on d 3 of BASE, on the day of CHAL, and on d 7 of REC1. Starting at the time of feeding (0900 h), samples were collected every 2 h for a total of 7 samples over 12 h. Due to an observed continued response to the challenge during block 1, the sampling protocol was extended to cover a 24 h duration on the challenge day for blocks 2 to 4. The extended sampling was used to collect 3 additional ruminal fluid samples with a 4-h interval between consecutive samples. Digesta was collected from 3 locations in the rumen (cranial, central-ventral, and caudal sacs) with an equal volume from each location used to prepare a composite (250 mL each for 750 mL total). Composited digesta was strained through PECAP polyester monofilament (pore size 355 µm; part 7-355/47; B & SH Thompson, Ville Mont-Royal, QC, Canada) and aliquots were stored at –20°C for determination of SCFA (1.5 mL), lactate (1.5 mL), and osmolality (2 mL). For SCFA and lactate determination, 1.5-mL aliquots were preserved with 0.3 mL frozen 25% (wt/vol) metaphosphoric acid. Ruminal fluid osmolality (mOsm/kg) was determined in duplicate by freezing-point depression. Samples were centrifuged at 16,100 × g for 30 min 4°C, and 250 μL of supernatant was loaded into the osmometer (Advanced Instruments 3250; Advanced Instruments, Norwood, MA). For quality control, double distilled H2O and standards (290 and 500 mOsm/kg) were analyzed at the beginning and end of each day. Ruminal fluid SCFA and lactate concentrations were determined by gas chromatography using a flame ionization detector. The gas chromatograph (Hewlett-Packard 5890; Hewlett-Packard, Santa Clara, CA) was fitted with a Zebron capillary column (ZB-FFAP; 30 m by 0.32 mm i.d. by 1.0 µm phase thickness; Phenomenex, Torrance, CA), and crotonic acid (trans-2-butenoic acid) and malonic acid (propanedioic acid) were used as internal standards for SCFA and lactate, respectively. In both cases, helium was used as carrier gas (28.5 cm/s). For SCFA determination, 1 μL was injected using a split ratio of 50:1. The injector temperature was set at 225°C and the column temperature was held at 150°C for 1 min followed by a 5°C/min increase in temperature until reaching 195°C, after which temperature was held for 5 min. The detector temperature was held constant at 250°C. For lactate determination, lactate methyl esters were prepared using BF3–MeOH as described by Supelco (1998) and a 1-μL splitless injection was used. The injector temperature was set at 225°C and the column temperature was held at 45°C for 1 min followed by a 30°C/min increase in temperature until reaching 160°C and then a 5°C/min increase in temperature until reaching 195°C, after which temperature was held for 5 min. The detector temperature was held constant at 250°C. Lactate concentrations below the lowest standard (0.21 mM) were assumed to be 0 mM. Statistical Analyses Data were analyzed as a randomized complete block design using the PROC MIXED procedure of SAS (version 9.2; SAS Inst. Inc., Cary, NC). Block was considered a fixed effect and was left in the model except when performing analysis of covariance as described below. Significance was declared when P ≤ 0.05 and tendencies are discussed when 0.05 < P ≤ 0.10. Mean separation was conducted for all variables using the Tukey's post hoc mean separation test and the SAS PDMIX800 macro (Saxton, 1998). The PROC UNIVARIATE procedure of SAS was used to determine if residual data were normally, identically, and independently distributed (NIID). If necessary (based on the normal probability plot and the Shapiro-Wilk test), outliers were removed in a stepwise fashion until the normal probability plot indicated that residual data were NIID. In total 3 observations were removed for serum lactate during BASE and 4 observations during CHAL. For serum L-lactate, 3 observations were removed for BASE, and for serum D-lactate, 3 and 7 observations were removed during BASE and CHAL, respectively. Three observations during both BASE and REC1 were removed for plasma insulin. Ruminal pH and DMI data were summarized by day and heifer. The fixed effect of treatment was investigated over the first 7 d on the high-grain diet. To compare daily means across periods with a different number of days, a split-plot design was used. In this manner the fixed effects of treatment, period, treatment × period, period × day, and treatment × period × day were investigated with the random effects of heifer × block and heifer × period × treatment × block. The effects of period × day and treatment × period × day were left in the model but not reported due to their lack of physiological importance. The effects of treatment and treatment × period were also not reported, as each sampling period was considered biologically distinct. The fixed effects of treatment, day, and treatment × day were then investigated within BASE using day as a repeated measure for the subject heifer × treatment × block. The fixed effect of treatment was then investigated within CHAL and regression analysis was conducted for each variable with respect to day using daily means from BASE and CHAL to determine if there were significant linear, quadratic, or cubic effects of day. To accomplish this, the fixed effects of treatment, day, treatment × day, day2, treatment × day2, day3, and treatment × day3 were tested while accounting for repeated measures with heifer × treatment as the subject. Starting with the highest order term, insignificant (P > 0.05) terms were removed from the model in a stepwise fashion until only significant (P ≤ 0.05) terms remained in the model. When an interaction was significant, the lower order term was removed. The same regression approach as listed above was used to evaluate the recovery response during REC1. The fixed effects of treatment, period, and treatment × period were investigated for ruminal fluid fermentation products by summarizing the data by heifer and period. Only the first 12 h of challenge sampling was used for ruminal fluid fermentation products (extended sampling on CHAL was ignored). Period was used as a repeated measure for the subject heifer × treatment × block. Then, within each sampling period the fixed effects of treatment, time, and treatment × time were investigated for ruminal fluid fermentation products by summarizing the data by heifer, period, and time. Time was used as a repeated measure for the subject heifer × treatment × block. One SA heifer was removed from the study before the acidosis induction due to low intake, frothy ruminal contents, and keratinized epithelia; however, baseline data from this heifer were used in statistical analysis. A failed pH electrode resulted in the loss of 6 d of pH data from 1 heifer (SA; BASE d 6 to d 8, CHAL, REC1 d 1 to d 2). Dry matter intake data were missing for 1 heifer (SA) on d 5 of REC2. RESULTS Confirmation of the Experimental Model The Duration of Time Fed the Backgrounding Diet did not Affect DMI or Ruminal Fermentation. The aim of this study was to evaluate whether duration of time that heifers were fed a high-grain diet affects the susceptibility to and recovery from an induced bout of ruminal acidosis. To determine if the duration of time on the backgrounding diet confounded the response, comparisons were made for BW, DMI, and ruminal pH during the first 7 d that heifers from each treatment were fed the high-grain diet (Table 2). The 26 additional d that the SA cattle were fed the backgrounding diet increased BW (P < 0.001) and tended to increase DMI (P = 0.094) during the first 7 d on the high-grain diet; however, there was no effect of treatment on DMI when reported as a percentage of BW (P = 0.70). There were also no differences between treatments for minimum and mean ruminal pH, nor were there differences for DUR. On the other hand, maximum pH was greater for LA than SA (6.68 vs. 6.49; P = 0.001). Overall, we interpret these data to suggest that the duration of time that heifers were fed the backgrounding diet did not have a significant impact on DMI or ruminal fermentation, and therefore responses observed for the subsequent tables can be attributed to the duration of time that heifers were fed the high-grain diet. Table 2. Mean daily BW, DMI, and ruminal pH for the first 7 d that long-adapted (LA) and short-adapted (SA) heifers were fed the high-grain diet1   Treatment      Item  LA  SA  SEM  P value  BW,2 kg  314  342  2.4  <0.001  DMI, kg  8.7  9.3  0.25  0.094  DMI, % BW  2.8  2.7  0.07  0.70  Minimum pH3  5.04  5.06  0.032  0.65  Mean pH  5.79  5.72  0.042  0.24  Maximum pH  6.68  6.49  0.037  0.001  pH < 5.5      Duration, min/d  475  522  50.3  0.52      Area, (min × pH)/d  153  166  22.5  0.67    Treatment      Item  LA  SA  SEM  P value  BW,2 kg  314  342  2.4  <0.001  DMI, kg  8.7  9.3  0.25  0.094  DMI, % BW  2.8  2.7  0.07  0.70  Minimum pH3  5.04  5.06  0.032  0.65  Mean pH  5.79  5.72  0.042  0.24  Maximum pH  6.68  6.49  0.037  0.001  pH < 5.5      Duration, min/d  475  522  50.3  0.52      Area, (min × pH)/d  153  166  22.5  0.67  1Forage:concentrate ratio = 9:91. 