Effects of abomasally infused rumen fluid from corn-challenged donor cows on production, metabolism, and inflammatory biomarkers in healthy recipient cows

Subacute rumen acidosis may cause postruminal intestinal barrier dysfunction, but this does not appear to be due to increased hindgut fermentation. Alterna-tively, intestinal hyperpermeability may be explained by the plethora of potentially harmful substances (e.g., ethanol, endotoxin, and amines) produced in the rumen during subacute rumen acidosis, which are difficult to isolate in traditional in vivo experiments. Therefore, objectives were to evaluate whether abomasal infusion of acidotic rumen fluid collected from donor (Donor) cows elicits systemic inflammation or alters metabolism or production in healthy recipients. Ten rumen-cannulated lactating dairy cows [249 ± 63 d in milk; 753 ± 32 kg of body weight (BW)] were randomly assigned to 1 of 2 abomasal infusion treatments: (1) healthy rumen fluid (HF; 5 L/h; n = 5) or (2) acidotic rumen fluid (AF; 5 L/h; n = 5) infused. Eight rumen-cannulated cows [4 dry, 4 lactating (lactating = 391 ± 220 d in milk); 760 ± 70 kg of BW] were used as Donor cows. All 18 cows were acclimated to a high-fiber diet (46% neutral detergent fiber; 14% starch) during an 11-d prefeeding period during which rumen fluid was collected for the eventual infusion into HF cows. During period (P) 1 (5 d), baseline data were obtained and on d 5 Donor were corn-challenged (2.75% BW ground corn after 16 h of 75% feed restriction). Cows were fasted until 36 h relative to rumen acidosis induction (RAI), and data were collected through 96 h RAI. At 12 h RAI, an additional 0.50% BW of ground corn was added, and acidotic fluid collections began (7 L/Donor every 2 h; 6 M HCl was added to collected fluid until pH was between 5.0 and 5.2). On d 1 of P2 (4 d), HF/AF cows were abomasally infused with their respective treatments for 16 h, and data were collected for 96 h relative to the first infusion. Data were analyzed in SAS (SAS Institute Inc.) using PROC MIXED. Following the corn challenge in the Donor cows, rumen pH only mildly decreased at nadir (pH = 5.64 at 8 h RAI) and remained above the desired threshold for both acute (5.2) and subacute (5.6) acidosis. In contrast, fecal and blood pH markedly decreased to acidotic levels (nadir = 4.65 and 7.28 at 36 and 30 h RAI, respectively), and fecal pH remained below 5 from 22 to 36 h RAI. In Donor cows, dry matter intake remained decreased through d 4 (36% relative to baseline) and serum amyloid A and lipopolysaccharide-binding protein markedly increased by 48 h RAI in Donor cows (30-and 3-fold, respectively). In cows that received the abomasal infusions, fecal pH decreased in AF from 6 to 12 h relative to the first infusion (7.07 vs. 6.33) compared with HF; however, milk yield, dry matter intake, energy-corrected milk, rectal temperature, serum amyloid A, and lipopolysaccharide-binding protein were unaffected. Overall, the corn challenge did not cause subacute rumen acidosis but markedly decreased fecal and blood pH and stimulated a delayed inflammatory response in the Donor cows. Abomasal infusion of rumen fluid from corn-challenged Donor cows decreased fecal pH but did not cause inflammation, nor did it create an immune-activated phenotype in recipient cows.