2Mean BW was estimated assuming linear growth between measurements separated by 20 d (LA) and 13 d (SA). 3Data for 7 d of indwelling ruminal pH measurement. View Large Table 2. Mean daily BW, DMI, and ruminal pH for the first 7 d that long-adapted (LA) and short-adapted (SA) heifers were fed the high-grain diet1   Treatment      Item  LA  SA  SEM  P value  BW,2 kg  314  342  2.4  <0.001  DMI, kg  8.7  9.3  0.25  0.094  DMI, % BW  2.8  2.7  0.07  0.70  Minimum pH3  5.04  5.06  0.032  0.65  Mean pH  5.79  5.72  0.042  0.24  Maximum pH  6.68  6.49  0.037  0.001  pH < 5.5      Duration, min/d  475  522  50.3  0.52      Area, (min × pH)/d  153  166  22.5  0.67    Treatment      Item  LA  SA  SEM  P value  BW,2 kg  314  342  2.4  <0.001  DMI, kg  8.7  9.3  0.25  0.094  DMI, % BW  2.8  2.7  0.07  0.70  Minimum pH3  5.04  5.06  0.032  0.65  Mean pH  5.79  5.72  0.042  0.24  Maximum pH  6.68  6.49  0.037  0.001  pH < 5.5      Duration, min/d  475  522  50.3  0.52      Area, (min × pH)/d  153  166  22.5  0.67  1Forage:concentrate ratio = 9:91. 2Mean BW was estimated assuming linear growth between measurements separated by 20 d (LA) and 13 d (SA). 3Data for 7 d of indwelling ruminal pH measurement. View Large Confirmation that Ruminal Acidosis was Induced. To illustrate the effects of the induced challenge on ruminal pH, mean pH (10-min intervals) was plotted for the 2 treatments over a 5-d duration surrounding the induced bout of ruminal acidosis (Fig. 1). From the rapid drop in pH and the extent to which pH dropped, it is clear that a bout of ruminal acidosis was successfully induced using the acidosis challenge protocol. Figure 1. View largeDownload slide Mean ruminal pH for the 5 d surrounding the induced challenge. Data were summarized by minute for each heifer, and means were calculated for every 10 min for long-adapted (LA) and short-adapted (SA) heifers during the last 2 d of baseline (BASE 7 and 8), during the dietary restriction (REST), during the day of the induced challenge (CHAL), and during the first day of recovery from the challenge (REC1). All (n = 8) LA heifers were used for all means shown while 7 (n = 7) SA heifers were used during BASE and 6 (n = 6) SA heifers were used during REST, CHAL, and REC1. One SA heifer was removed from the study after BASE, and another had a failed pH electrode. Figure 1. View largeDownload slide Mean ruminal pH for the 5 d surrounding the induced challenge. Data were summarized by minute for each heifer, and means were calculated for every 10 min for long-adapted (LA) and short-adapted (SA) heifers during the last 2 d of baseline (BASE 7 and 8), during the dietary restriction (REST), during the day of the induced challenge (CHAL), and during the first day of recovery from the challenge (REC1). All (n = 8) LA heifers were used for all means shown while 7 (n = 7) SA heifers were used during BASE and 6 (n = 6) SA heifers were used during REST, CHAL, and REC1. One SA heifer was removed from the study after BASE, and another had a failed pH electrode. Comparisons by period were conducted to evaluate whether the acidosis protocol affected DMI and ruminal pH. It is important to note that there were no interactions between treatment and period for DMI, ruminal pH, SCFA, lactate, or osmolality (P ≥ 0.12; data not shown). Dry matter intake tended (P = 0.072) to be affected by period, whereby DMI in CHAL tended to be greater than during the other periods. The acidosis challenge reduced minimum (P < 0.001) and mean pH (P < 0.001) relative to BASE with the effect reversed during REC1 and REC2, such that minimum and mean pH were greater during REC1 and REC2 than during BASE and CHAL. Maximum pH increased on the day of CHAL due to the elevated pH caused by the dietary restriction (P < 0.001; Fig. 1). This elevated pH persisted for approximately 1 h following the induced challenge. The DUR (P < 0.001) and AREA (P < 0.001) was greater during CHAL relative to BASE. Specifically, the CHAL nearly doubled the DUR relative to BASE (1,020 vs. 531 min/d; Table 3). The increases for DUR and AREA during CHAL were both reversible with reduced DUR and AREA observed during REC1 and REC2 than during BASE, and the AREA during REC1 and REC2 were not different than during BASE but less than CHAL. Table 3. Mean daily DMI, ruminal pH, short-chain fatty acids, lactate, and osmolality across measurement periods when heifers were fed the high-grain diet.1 There were no treatment and treatment × period interactions (P ≥ 0.12).2   Period2      Item  BASE  CHAL  REC1  REC2  SEM  P value  Number of days, d  7  1  7  7      DMI, kg  9.3  10.5  9.2  9.6  0.46  0.072  Minimum pH  5.03b  4.57c  5.20a  5.26a  0.03  <0.001  Mean pH  5.73b  5.27c  5.86a  5.93a  0.043  <0.001  Maximum pH  6.54b  6.96a  6.52b  6.60b  0.038  <0.001  pH < 5.5      Duration, min/d  531b  1,020a  294c  259c  55.7  <0.001      Area, (pH × min)/d  176b  595a  72b  58b  36  <0.001  Total SCFA,3 mM      Mean  128.2  133  122  –  4.32  0.14      Maximum  157.7ab  165.7a  143.2b  –  5  0.011  Acetate, mM      Mean  62.2  67.8  63.6  –  1.62  0.22      Maximum  75.5  80  73.6  –  1.98  0.08  Propionate, mM      Mean  50.4  50  42.8  –  3.88  0.12      Maximum  65.0a  61.7ab  51.0b  –  4.35  0.018  Butyrate, mM      Mean  15.2  17.9  15  –  1.55  0.23      Maximum  20.1  24.5  19.4  –  2.1  0.20  Lactate, mM      Mean  0.3b  11.4a  0.1b  –  1.04  0.013      Maximum  1.3b  29.3a  0.5b  –  2.55  0.008  Osmolality, mOsm/kg      Mean  369  369  369  –  5.3  0.99      Maximum  412ab  432a  407b  –  6.9  0.030    Period2      Item  BASE  CHAL  REC1  REC2  SEM  P value  Number of days, d  7  1  7  7      DMI, kg  9.3  10.5  9.2  9.6  0.46  0.072  Minimum pH  5.03b  4.57c  5.20a  5.26a  0.03  <0.001  Mean pH  5.73b  5.27c  5.86a  5.93a  0.043  <0.001  Maximum pH  6.54b  6.96a  6.52b  6.60b  0.038  <0.001  pH < 5.5      Duration, min/d  531b  1,020a  294c  259c  55.7  <0.001      Area, (pH × min)/d  176b  595a  72b  58b  36  <0.001  Total SCFA,3 mM      Mean  128.2  133  122  –  4.32  0.14      Maximum  157.7ab  165.7a  143.2b  –  5  0.011  Acetate, mM      Mean  62.2  67.8  63.6  –  1.62  0.22      Maximum  75.5  80  73.6  –  1.98  0.08  Propionate, mM      Mean  50.4  50  42.8  –  3.88  0.12      Maximum  65.0a  61.7ab  51.0b  –  4.35  0.018  Butyrate, mM      Mean  15.2  17.9  15  –  1.55  0.23      Maximum  20.1  24.5  19.4  –  2.1  0.20  Lactate, mM      Mean  0.3b  11.4a  0.1b  –  1.04  0.013      Maximum  1.3b  29.3a  0.5b  –  2.55  0.008  Osmolality, mOsm/kg      Mean  369  369  369  –  5.3  0.99      Maximum  412ab  432a  407b  –  6.9  0.030  a,b,cMeans within a dependent variable differ (P ≤ 0.05). The Tukey post hoc mean separation test was used for mean separation. 1Forage:concentrate ratio = 9:91. 2For ruminal pH BASE = baseline measurement period that consisted of 8 d on the final diet before dietary restriction (50% DMI/BW). CHAL = challenge day that occurred 1 d after dietary restriction and consisted of an intraruminal infusion of ground barley grain (10% DMI/BW) followed by full diet allocation. REC = recovery period that started 24 h after the challenge and is separated into 2 consecutive 8 d periods (REC1 and REC2). Data from days where the temporarily isolated and washed reticulorumen technique was conducted during BASE, REC1, and REC2 have been omitted. For ruminal fluid, BASE sampling occurred 7 d prior to CHAL, CHAL sampling occurred on d of induced challenge, REC1 sampling occurred 7 d after challenge. Daily mean and maximum values were determined from samples collected at the time of feeding and every 2 hours for 12 h after feeding. 3SCFA = short-chain fatty acid. View Large Table 3. Mean daily DMI, ruminal pH, short-chain fatty acids, lactate, and osmolality across measurement periods when heifers were fed the high-grain diet.1 There were no treatment and treatment × period interactions (P ≥ 0.12).2   Period2      Item  BASE  CHAL  REC1  REC2  SEM  P value  Number of days, d  7  1  7  7      DMI, kg  9.3  10.5  9.2  9.6  0.46  0.072  Minimum pH  5.03b  4.57c  5.20a  5.26a  0.03  <0.001  Mean pH  5.73b  5.27c  5.86a  5.93a  0.043  <0.001  Maximum pH  6.54b  6.96a  6.52b  6.60b  0.038  <0.001  pH < 5.5      Duration, min/d  531b  1,020a  294c  259c  55.7  <0.001      Area, (pH × min)/d  176b  595a  72b  58b  36  <0.001  Total SCFA,3 mM      Mean  128.2  133  122  –  4.32  0.14      Maximum  157.7ab  165.7a  143.2b  –  5  0.011  Acetate, mM      Mean  62.2  67.8  63.6  –  1.62  0.22      Maximum  75.5  80  73.6  –  1.98  0.08  Propionate, mM      Mean  50.4  50  42.8  –  3.88  0.12      Maximum  65.0a  61.7ab  51.0b  –  4.35  0.018  Butyrate, mM      Mean  15.2  17.9  15  –  1.55  0.23      Maximum  20.1  24.5  19.4  –  2.1  0.20  Lactate, mM      Mean  0.3b  11.4a  0.1b  –  1.04  0.013      Maximum  1.3b  29.3a  0.5b  –  2.55  0.008  Osmolality, mOsm/kg      Mean  369  369  369  –  5.3  0.99      Maximum  412ab  432a  407b  –  6.9  0.030    Period2      Item  BASE  CHAL  REC1  REC2  SEM  P value  Number of days, d  7  1  7  7      DMI, kg  9.3  10.5  9.2  9.6  0.46  0.072  Minimum pH  5.03b  4.57c  5.20a  5.26a  0.03  <0.001  Mean pH  5.73b  5.27c  5.86a  5.93a  0.043  <0.001  Maximum pH  6.54b  6.96a  6.52b  6.60b  0.038  <0.001  pH < 5.5      Duration, min/d  531b  1,020a  294c  259c  55.7  <0.001      Area, (pH × min)/d  176b  595a  72b  58b  36  <0.001  Total SCFA,3 mM      Mean  128.2  133  122  –  4.32  0.14      Maximum  157.7ab  165.7a  143.2b  –  5  0.011  Acetate, mM      Mean  62.2  67.8  63.6  –  1.62  0.22      Maximum  75.5  80  73.6  –  1.98  0.08  Propionate, mM      Mean  50.4  50  42.8  –  3.88  0.12      Maximum  65.0a  61.7ab  51.0b  –  4.35  0.018  Butyrate, mM      Mean  15.2  17.9  15  –  1.55  0.23      Maximum  20.1  24.5  19.4  –  2.1  0.20  Lactate, mM      Mean  0.3b  11.4a  0.1b  –  1.04  0.013      Maximum  1.3b  29.3a  0.5b  –  2.55  0.008  Osmolality, mOsm/kg      Mean  369  369  369  –  5.3  0.99      Maximum  412ab  432a  407b  –  6.9  0.030  a,b,cMeans within a dependent variable differ (P ≤ 0.05). The Tukey post hoc mean separation test was used for mean separation. 1Forage:concentrate ratio = 9:91. 2For ruminal pH BASE = baseline measurement period that consisted of 8 d on the final diet before dietary restriction (50% DMI/BW). CHAL = challenge day that occurred 1 d after dietary restriction and consisted of an intraruminal infusion of ground barley grain (10% DMI/BW) followed by full diet allocation. REC = recovery period that started 24 h after the challenge and is separated into 2 consecutive 8 d periods (REC1 and REC2). Data from days where the temporarily isolated and washed reticulorumen technique was conducted during BASE, REC1, and REC2 have been omitted. For ruminal fluid, BASE sampling occurred 7 d prior to CHAL, CHAL sampling occurred on d of induced challenge, REC1 sampling occurred 7 d after challenge. Daily mean and maximum values were determined from samples collected at the time of feeding and every 2 hours for 12 h after feeding. 3SCFA = short-chain fatty acid. View Large No differences were detected for mean SCFA, mean acetate, mean propionate, mean and maximum butyrate concentrations, and mean osmolality when compared across periods (Table 3). Maximum acetate concentration tended (P = 0.08) to increase during CHAL and decrease during REC1. Maximum propionate concentration was greatest during BASE and least during REC1 (P = 0.018) while both maximum total SCFA (165.7 vs. 143.2 mM; P = 0.011) and maximum ruminal fluid osmolality (432 vs. 407 mOsm/kg; P = 0.03) were greatest during the CHAL and least during REC1. Mean (P = 0.013) and maximum (P = 0.008) ruminal fluid lactate concentration increased during the CHAL (11.4 and 29.3 mM) relative to BASE (0.3 and 1.3 mM) but BASE and REC1 (0.1 and 0.5 mM; Table 3) were not different. Ruminal lactate concentration >5.0 mM was observed in 12 of the 15 heifers during CHAL. In contrast, none of the heifers experienced a maximal daily ruminal lactate concentration ≥5.0 mM during either BASE or REC1 (data is not shown). Although there was significant between-heifer variability in response to the challenge, we were able to successfully induce an average bout of acute ruminal acidosis when data from all heifers were considered (Table 3). By implementing our challenge model, we observed a significant increase in ruminal fluid lactate concentration (Table 3) with a peak mean lactate concentration of 19.0 mM observed 8 h after providing the challenge dose. The daily mean lactate concentration of 11.4 mM, and daily maximum lactate concentration of 29.3 mM are both above the suggested threshold of 5.0 mM to indicate acute ruminal lactic acidosis (Aschenbach et al., 2011). Dry Matter Intake and Ruminal Fermentation during the Baseline Measurement Period To evaluate the effect of treatment and day on DMI and pH and the effect of treatment and time on ruminal SCFA and osmolality before CHAL, the data were investigated within BASE (Tables 4 and 5). Differences observed during BASE are deemed to be part of the adaptation response. There was no effect of treatment on DMI or pH variables during BASE (P ≥ 0.088). Dry matter intake was greatest on the first day of BASE (P = 0.038); however, minimum (P = 0.01), mean (P = 0.03), and maximum (P = 0.027) pH all reached the lowest values on d 6 of BASE, during which time both DUR (P = 0.064) and AREA (pH × min/d; P = 0.075) tended to be greatest. A tendency for a treatment × day interaction for maximum pH (P = 0.063) indicated that the LA heifers tended to experience an increase in maximum pH between d 1 and 4 during BASE while the SA heifers tended to experience a decrease in maximum pH during this time (data not shown). Table 4. Mean daily DMI and pH variables for the 8 d before the induced acidosis challenge (BASE1). Long-adapted (LA) heifers were fed the high-grain diet2 for 26 additional d before this measurement period; short-adapted (SA) heifers received the high-grain diet on d 1 of the BASE period.   Treatment    Day of baseline period (BASE)    P value3  Item  LA  SA  SEM  1  2  3  4  6  7  8  SEM  T  D  T × D  DMI, kg  9.3  9.3  0.64  10.5  9.5  9.6  8.3  8.8  8.5  9.6  0.58  1.00  0.038  0.97  Minimum pH  4.99  5.06  0.037  5.11ab  5.02ab  4.93ab  5.04ab  4.88b  5.12a  5.09ab  0.058  0.20  0.010  0.69  Mean pH  5.73  5.72  0.056  5.78  5.66  5.63  5.82  5.58  5.82  5.78  0.078  0.93  0.030  0.56  Maximum pH  6.59  6.48  0.043  6.65a  6.56ab  6.52ab  6.55ab  6.45b  6.46ab  6.56ab  0.060  0.088  0.027  0.063  Ruminal pH < 5.5      Duration, min/d  547  516  72.6  474  581  639  485  680  397  465  94.3  0.77  0.064  0.29      Area, (min × pH)/d  190  167  31.2  138  210  223  147  261  127  141  44.2  0.60  0.075  0.53    Treatment    Day of baseline period (BASE)    P value3  Item  LA  SA  SEM  1  2  3  4  6  7  8  SEM  T  D  T × D  DMI, kg  9.3  9.3  0.64  10.5  9.5  9.6  8.3  8.8  8.5  9.6  0.58  1.00  0.038  0.97  Minimum pH  4.99  5.06  0.037  5.11ab  5.02ab  4.93ab  5.04ab  4.88b  5.12a  5.09ab  0.058  0.20  0.010  0.69  Mean pH  5.73  5.72  0.056  5.78  5.66  5.63  5.82  5.58  5.82  5.78  0.078  0.93  0.030  0.56  Maximum pH  6.59  6.48  0.043  6.65a  6.56ab  6.52ab  6.55ab  6.45b  6.46ab  6.56ab  0.060  0.088  0.027  0.063  Ruminal pH < 5.5      Duration, min/d  547  516  72.6  474  581  639  485  680  397  465  94.3  0.77  0.064  0.29      Area, (min × pH)/d  190  167  31.2  138  210  223  147  261  127  141  44.2  0.60  0.075  0.53  a,bMeans within a dependent variable differ (P ≤ 0.050). The Tukey post hoc mean separation test was used for mean separation. For DMI and mean pH, mean separation was not achieved despite a significant effect of day. 1BASE = the baseline measurement period consisted of 8 d on the final diet before dietary restriction and the acidosis challenge. Day 5 was the day of the temporarily isolated and washed reticulorumen technique. Due to manipulation of the rumen, DMI and pH data were not included. 2Forage:concentrate ratio = 9:91. 3T = treatment; D = day of baseline period. View Large Table 4. Mean daily DMI and pH variables for the 8 d before the induced acidosis challenge (BASE1). Long-adapted (LA) heifers were fed the high-grain diet2 for 26 additional d before this measurement period; short-adapted (SA) heifers received the high-grain diet on d 1 of the BASE period.   