INTRODUCTION
The pathological etiology involved in the negative sequelae associated with SARA (e.g., reduced feed intake and systemic inflammation; Kleen et al., 2003;Plaizier et al., 2022) remains mechanistically undetermined.Several reports have established a relationship between SARA and intestinal hyperpermeability as indicated by an increase in circulating endotoxin concentrations (i.e., LPS; Gozho et al., 2005;Khafipour et al., 2009b;Li et al., 2016).However, to date there remains uncertainty about which GIT segments become more porous during Effects of abomasally infused rumen fluid from corn-challenged donor cows on production, metabolism, and inflammatory biomarkers in healthy recipient cows SARA (Gressley et al., 2011;Plaizier et al., 2022).The development of hyperkeratinization or lesions in the stratified squamous epithelium of the rumen suggests the rumen wall may become compromised with acute or prolonged acidosis (Steele et al., 2009;Minuti et al., 2014).However, responses of the rumen barrier to SARA are inconsistent (Khafipour et al., 2009a;Penner et al., 2010), and others hypothesize it may be less permeable to antigens than other regions of the gastrointestinal tract (GIT) (Nagaraja and Titgemeyer, 2007;Sanz-Fernandez et al., 2020).Further, the distal GIT lacks accessibility to major buffering mechanisms afforded to the rumen (i.e., saliva, protozoa) and is seemingly at an anatomical disadvantage considering its columnar epithelial structure (Gressley et al., 2011;Steele et al., 2016).These discrepancies were foundational for the hypothesis that hindgut acidosis (HGA) associated with increased postruminal starch flow may be an important contributor to inflammation during SARA (Khafipour et al., 2009a,b;Gressley et al., 2011;Sanz-Fernandez et al., 2020).However, despite some inconsistencies (Zust et al., 2000;Bissell and Hall, 2010), the majority of efforts to confirm HGA as a source of intestinal barrier dysfunction indicate that excessive hindgut fermentation in isolation from SARA does not cause an immune-activated phenotype (Mainardi et al., 2011;van Gastelen et al., 2021a,b;Abeyta et al., 2023a,b,c,d;Piantoni et al., 2022).
Notwithstanding the apparent tolerance of dairy cows to isolated HGA, there are reports of SARA causing postruminal barrier damage in ruminants.For instance, Lai and colleagues (2022) demonstrated increased cytokine gene expression, decreased tight junction protein expression, and a damaged small intestine epithelium during SARA.Additionally, structural damage, apoptosis, goblet cell loss, increased cytokine gene expression, and decreased tight junction protein expression have been reported in the colonic epithelium of high-concentrate-fed goats (Tao et al., 2014;Samo et al., 2020;Ma et al., 2022).These findings and those from the aforementioned isolated HGA experiments suggest that a mechanism other than increased hindgut fermentation may compromise the small and large bowel barrier during SARA.There are multiple potentially toxic compounds (e.g., ethanol, endotoxin, and bioactive amines; Slyter, 1976;Saleem et al., 2012) produced in acidotic rumen fluid that are capable of damaging the intestinal barrier (Aschenbach et al., 1998;Bala et al., 2014).These molecules may influence postruminal barrier function and compromise cow welfare and production, particularly if rumen acidosis were to develop in early lactation when cows are seemingly more vulnerable to inflammatory stressors (Trevisi and Minuti, 2018;Horst et al., 2021).Considering that immune activation diverts nutrients away from productive purposes (Johnson, 2012;Kvidera et al., 2017), there is economic merit in better characterizing the etiology of SARA-induced GIT barrier dysfunction.However, it is difficult to isolate the postruminal effects of acidosis using traditional in vivo SARA models.Therefore, we designed an experiment to evaluate the effects of abomasally infused acidotic rumen fluid collected from a subset of corn-challenged donor cows in a group of healthy, non-rumen-acidotic recipients.We hypothesized that the acidotic rumen fluid infusion would cause postruminal intestinal barrier dysfunction in the receiving cows, resulting in a systemic inflammatory response, altered metabolism, and reduced production.