Treatment    Day of baseline period (BASE)    P value3  Item  LA  SA  SEM  1  2  3  4  6  7  8  SEM  T  D  T × D  DMI, kg  9.3  9.3  0.64  10.5  9.5  9.6  8.3  8.8  8.5  9.6  0.58  1.00  0.038  0.97  Minimum pH  4.99  5.06  0.037  5.11ab  5.02ab  4.93ab  5.04ab  4.88b  5.12a  5.09ab  0.058  0.20  0.010  0.69  Mean pH  5.73  5.72  0.056  5.78  5.66  5.63  5.82  5.58  5.82  5.78  0.078  0.93  0.030  0.56  Maximum pH  6.59  6.48  0.043  6.65a  6.56ab  6.52ab  6.55ab  6.45b  6.46ab  6.56ab  0.060  0.088  0.027  0.063  Ruminal pH < 5.5      Duration, min/d  547  516  72.6  474  581  639  485  680  397  465  94.3  0.77  0.064  0.29      Area, (min × pH)/d  190  167  31.2  138  210  223  147  261  127  141  44.2  0.60  0.075  0.53    Treatment    Day of baseline period (BASE)    P value3  Item  LA  SA  SEM  1  2  3  4  6  7  8  SEM  T  D  T × D  DMI, kg  9.3  9.3  0.64  10.5  9.5  9.6  8.3  8.8  8.5  9.6  0.58  1.00  0.038  0.97  Minimum pH  4.99  5.06  0.037  5.11ab  5.02ab  4.93ab  5.04ab  4.88b  5.12a  5.09ab  0.058  0.20  0.010  0.69  Mean pH  5.73  5.72  0.056  5.78  5.66  5.63  5.82  5.58  5.82  5.78  0.078  0.93  0.030  0.56  Maximum pH  6.59  6.48  0.043  6.65a  6.56ab  6.52ab  6.55ab  6.45b  6.46ab  6.56ab  0.060  0.088  0.027  0.063  Ruminal pH < 5.5      Duration, min/d  547  516  72.6  474  581  639  485  680  397  465  94.3  0.77  0.064  0.29      Area, (min × pH)/d  190  167  31.2  138  210  223  147  261  127  141  44.2  0.60  0.075  0.53  a,bMeans within a dependent variable differ (P ≤ 0.050). The Tukey post hoc mean separation test was used for mean separation. For DMI and mean pH, mean separation was not achieved despite a significant effect of day. 1BASE = the baseline measurement period consisted of 8 d on the final diet before dietary restriction and the acidosis challenge. Day 5 was the day of the temporarily isolated and washed reticulorumen technique. Due to manipulation of the rumen, DMI and pH data were not included. 2Forage:concentrate ratio = 9:91. 3T = treatment; D = day of baseline period. View Large Table 5. Ruminal short-chain fatty acid (SCFA) concentration and osmolality before the induced acidosis challenge (BASE1). Long-adapted (LA) heifers were fed the high-grain diet2 for 26 additional d before this measurement period; short-adapted (SA) heifers received the high-grain diet on d 1 of the BASE2 period.   Treatment    Hour relative to feeding (h)    P value2  LA  SA  SEM  0  2  4  6  8  10  12  SEM  T  h  T × h  Total SCFA, mM  133.5  122.1  4.12  94.7c  109.1bc  125.8ab  139.9a  144.8a  133.7a  146.8a  4.12  0.065  <0.001  0.59  Acetate, mM  63.7  60.8  1.58  47.6c  56.5b  62.5ab  68.0a  68.9a  63.4ab  68.9a  2.31  0.23  <0.001  0.70  Propionate, mM  54.1  46.6  2.93  35.2c  39.7c  48.1b  55.5ab  58.6a  54.6ab  60.9a  3.08  0.093  <0.001  0.52  Butyrate, mM  15.8  14.7  1.89  11.9ab  13.0ab  15.2b  16.5ab  17.4a  15.8ab  17.1ab  1.62  0.71  0.015  0.53  Osmolality, mOsm/kg  380a  358b  6.6  321c  350b  369ab  384a  390a  376ab  391a  7.3  0.038  <0.001  0.42    Treatment    Hour relative to feeding (h)    P value2  LA  SA  SEM  0  2  4  6  8  10  12  SEM  T  h  T × h  Total SCFA, mM  133.5  122.1  4.12  94.7c  109.1bc  125.8ab  139.9a  144.8a  133.7a  146.8a  4.12  0.065  <0.001  0.59  Acetate, mM  63.7  60.8  1.58  47.6c  56.5b  62.5ab  68.0a  68.9a  63.4ab  68.9a  2.31  0.23  <0.001  0.70  Propionate, mM  54.1  46.6  2.93  35.2c  39.7c  48.1b  55.5ab  58.6a  54.6ab  60.9a  3.08  0.093  <0.001  0.52  Butyrate, mM  15.8  14.7  1.89  11.9ab  13.0ab  15.2b  16.5ab  17.4a  15.8ab  17.1ab  1.62  0.71  0.015  0.53  Osmolality, mOsm/kg  380a  358b  6.6  321c  350b  369ab  384a  390a  376ab  391a  7.3  0.038  <0.001  0.42  a,b,cMeans within a dependent variable differ (P ≤ 0.05). The Tukey post hoc mean separation test was used for mean separation. 1BASE = the baseline measurement period consisted of 8 d on the final diet before dietary restriction and the acidosis challenge. Day 5 was the day of the temporarily isolated and washed reticulorumen technique. Due to manipulation of the rumen, DMI and pH data were not included. 2Forage:concentrate ratio = 9:91. 3T = treatment. View Large Table 5. Ruminal short-chain fatty acid (SCFA) concentration and osmolality before the induced acidosis challenge (BASE1). Long-adapted (LA) heifers were fed the high-grain diet2 for 26 additional d before this measurement period; short-adapted (SA) heifers received the high-grain diet on d 1 of the BASE2 period.   Treatment    Hour relative to feeding (h)    P value2  LA  SA  SEM  0  2  4  6  8  10  12  SEM  T  h  T × h  Total SCFA, mM  133.5  122.1  4.12  94.7c  109.1bc  125.8ab  139.9a  144.8a  133.7a  146.8a  4.12  0.065  <0.001  0.59  Acetate, mM  63.7  60.8  1.58  47.6c  56.5b  62.5ab  68.0a  68.9a  63.4ab  68.9a  2.31  0.23  <0.001  0.70  Propionate, mM  54.1  46.6  2.93  35.2c  39.7c  48.1b  55.5ab  58.6a  54.6ab  60.9a  3.08  0.093  <0.001  0.52  Butyrate, mM  15.8  14.7  1.89  11.9ab  13.0ab  15.2b  16.5ab  17.4a  15.8ab  17.1ab  1.62  0.71  0.015  0.53  Osmolality, mOsm/kg  380a  358b  6.6  321c  350b  369ab  384a  390a  376ab  391a  7.3  0.038  <0.001  0.42    Treatment    Hour relative to feeding (h)    P value2  LA  SA  SEM  0  2  4  6  8  10  12  SEM  T  h  T × h  Total SCFA, mM  133.5  122.1  4.12  94.7c  109.1bc  125.8ab  139.9a  144.8a  133.7a  146.8a  4.12  0.065  <0.001  0.59  Acetate, mM  63.7  60.8  1.58  47.6c  56.5b  62.5ab  68.0a  68.9a  63.4ab  68.9a  2.31  0.23  <0.001  0.70  Propionate, mM  54.1  46.6  2.93  35.2c  39.7c  48.1b  55.5ab  58.6a  54.6ab  60.9a  3.08  0.093  <0.001  0.52  Butyrate, mM  15.8  14.7  1.89  11.9ab  13.0ab  15.2b  16.5ab  17.4a  15.8ab  17.1ab  1.62  0.71  0.015  0.53  Osmolality, mOsm/kg  380a  358b  6.6  321c  350b  369ab  384a  390a  376ab  391a  7.3  0.038  <0.001  0.42  a,b,cMeans within a dependent variable differ (P ≤ 0.05). The Tukey post hoc mean separation test was used for mean separation. 1BASE = the baseline measurement period consisted of 8 d on the final diet before dietary restriction and the acidosis challenge. Day 5 was the day of the temporarily isolated and washed reticulorumen technique. Due to manipulation of the rumen, DMI and pH data were not included. 2Forage:concentrate ratio = 9:91. 3T = treatment. View Large No interactions between treatment and hour of sampling were observed for ruminal SCFA concentration or osmolality during BASE (Table 5). The concentration of SCFA and ruminal fluid osmolality were all affected by time such that the greatest concentrations and osmolality were generally observed 8 to 12 h postfeeding. Dry Matter Intake and Ruminal Fermentation during an Induced Bout of Ruminal Acidosis The impact of the duration of dietary adaptation on the susceptibility to an induced bout of acidosis was investigated solely using data within CHAL (Table 6). As with BASE, there was no effect of treatment on DMI, pH variables, SCFA concentrations, lactate concentration, or osmolality during CHAL (P ≥ 0.44; Table 6). Table 6. Mean DMI, ruminal pH, short-chain fatty acid (SCFA) concentration, and osmolality variables on the challenge day. Long-adapted (LA) heifers were fed the high-grain diet1 for 34 d compared to 8 d for the short-adapted (SA) heifers.   Treatment    Hour relative to feeding    P value2  Item  LA  SA  SEM  0  2  4  6  8  10  12  16  20  24  SEM  T  h  T × h  DMI, kg  11  10.1  0.82  –  –  –  –  –  –  –  –  –  –    0.47  –  –  Minimum pH  4.6  4.58  0.134  –  –  –  –  –  –  –  –  –  –    0.9  –  –  Mean pH  5.3  5.31  0.095  –  –  –  –  –  –  –  –  –  –    0.94  –  –  Maximum pH  6.95  6.96  0.066  –  –  –  –  –  –  –  –  –  –    0.93  –  –  Ruminal pH < 5.5      Duration, min/d  951  1,013  58.6  –  –  –  –  –  –  –  –  –  –    0.51  –  –      Area, (min × pH)/d  583  545  123.1  –  –  –  –  –  –  –  –  –  –    0.85  –  –      Total SCFA, mM  121.6  122.7  6.71  47.8e  88.3d  123.5c  149.9ab  156.3a  136.6bc  136.5abc  135.7abc  130.4abc  116.2bcd  7.43  0.91  <0.001  0.34      Acetate, mM  61.6  63.1  2.66  28.4g  51.7f  66.4cde  74.8ab  74.8ac  66.0bde  67.9abcd  69.0abcd  66.0abcdf  59.1df  3.24  0.7  <0.001  0.39      Propionate, mM  45.7  43.4  4.77  12.8e  25.5d  42.3c  54.1ab  59.1a  51.7bc  51.1abc  52.1abc  51.4abc  45.3abc  3.95  0.74  <0.001  0.17      Butyrate, mM  14.2  16.3  1.82  6.4d  11.1c  14.8bc  20.8ab  22.3a  18.8ab  17.6abc  14.8bc  13.6bcd  12.2bc  1.75  0.44  <0.001  0.41      Lactate, mM  12.7  11.9  5.22  ND4  5.3bc  3.6bc  10.0b  19.0a  20.5abc  21.4abc  22.4abc  8.2abc  0.7c  4.29  0.92  0.027  0.53      Osmolality, mOsm/kg  376  376  6.0  285d  325c  367b  404a  414a  386ab  388ab  416a  398ab  375ab  9.8  0.98  <0.001  0.075    Treatment    Hour relative to feeding    P value2  Item  LA  SA  SEM  0  2  4  6  8  10  12  16  20  24  SEM  T  h  T × h  DMI, kg  11  10.1  0.82  –  –  –  –  –  –  –  –  –  –    0.47  –  –  Minimum pH  4.6  4.58  0.134  –  –  –  –  –  –  –  –  –  –    0.9  –  –  Mean pH  5.3  5.31  0.095  –  –  –  –  –  –  –  –  –  –    0.94  –  –  Maximum pH  6.95  6.96  0.066  –  –  –  –  –  –  –  –  –  –    0.93  –  –  Ruminal pH < 5.5      Duration, min/d  951  1,013  58.6  –  –  –  –  –  –  –  –  –  –    0.51  –  –      Area, (min × pH)/d  583  545  123.1  –  –  –  –  –  –  –  –  –  –    0.85  –  –      Total SCFA, mM  121.6  122.7  6.71  47.8e  88.3d  123.5c  149.9ab  156.3a  136.6bc  136.5abc  135.7abc  130.4abc  116.2bcd  7.43  0.91  <0.001  0.34      Acetate, mM  61.6  63.1  2.66  28.4g  51.7f  66.4cde  74.8ab  74.8ac  66.0bde  67.9abcd  69.0abcd  66.0abcdf  59.1df  3.24  0.7  <0.001  0.39      Propionate, mM  45.7  43.4  4.77  12.8e  25.5d  42.3c  54.1ab  59.1a  51.7bc  51.1abc  52.1abc  51.4abc  45.3abc  3.95  0.74  <0.001  0.17      Butyrate, mM  14.2  16.3  1.82  6.4d  11.1c  14.8bc  20.8ab  22.3a  18.8ab  17.6abc  14.8bc  13.6bcd  12.2bc  1.75  0.44  <0.001  0.41      Lactate, mM  12.7  11.9  5.22  ND4  5.3bc  3.6bc  10.0b  19.0a  20.5abc  21.4abc  22.4abc  8.2abc  0.7c  4.29  0.92  0.027  0.53      Osmolality, mOsm/kg  376  376  6.0  285d  325c  367b  404a  414a  386ab  388ab  416a  398ab  375ab  9.8  0.98  <0.001  0.075  a,b,c,d,e,fMeans within a dependent variable differ (P ≤ 0.05). The Tukey post hoc mean separation test was used for mean separation. 1Forage:concentrate ratio = 9:91. 2T = treatment. View Large Table 6. Mean DMI, ruminal pH, short-chain fatty acid (SCFA) concentration, and osmolality variables on the challenge day. Long-adapted (LA) heifers were fed the high-grain diet1 for 34 d compared to 8 d for the short-adapted (SA) heifers.   Treatment    Hour relative to feeding    P value2  Item  LA  SA  SEM  0  2  4  6  8  10  12  16  20  24  SEM  T  h  T × h  DMI, kg  11  10.1  0.82  –  –  –  –  –  –  –  –  –  –    0.47  –  –  Minimum pH  4.6  4.58  0.134  –  –  –  –  –  –  –  –  –  –    0.9  –  –  Mean pH  5.3  5.31  0.095  –  –  –  –  –  –  –  –  –  –    0.94  –  –  Maximum pH  6.95  6.96  0.066  –  –  –  –  –  –  –  –  –  –    0.93  –  –  Ruminal pH < 5.5      Duration, min/d  951  1,013  58.6  –  –  –  –  –  –  –  –  –  –    0.51  –  –      Area, (min × pH)/d  583  545  123.1  –  –  –  –  –  –  –  –  –  –    0.85  –  –      Total SCFA, mM  121.6  122.7  6.71  47.8e  88.3d  123.5c  149.9ab  156.3a  136.6bc  136.5abc  135.7abc  130.4abc  116.2bcd  7.43  0.91  <0.001  0.34      Acetate, mM  61.6  63.1  2.66  28.4g  51.7f  66.4cde  74.8ab  74.8ac  66.0bde  67.9abcd  69.0abcd  66.0abcdf  59.1df  3.24  0.7  <0.001  0.39      Propionate, mM  45.7  43.4  4.77  12.8e  25.5d  42.3c  54.1ab  59.1a  51.7bc  51.1abc  52.1abc  51.4abc  45.3abc  3.95  0.74  <0.001  0.17      Butyrate, mM  14.2  16.3  1.82  6.4d  11.1c  14.8bc  20.8ab  22.3a  18.8ab  17.6abc  14.8bc  13.6bcd  12.2bc  1.75  0.44  <0.001  0.41      Lactate, mM  12.7  11.9  5.22  ND4  5.3bc  3.6bc  10.0b  19.0a  20.5abc  21.4abc  22.4abc  8.2abc  0.7c  4.29  0.92  0.027  0.53      Osmolality, mOsm/kg  376  376  6.0  285d  325c  367b  404a  414a  386ab  388ab  416a  398ab  375ab  9.8  0.98  <0.001  0.075    Treatment    Hour relative to feeding    P value2  Item  LA  SA  SEM  0  2  4  6  8  10  12  16  20  24  SEM  T  h  T × h  DMI, kg  11  10.1  0.82  –  –  –  –  –  –  –  –  –  –    0.47  –  –  Minimum pH  4.6  4.58  0.134  –  –  –  –  –  –  –  –  –  –    0.9  –  –  Mean pH  5.3  5.31  0.095  –  –  –  –  –  –  –  –  –  –    0.94  –  –  Maximum pH  6.95  6.96  0.066  –  –  –  –  –  –  –  –  –  –    0.93  –  –  Ruminal pH < 5.5      Duration, min/d  951  1,013  58.6  –  –  –  –  –  –  –  –  –  –    0.51  –  –      Area, (min × pH)/d  583  545  123.1  –  –  –  –  –  –  –  –  –  –    0.85  –  –      Total SCFA, mM  121.6  122.7  6.71  47.8e  88.3d  123.5c  149.9ab  156.3a  136.6bc  136.5abc  135.7abc  130.4abc  116.2bcd  7.43  0.91  <0.001  0.34      Acetate, mM  61.6  63.1  2.66  28.4g  51.7f  66.4cde  74.8ab  74.8ac  66.0bde  67.9abcd  69.0abcd  66.0abcdf  59.1df  3.24  0.7  <0.001  0.39      Propionate, mM  45.7  43.4  4.77  12.8e  25.5d  42.3c  54.1ab  59.1a  51.7bc  51.1abc  52.1abc  51.4abc  45.3abc  3.95  0.74  <0.001  0.17      Butyrate, mM  14.2  16.3  1.82  6.4d  11.1c  14.8bc  20.8ab  22.3a  18.8ab  17.6abc  14.8bc  13.6bcd  12.2bc  1.75  0.44  <0.001  0.41      Lactate, mM  12.7  11.9  5.22  ND4  5.3bc  3.6bc  10.0b  19.0a  20.5abc  21.4abc  22.4abc  8.2abc  0.7c  4.29  0.92  0.027  0.53      Osmolality, mOsm/kg  376  376  6.0  285d  325c  367b  404a  414a  386ab  388ab  416a  398ab  375ab  9.8  0.98  <0.001  0.075  a,b,c,d,e,fMeans within a dependent variable differ (P ≤ 0.05). The Tukey post hoc mean separation test was used for mean separation. 1Forage:concentrate ratio = 9:91. 2T = treatment. View Large However, SCFA concentrations increased from the time of feeding achieving peak concentrations between 6 and 8 h postchallenge (P < 0.001). Ruminal acetate concentrations remained elevated between 6 and 20 h after challenge induction (Table 6; P < 0.001). Ruminal propionate (P < 0.001), butyrate (P < 0.001), total SCFA (P < 0.001), and lactate concentrations (P = 0.027) all peaked at 8 h after challenge induction. Ruminal fluid osmolality reached a maximum at h 6, 8, and 16 following the barley infusion (P < 0.001). The mean and maximum daily ruminal lactate concentrations ranged from 0.3 to 30.5 mM and 1.0 to 76.4 mM, respectively. Dry Matter Intake and Ruminal Fermentation during the First and Second Recovery Periods The effect of treatment and day on DMI and pH after the CHAL was investigated within REC1 (Table 7) to determine whether the duration of time fed a high-concentrate diet influenced the recovery response from an induced bout of ruminal acidosis. There was no effect of treatment on DMI or pH variables during REC1 (P > 0.10). Table 7. Mean daily DMI and ruminal pH during recovery (REC1) after an induced acidosis challenge. Long-adapted (LA) heifers were fed the high-grain diet1 for 34 d compared to 8 d for the short-adapted (SA) heifers.   Treatment    Day of recovery (REC1)2    P value3  Item  LA  SA  SEM  1  3  4  5  6  7  8  SEM  T  D  T × D  DMI, kg  9.1  9.5  0.56  8.0cd  8.6d  8.8bcd  9.9abc  9.5abcd  10.0ab  10.2a  0.48  0.69  0.001  0.38  Minimum pH  5.16  5.25  0.043  5.07  5.26  5.18  5.26  5.28  5.2  5.19  0.064  0.17  0.21  0.088  Mean pH  5.87  5.84  0.039  5.75  5.86  5.78  5.94  5.94  5.91  5.81  0.058  0.64  0.012  0.31  Maximum pH  6.56  6.49  0.053  6.44  6.51  6.52  6.60  6.56  6.53  6.54  0.051  0.39  0.54  1.00  pH < 5.5      Duration, min/d  297  274  56.0  458  281  407  192  167  167  326  75.8  0.78  0.019  0.085      Area, (min × pH)/d  83  71  23.2  178ab  61ab  106a  42b  32ab  44ab  76ab  28.6  0.70  0.018  0.37    Treatment    Day of recovery (REC1)2    P value3  Item  LA  SA  SEM  1  3  4  5  6  7  8  SEM  T  D  T × D  DMI, kg  9.1  9.5  0.56  8.0cd  8.6d  8.8bcd  9.9abc  9.5abcd  10.0ab  10.2a  0.48  0.69  0.001  0.38  Minimum pH  5.16  5.25  0.043  5.07  5.26  5.18  5.26  5.28  5.2  5.19  0.064  0.17  0.21  0.088  Mean pH  5.87  5.84  0.039  5.75  5.86  5.78  5.94  5.94  5.91  5.81  0.058  0.64  0.012  0.31  Maximum pH  6.56  6.49  0.053  6.44  6.51  6.52  6.60  6.56  6.53  6.54  0.051  0.39  0.54  1.00  pH < 5.5      Duration, min/d  297  274  56.0  458  281  407  192  167  167  326  75.8  0.78  0.019  0.085      Area, (min × pH)/d  83  71  23.2  178ab  61ab  106a  42b  32ab  44ab  76ab  28.6  0.70  0.018  0.37  a,b,c,dMeans within a dependent variable differ (P ≤ 0.050). Tukey was used for mean separation. For mean pH and duration of pH < 5.5, mean separation was not achieved despite a significant effect of day. 1Forage:concentrate ratio = 9:91 2REC1 = recovery 1 measurement period that started 24 h after the acidosis challenge and consisted of 8 d. Day 2 was the day of the temporarily isolated and washed reticulorumen technique. Due to manipulation of the rumen, DMI and pH data were not included. 3T = treatment; D = day of recovery. View Large Table 7. Mean daily DMI and ruminal pH during recovery (REC1) after an induced acidosis challenge. Long-adapted (LA) heifers were fed the high-grain diet1 for 34 d compared to 8 d for the short-adapted (SA) heifers.   