MATERIALS AND METHODS
All procedures were approved by the Iowa State University Institutional Animal Care and Use Committee and were conducted at the Iowa State University Dairy Farm (Ames) in April 2021.Ten rumen-cannulated lactating Holstein cows (753 ± 32 kg of BW, 249 ± 63 DIM; parity 2.8 ± 0.6) were balanced by milk yield, DIM, parity, and presumed pregnancy status and assigned to 1 of 2 abomasal infusion treatments: (1) healthy rumen fluid controls (HF; 5 L of nonacidotic rumen fluid per hour; n = 5) or (2) acidotic rumen fluid (AF; 5 L of acidotic rumen fluid per hour; n = 5).Respective abomasal infusion treatments were infused for 16 h (see later discussion).Acidotic rumen fluid for the AF group was collected from a cohort of 8 rumencannulated Holstein cows [4 dry, 4 lactating (lactating: 391 ± 220 DIM); 760 ± 70 kg of BW; parity 2.3 ± 0.9], and these animals were designated as the donor (Donor) group.A detailed schematic of the experimental design and chronological order of infusions is provided in Figure 1.Throughout the experiment, all 18 cows were fed a high-forage diet primarily consisting of corn silage, straw, and alfalfa hay formulated to meet or exceed the predicted requirements (NRC, 2001; Table 1) of energy, protein, vitamins, and minerals for gestating dry cows, and water was provided ad libitum.Feed samples were collected weekly and stored at −20°C until they were later composited and submitted for chemical analysis using near-infrared spectroscopy (Dairyland Laboratories, Arcadia, WI).

Healthy Rumen Fluid Collections for Cows Destined to Receive the HF
All rumen fluid collections (healthy and acidotic) were facilitated using a transfaun-aider rumen fluid collection pump (MWA Manufacturing).Healthy (i.e., nonacidotic) rumen fluid was collected from all 18 cannulated cows during the prefeeding and acclimation periods (7 d total; Figure 1).We determined that collecting 8 L/d per cow was necessary to acquire an estimated 900 L of healthy fluid for our originally intended 36 h of abomasal infusions.Therefore, on each collection day, 8 L of rumen fluid was obtained from every cow and strained through a paint strainer (Reaves & Co. Inc.).Strained rumen contents were homogenized via manual mixing and stored at 4°C.Directly before infusions, pH was determined from the homogenized batch of healthy rumen fluid (pH = 6.81).

Experimental Timeline and Methods: Donor Cows
The Donor cows were housed in a free stall barn and fed using the Calan Broadbent feeding system (American Calan) to collect individual feed intake.The 4 lactating cows were milked twice daily (0700 and 1900 h) and yield was recorded.The Donor cows were acclimated to the high-fiber diet during an 11-d prefeeding period (P) before P1.Period 1 (5 d) generated baseline data during which ad libitum feed intake was determined for use during the acidosis challenge.On the fifth day of P1, Donor cows were provided only 25% of their baseline feed intake (calculated from d Baseline data were collected during P1, and on d 5 Donor cows were given a corn challenge (Acidosis Day) during which they were feed restricted to 25% of their ad libitum feed intake for 16 h before manual insertion of 2.75% BW finely ground corn at 0000 h on P2 d 1.After their initial feed allotment was consumed, Donor cows were fasted until 36 h and data were collected until 96 h relative to acidosis induction (RAI).At 12 h RAI, an additional 0.5% BW finely ground corn was manually inserted into the rumen, and rumen fluid collections (approximately 7 L every other hour) began to facilitate abomasal infusions for AF cows.Starting at 1330 h on d 1 of P2, HF and AF cows were abomasally infused with their respective treatments for 16 h and data were collected through 96 h relative to the first infusion (RFI).*Indicates when blood samples were obtained for respective groups.
1 to 4) at 0700 h.After 16 h of 25% baseline feed intake (until 0000 h on d 1 of P2), Donor cows were administered a corn challenge via the manual insertion of 2.75% BW of dry finely ground corn into the rumen fistula based on a modified protocol from Nagaraja et al. (1981).Once the provided allotment of feed was consumed, cows were fasted through 36 h relative to acidosis induction (RAI).Rumen fluid for pH monitoring was collected hourly from 0 to 36 h RAI, and again at 48, 60, 72, 84, and 96 h RAI.Fluid was collected manually from the cranial ventral, ventral, and caudal ventral regions of the rumen using a 50-mL conical tube and was thoroughly mixed in a plastic container before pH was measured with a calibrated portable pH meter (Orion Star A121, Thermo Fisher Scientific).