Treatment    Day of recovery (REC1)2    P value3  Item  LA  SA  SEM  1  3  4  5  6  7  8  SEM  T  D  T × D  DMI, kg  9.1  9.5  0.56  8.0cd  8.6d  8.8bcd  9.9abc  9.5abcd  10.0ab  10.2a  0.48  0.69  0.001  0.38  Minimum pH  5.16  5.25  0.043  5.07  5.26  5.18  5.26  5.28  5.2  5.19  0.064  0.17  0.21  0.088  Mean pH  5.87  5.84  0.039  5.75  5.86  5.78  5.94  5.94  5.91  5.81  0.058  0.64  0.012  0.31  Maximum pH  6.56  6.49  0.053  6.44  6.51  6.52  6.60  6.56  6.53  6.54  0.051  0.39  0.54  1.00  pH < 5.5      Duration, min/d  297  274  56.0  458  281  407  192  167  167  326  75.8  0.78  0.019  0.085      Area, (min × pH)/d  83  71  23.2  178ab  61ab  106a  42b  32ab  44ab  76ab  28.6  0.70  0.018  0.37    Treatment    Day of recovery (REC1)2    P value3  Item  LA  SA  SEM  1  3  4  5  6  7  8  SEM  T  D  T × D  DMI, kg  9.1  9.5  0.56  8.0cd  8.6d  8.8bcd  9.9abc  9.5abcd  10.0ab  10.2a  0.48  0.69  0.001  0.38  Minimum pH  5.16  5.25  0.043  5.07  5.26  5.18  5.26  5.28  5.2  5.19  0.064  0.17  0.21  0.088  Mean pH  5.87  5.84  0.039  5.75  5.86  5.78  5.94  5.94  5.91  5.81  0.058  0.64  0.012  0.31  Maximum pH  6.56  6.49  0.053  6.44  6.51  6.52  6.60  6.56  6.53  6.54  0.051  0.39  0.54  1.00  pH < 5.5      Duration, min/d  297  274  56.0  458  281  407  192  167  167  326  75.8  0.78  0.019  0.085      Area, (min × pH)/d  83  71  23.2  178ab  61ab  106a  42b  32ab  44ab  76ab  28.6  0.70  0.018  0.37  a,b,c,dMeans within a dependent variable differ (P ≤ 0.050). Tukey was used for mean separation. For mean pH and duration of pH < 5.5, mean separation was not achieved despite a significant effect of day. 1Forage:concentrate ratio = 9:91 2REC1 = recovery 1 measurement period that started 24 h after the acidosis challenge and consisted of 8 d. Day 2 was the day of the temporarily isolated and washed reticulorumen technique. Due to manipulation of the rumen, DMI and pH data were not included. 3T = treatment; D = day of recovery. View Large Dry matter intake increased linearly (P < 0.001; data not shown) from d 1 to d 8, culminating in the greatest value on d 8 (P = 0.001; Table 7). A tendency for a treatment × day interaction for minimum pH (P = 0.088) indicated that minimum pH tended to increase to a greater extent between d 1 and d 3 for LA cattle but reached a greater value for SA cattle on d 6 and 7 (data not shown). Although there was no effect of day for maximum pH, mean pH reached its greatest values on d 5 and 6 (P = 0.012) while minimum values for DUR (P = 0.019) and AREA (P = 0.018) were observed on d 7 and 5, respectively. A tendency for a treatment × day interaction for DUR (P = 0.085) indicated that DUR tended to decrease between d 1 and d 3 for LA cattle and instead increased from d 1 to d 4 for SA cattle (data not shown). Ruminal SCFA concentrations were not affected by treatment or the treatment × hour interaction during REC1 (Table 8). However, SCFA concentration and osmolality increased over time after feeding with peak concentrations observed between 6 and 12 h postfeeding. Ruminal osmolality reached a peak at 6 and 12 h postfeeding. Table 8. Ruminal short-chain fatty acid (SCFA) and osmolality during recovery (REC1) after an induced acidosis challenge. Long-adapted (LA) heifers were fed the high-grain diet1 for 34 d compared to 8 d for the short-adapted (SA) heifers.   Treatment    Hour relative to feeding    P value2  Item  LA  SA  SEM  0  2  4  6  8  10  12  SEM  T  h  T × h  Total SCFA, mM  120.8  124.6  3.15  104.2c  117.7b  126.0ab  130.5ab  122.6b  120.6b  137.2a  3.80  0.41  <0.001  0.63  Acetate, mM  64  63  1.3  54.7c  62.7ab  65.5ab  67.3ab  62.6ab  62.2bc  69.3a  1.77  0.58  <0.001  0.65  Propionate, mM  42.4  45.9  5.17  37.5d  41.2bde  45.7ac  47.0abc  44.8abcd  42.9cde  49.9ab  3.95  0.64  <0.001  0.35  Butyrate, mM  14.4  15.9  2.21  12.0bd  13.9abc  14.9abc  16.3ac  15.4abc  15.6cd  18.0ab  1.77  0.64  <0.001  0.97  Osmolality, mOsm/kg  368  372  7.3  344b  368ab  373ab  383a  367ab  369ab  387a  8.4  0.73  0.002  0.68    Treatment    Hour relative to feeding    P value2  Item  LA  SA  SEM  0  2  4  6  8  10  12  SEM  T  h  T × h  Total SCFA, mM  120.8  124.6  3.15  104.2c  117.7b  126.0ab  130.5ab  122.6b  120.6b  137.2a  3.80  0.41  <0.001  0.63  Acetate, mM  64  63  1.3  54.7c  62.7ab  65.5ab  67.3ab  62.6ab  62.2bc  69.3a  1.77  0.58  <0.001  0.65  Propionate, mM  42.4  45.9  5.17  37.5d  41.2bde  45.7ac  47.0abc  44.8abcd  42.9cde  49.9ab  3.95  0.64  <0.001  0.35  Butyrate, mM  14.4  15.9  2.21  12.0bd  13.9abc  14.9abc  16.3ac  15.4abc  15.6cd  18.0ab  1.77  0.64  <0.001  0.97  Osmolality, mOsm/kg  368  372  7.3  344b  368ab  373ab  383a  367ab  369ab  387a  8.4  0.73  0.002  0.68  a,b,c,dMeans within a dependent variable differ (P ≤ 0.050). The Tukey post hoc test was used for mean separation. 1Forage:concentrate ratio = 9:91. 2T = treatment. View Large Table 8. Ruminal short-chain fatty acid (SCFA) and osmolality during recovery (REC1) after an induced acidosis challenge. Long-adapted (LA) heifers were fed the high-grain diet1 for 34 d compared to 8 d for the short-adapted (SA) heifers.   Treatment    Hour relative to feeding    P value2  Item  LA  SA  SEM  0  2  4  6  8  10  12  SEM  T  h  T × h  Total SCFA, mM  120.8  124.6  3.15  104.2c  117.7b  126.0ab  130.5ab  122.6b  120.6b  137.2a  3.80  0.41  <0.001  0.63  Acetate, mM  64  63  1.3  54.7c  62.7ab  65.5ab  67.3ab  62.6ab  62.2bc  69.3a  1.77  0.58  <0.001  0.65  Propionate, mM  42.4  45.9  5.17  37.5d  41.2bde  45.7ac  47.0abc  44.8abcd  42.9cde  49.9ab  3.95  0.64  <0.001  0.35  Butyrate, mM  14.4  15.9  2.21  12.0bd  13.9abc  14.9abc  16.3ac  15.4abc  15.6cd  18.0ab  1.77  0.64  <0.001  0.97  Osmolality, mOsm/kg  368  372  7.3  344b  368ab  373ab  383a  367ab  369ab  387a  8.4  0.73  0.002  0.68    Treatment    Hour relative to feeding    P value2  Item  LA  SA  SEM  0  2  4  6  8  10  12  SEM  T  h  T × h  Total SCFA, mM  120.8  124.6  3.15  104.2c  117.7b  126.0ab  130.5ab  122.6b  120.6b  137.2a  3.80  0.41  <0.001  0.63  Acetate, mM  64  63  1.3  54.7c  62.7ab  65.5ab  67.3ab  62.6ab  62.2bc  69.3a  1.77  0.58  <0.001  0.65  Propionate, mM  42.4  45.9  5.17  37.5d  41.2bde  45.7ac  47.0abc  44.8abcd  42.9cde  49.9ab  3.95  0.64  <0.001  0.35  Butyrate, mM  14.4  15.9  2.21  12.0bd  13.9abc  14.9abc  16.3ac  15.4abc  15.6cd  18.0ab  1.77  0.64  <0.001  0.97  Osmolality, mOsm/kg  368  372  7.3  344b  368ab  373ab  383a  367ab  369ab  387a  8.4  0.73  0.002  0.68  a,b,c,dMeans within a dependent variable differ (P ≤ 0.050). The Tukey post hoc test was used for mean separation. 1Forage:concentrate ratio = 9:91. 2T = treatment. View Large Data for REC2 is not shown as there were no treatment × day or treatment effects and mean values for REC2 did not differ from REC1 (Table 3). Regression Analysis Detects Differences in the Recovery Response after Ruminal Acidosis but not for the Susceptibility to Ruminal Acidosis Although treatment differences were not detected within BASE or CHAL, regression analysis was used to comprehensively evaluate the effect of treatment on the slope of the responses for the DUR before and leading into the CHAL and during REC1. Regression analysis, accounting for repeated measures, is a more appropriate method of detecting a change in a variable over time than mean separation. From Fig. 2, it is evident that over the 9 d BASE and CHAL, DUR was more variable in SA than LA heifers among days. The fitted data indicated significant treatment differences between the y-intercepts (P = 0.047), linear slopes (P = 0.007), quadratic coefficients (P = 0.003), and cubic coefficients (P = 0.001) with trend line equations of y = 567.4 + 48.7 d – 23.1 d2 + 2.2 d3 for LA and y = –173.6 + 653.1 d – 150.5 d2 + 9.8 d3 for SA. In support of this observed increase in between-day variability in pH, SA heifers also tended to have a greater SD in the duration of time that pH was <5.5 over the 8 d considered in this analysis when compared to LA heifers (data not shown; P = 0.096). The negative intercept observed for SA heifers is likely a response arising from being fed the high-grain diet for fewer days (i.e., d 1 was the first day SA heifers were fed the high-grain diet) and greater pH when fed the previous diet. Figure 2. View largeDownload slide Change in the duration of time that pH < 5.5 over the days leading up to and including the induced challenge. Long-adapted (LA) heifers were fed the high-grain diet for 26 d before this measurement period; short-adapted (SA) heifers received the high-grain diet on d 1 of the baseline period. Trend lines were constructed using analysis of covariance. Daily means were used from the 7 d during baseline (BASE; d 5 = day where the temporarily isolated and washed reticulo-rumen technique was performed and as a result pH data were removed.) and the challenge day depicted as C in the figure above.. Data from the day of restriction (R) was not included in the analysis. Significant treatment differences were found between y-intercepts (P = 0.047), linear slopes (P = 0.007), quadratic coefficients (P = 0.003), and cubic coefficients (P = 0.001). The resulting equations, therefore, account for a significant amount of variation in the dependant variable and were used in constructing the trend lines: y = 567.4 + 48.7 d – 23.1 d2 + 2.2 d3 (LA) and y = –173.6 + 653.1 d – 150.5 d2 + 9.8 d3 (SA). Figure 2. View largeDownload slide Change in the duration of time that pH < 5.5 over the days leading up to and including the induced challenge. Long-adapted (LA) heifers were fed the high-grain diet for 26 d before this measurement period; short-adapted (SA) heifers received the high-grain diet on d 1 of the baseline period. Trend lines were constructed using analysis of covariance. Daily means were used from the 7 d during baseline (BASE; d 5 = day where the temporarily isolated and washed reticulo-rumen technique was performed and as a result pH data were removed.) and the challenge day depicted as C in the figure above.. Data from the day of restriction (R) was not included in the analysis. Significant treatment differences were found between y-intercepts (P = 0.047), linear slopes (P = 0.007), quadratic coefficients (P = 0.003), and cubic coefficients (P = 0.001). The resulting equations, therefore, account for a significant amount of variation in the dependant variable and were used in constructing the trend lines: y = 567.4 + 48.7 d – 23.1 d2 + 2.2 d3 (LA) and y = –173.6 + 653.1 d – 150.5 d2 + 9.8 d3 (SA). The tendencies observed for the treatment × day interaction for minimum pH (P = 0.088) and DUR (P = 0.085) during REC1 can both be interpreted to suggest that the LA heifers recovered more quickly from the CHAL. During REC1 (Fig. 3) the LA cattle experienced a greater linear reduction in the duration of time that pH was <5.5 over the first few days after the challenge (Fig. 3; –588 vs. 369 min/d; P = 0.019). Figure 3. View largeDownload slide Change in the duration of time that pH < 5.5 over the days following the induced challenge. Long-adapted (LA) heifers were fed the high-grain diet for 34 d compared to 8 d for the short-adapted (SA) heifers. Trend lines were constructed using analysis of covariance. Daily means were used from the first 7 d following the induced challenge (REC1; d 2 = the day that the temporarily isolated and washed reticulo-rumen technique was performed and therefore pH data were removed.). Significant treatment differences were found between y-intercepts (P = 0.048), linear slopes (P = 0.019), quadratic coefficients (P = 0.026), and cubic coefficients (P = 0.037). The resulting equations, therefore, account for a significant amount of variation in the dependant variable and were used in constructing the trend lines: y = 1,176.4 – 587.8 d + 100.9d2 – 5.06 d3 (LA) and y = 162.8 + 369.4 d – 123.7 d2 + 10.0 d3 (SA). Figure 3. View largeDownload slide Change in the duration of time that pH < 5.5 over the days following the induced challenge. Long-adapted (LA) heifers were fed the high-grain diet for 34 d compared to 8 d for the short-adapted (SA) heifers. Trend lines were constructed using analysis of covariance. Daily means were used from the first 7 d following the induced challenge (REC1; d 2 = the day that the temporarily isolated and washed reticulo-rumen technique was performed and therefore pH data were removed.). Significant treatment differences were found between y-intercepts (P = 0.048), linear slopes (P = 0.019), quadratic coefficients (P = 0.026), and cubic coefficients (P = 0.037). The resulting equations, therefore, account for a significant amount of variation in the dependant variable and were used in constructing the trend lines: y = 1,176.4 – 587.8 d + 100.9d2 – 5.06 d3 (LA) and y = 162.8 + 369.4 d – 123.7 d2 + 10.0 d3 (SA). DISCUSSION In North American feedlots, beef cattle are typically fed diets with a high proportion of grain to maximize energy intake and improve performance and feed efficiency (Owens et al., 1997). However, feeding highly fermentable diets increases the risk for ruminal acidosis (Aschenbach et al., 2011) as rates of acid production in the rumen may exceed rates of acid removal (Penner et al., 2009c). While prevalence rates for ruminal acidosis in feedlot cattle are not currently available, several studies using continuous ruminal pH measurement in feedlot cattle indicate that based on a threshold pH value of 5.5, the risk for ruminal acidosis and likelihood of cattle experiencing ruminal acidosis is high (Wierenga et al., 2010; Li et al., 2011; Moya et al., 2011). In addition, acute ruminal acidosis is primarily characterized by an increase in ruminal lactate concentration (Nocek, 1997; Nagaraja and Titgemeyer, 2007; Aschenbach et al., 2011). For these reasons, we used the DUR and AREA that pH was <5.5 to detect ruminal acidosis (both subacute and acute) and ruminal fluid lactate concentrations to detect acute ruminal acidosis. A number of studies have investigated changes in ruminal pH, SCFA, and lactate concentrations that occur in beef cattle subjected to an induced bout of ruminal acidosis (Hibbard et al., 1995; Goad et al., 1998; Brown et al., 2000). However, none of those studies have examined the DUR and AREA of ruminal pH depression in beef cattle following feed restriction and a grain challenge using a continuous indwelling ruminal pH measurement system (Dado and Allen, 1993; Penner et al., 2006, 2009b). Continuous measurement of pH has been widely used in dairy cattle (Krause and Oetzel, 2005; Dohme et al., 2008; Khafipour et al., 2009) and has facilitated the growing knowledge gap between the characterization of ruminal acidosis in dairy cattle (Nocek, 1997; Krause and Oetzel, 2006; Kleen and Cannizzo, 2012) and beef cattle (Huber, 1976; Owens et al., 1998; Nagaraja and Titgemeyer, 2007). As the dietary inclusion rates of cereal grains are much higher in finishing diets for beef cattle relative to diets fed to lactating dairy cattle, it is important that comprehensive measurement approaches are used to improve our understanding of ruminal acidosis in general and to improve our knowledge regarding the severity and prevalence of ruminal acidosis in beef cattle. Acute Ruminal Acidosis was Effectively Induced Our challenge model was successful in decreasing mean and minimum ruminal pH and increasing both the duration and area that pH was <5.5. However, the extent of ruminal pH depression and lactate accumulation was more severe than most previous studies implementing a grain challenge to induce ruminal acidosis in dairy (Krause and Oetzel, 2005; Dohme et al., 2008; Khafipour et al., 2009) and beef (Burrin and Britton, 1986; Goad et al., 1998) cattle. In fact, the challenge in the current study was more in line with a challenge model using ruminal glucose infusion (Harmon et al., 1985) or consecutive challenges (Nagaraja et al., 1985; Coe et al., 1999). The severe challenge induced in the current study was likely the result of low ruminal pH for heifers when fed the basal diet. In fact, the mean duration pH was <5.5 was 531 min/d during BASE. This prolonged duration of pH depression during BASE was greater than the severity of subacute ruminal acidosis induced in dairy cattle by Keunen et al. (2002), Osborne et al. (2004), and Gozho et al. (2007) and was similar in severity to the grain challenge imposed on hay-adapted and grain-adapted steers by Goad et al. (1998) as well as the most severe challenge induced by Dohme et al. (2008) in lactating dairy cattle (9.5 h/d that pH was <5.5). Therefore, it is clear that the heifers in our study were already coping with a substantial acidotic challenge before the induced bout of acidosis. The severity of ruminal acidosis observed during BASE was caused primarily by the high fermentability of the high-grain diet and was similar to the severity of acidosis observed in other studies feeding similar high-grain barley-based finishing diets to beef cattle (Bevans et al., 2005; Wierenga et al., 2010; Moya et al., 2011). This severely depressed pH can greatly increase the risk of an acute bout of ruminal acidosis, as is evident by the severity of our challenge and studies that have investigated multiple challenges as part of an acidosis induction protocol in cattle (Nagaraja et al., 1985; Coe et al., 1999; Dohme et al., 2008). The current findings in combination with past studies underline the importance of continued work with indwelling pH measurement systems and beef cattle fed finishing feedlot diets. It is evident that we induced acute acidosis. The nearly doubling of the DUR during CHAL relative to that during BASE and the observed minimum pH of 4.57 are supported by the high ruminal fluid lactate concentrations. Because the observed minimum pH was below the pKa of SCFA (4.8 to 4.9; Aschenbach et al., 2011), the SCFA would have actually been acting as bases by stabilizing pH to approximately 4.8. However, the observed concomitant increase in lactate concentration (pKa = 3.9; Aschenbach et al., 2011) ensured that pH continued to plummet after maximum SCFA and lactate concentrations were observed. When both SCFA and lactate concentrations peaked at 8 h after the challenge, SCFA concentrations were eightfold greater than lactate concentrations. Over the next 8 h, a shift in organic acid accumulation and presumably production resulted in a decrease in SCFA concentrations while lactate concentration remained elevated. However, the concentration of SCFA in the ruminal fluid remained sixfold greater than lactate at 16 h after the challenge. While for subacute ruminal acidosis, high production rates and dissociation of SCFA is clearly the driving factor for low ruminal pH, the observed response is an example of when ruminal lactate becomes a more effective organic acid at reducing ruminal pH than the more abundant SCFA. Duration of Time Cattle are Fed the High-Grain Diet Influences Variability in Ruminal pH One major challenge with feeding cattle high-grain diets is the variation in the ruminal pH response among cattle, even when fed the same diet (Brown et al., 2000; Bevans et al., 2005; Nagaraja and Titgemeyer, 2007). Leading up to and including the challenge, we observed that SA heifers had greater variability for DUR across days within BASE (based on the quadratic and cubic regression coefficients), suggesting that the short duration on the high-grain diet may have increased the between-day variability in ruminal pH buffering, SCFA production, or eating behavior. Greater day-to-day variation in ruminal pH for the SA cattle was observed despite a lack of a treatment effect on the between-day rate of change for DMI. Bevans et al. (2005) reported that dramatic differences in the variation in ruminal pH occurred in feedlot cattle during a dietary transition protocol to a high-grain diet. They also demonstrated that accelerating the rate of dietary transition to a high-grain finishing diet increased the variability in the ruminal pH response without affecting daily DMI. The decreased pH variability for LA relative to SA heifers observed in our study and that of Bevans et al. (2005) for cattle that were provided a gradual dietary adaptation suggests that extending the time on feed may improve the ability to resist dramatic changes in ruminal pH that are inherent when consuming a high-grain diet. However, the day-to-day variation in pH does not appear to be linked to similar variation in DMI indicating that ruminal acid production, buffering strategies, or meal patterns and amounts may differ for cattle exposed to SA and LA. This finding also presents a new opportunity to assess the between day variability in ruminal pH as an appropriate indicator for cattle experiencing ruminal acidosis. Increasing the Duration of Time Fed a High Grain Diet does not Affect Risk for Ruminal Acidosis but Hastens the Recovery Response While numerous studies have investigated ruminal acidosis (Dohme et al., 2008; Khafipour et al., 2009; Penner et al., 2009a), few have evaluated risk for ruminal acidosis as affected by dietary adaptation or the recovery response following an episode of ruminal acidosis (Gaebel and Martens, 1988; Krehbiel et al., 1995; Zhang et al., 2013). Our results clearly show that while variation in DUR was greater for SA than LA, the severity of the induced acidosis did not differ by treatment. This indicates that the duration of time for adaptation to the high-grain diet does not influence the risk for ruminal acidosis. Based on the recovery pattern following the induced bout of acidosis, LA cattle experienced their lowest daily DUR after approximately 4 d while it took the SA cattle approximately 6 d to experience their lowest daily DUR. The mean daily DUR was 294 min/d when averaged over both treatment groups; however, it took the LA heifers 3 d to reduce the DUR below 294 min/d while SA heifers required 5 d. The reduced amount of time required for the LA heifers to reach mean and minimum values for DUR during the first week of recovery from the induced challenge can be appropriately quantified by their greater linear decrease for DUR during this time (Fig. 3). This is the first study, known to the authors, to suggest that time on high-grain feed may decrease the time necessary for beef cattle to recover from an induced bout of acute ruminal acidosis. Epithelial adaptation to high-grain diets (Thorlacius and Lodge, 1973; Gäbel et al., 1991; Sehested et al., 2000) has been suggested to increase the buffering capacity of the rumen. Therefore, increased rates of SCFA absorption with advancing days on the high-grain diet offers one potential explanation for the observed reduced recovery time in LA cattle, and is addressed in our companion study (Schwaiger et al., 2013). In addition, it is also possible that advancing days on high-grain feed may have stabilized the microbial communities (Mackie et al., 1978; Mackie and Gilchrist, 1979; Counotte et al., 1981) or altered eating behavior. For example, the populations of lactate-utilizing bacteria may have increased to help prevent lactate accumulation during the days following CHAL (Goad et al., 1998); however, we did not evaluate whether microbial composition differed among treatments and over time and therefore cannot confirm or refute this mechanism. It was recently reported by Zhang et al. (2013) that the severity of feed restriction influences the rate of recovery for DMI and ruminal pH when beef cattle resume ad libitum consumption of a diet containing 40% concentrate. In that study, heifers that were subjected to an imposed feed restriction experienced elevated DUR and AREA when ad libitum feeding resumed. The recovery, with respect to DMI and ruminal pH, from the feed restriction challenge imposed by Zhang et al. (2013) was complete by the second week of ad libitum feeding and was greatest for those cattle who were subjected to the greatest severity of imposed feed restriction. The challenge induced in the current study was much more severe than that of Zhang et al. (2013), as was the time necessary for pH to stabilize. Taken together with the results of the current study, it can be hypothesized that the severity of the ruminal acidosis bout may positively correlate to the number of days required for recovery of ruminal pH and that cattle exposed to more time for adaptation before acute ruminal acidosis may have a greater ability to recover following a bout of ruminal acidosis. Conclusions Although the severity of acidosis was equivalent across treatments, heifers that spent more time on a high-concentrate diet before the bout of imposed ruminal acidosis exhibited decreased between-day variation in ruminal pH (based on the quadratic and cubic regression coefficients) without differences in DMI. However, the observed increased stability in ruminal pH leading up to the challenge did not influence the susceptibility to a bout of induced ruminal acidosis. 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Google Scholar CrossRef Search ADS PubMed  Footnotes 1 Funding for the project was provided through the Alberta Livestock and Meat Agency and Agriculture and Agri-Food Canada. American Society of Animal Science

Journal

Journal of Animal Science – Oxford University Press

Published: Dec 1, 2013

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