Our original plan was to monitor the rumen pH decline in Donor cows until at least 4 of the 8 Donor cows reached a rumen pH of ≤5.2, which has been described as one of several thresholds for acute rumen acidosis (Owens et al., 1998), and thereafter begin rumen fluid collections to facilitate abomasal infusions in AF cows.Based on our preliminary trial (Abeyta et al., 2021) and previous literature (Nagaraja et al., 1981), we anticipated this pH drop would occur within the first 4 to 10 h RAI.However, none of the Donor cows had a rumen pH of ≤5.2 by 11 h RAI.Thus, an additional 0.50% BW finely ground dry corn was delivered via the rumen fistula and we initiated rumen fluid collections regardless of rumen pH at 12 h RAI.
Starting at 12 h RAI, approximately 7 L of rumen fluid was collected from each Donor cow (a total of 56 L each collection) every other hour until 28 h RAI.A minimum of 50 L was required to facilitate 2 abomasal infusions in AF cows, which were being abomasally infused hourly (discussed later).Thus, if a cow was unable to donate the full 7 L, extra fluid was collected from the other Donor cows to equate to approximately 50 L total, and 3 L of a 1% NaCl solution (pH adjusted to between 5.0 and 5.2 using 2 M HCl) was immediately administered into the rumen of cows unable to make their complete donation (approximately 2 h before their next donation).Rumen fluid from all 8 Donor cows was strained through a paint strainer (Reaves & Co.) and thoroughly homogenized in a separate container, where pH was checked and recorded.If the pH was >5.2, 6 M HCl was added until pH was 5.20 ± 0.02.One-half of the rumen fluid (approximately 25 L) was then immediately abomasally infused into the AF cows.The remaining second half (approximately 25 L) was stored for 1 h, after which pH was checked and adjusted again (if necessary) before abomasal infusion.At 28 h RAI, the 8 Donor cows were unable to donate the full approximately 50 L required to facilitate abomasal infusions in AF cows; thus, rumen fluid collections ceased.

Experimental Timeline and Methods: HF and AF Cows
The HF and AF cows were housed in a free stall barn and acclimated to the high-fiber diet for 8 d.Following prefeeding, cows were moved to individual box stalls (4.57× 4.57 m) where they were allowed 3 d to acclimate to new housing conditions.Period 1 (5 d) generated baseline data for use as a covariate during P2.Starting at approximately 1330 h on d 1 of P2 (4 d), HF and AF cows were abomasally infused with 5 L of their respective rumen fluid treatments hourly for 16 h, after which production, blood, and pH metrics were obtained until 96 h relative to the first infusion (RFI).Abomasal infusion lines were composed of approximately 3 m of PVC-reinforced braided vinyl tubing (1.27 cm o.d., 0.635 cm i.d., Eastman Chemical Company) fitted with a plastisol flange at the end (approximately 11 cm diameter) to allow for continuous placement (Plastisol; Bar Diamond Inc.).The flange had several drilled holes (approximately 2 cm) to allow for fluid passage.Infusion lines were manually inserted into the reticulo-omasal orifice on d 2 of acclimation, where they remained throughout the experiment, and their positions were confirmed every other day and then immediately before the first infusion during P2.Infusions were facilitated using a 600-mL drench syringe (Valley Vet Supply).Following each infusion, 300 mL of tap water was infused to flush residual rumen fluid out of the lines.Each infusion lasted approximately 7 to 10 min.Throughout the experiment, HF and AF cows were milked twice daily (0600 and 1800 h) in their stalls and yield was recorded.Further, milk samples were collected following each milking during P1 and P2 and stored at 4°C with a preservative (bronopol; D & F Control System) until composition analysis by Dairy Lab Services (Dubuque, IA) using AOAC-approved infrared analysis equipment and procedures (AOAC International, 1995).Respiration rate and rectal temperature were collected following each milking.Respiration rate was determined by measuring flank movements for 15 s and multiplying by 4 to calculate breaths per minute.Rectal temperature was collected using a digital thermometer (GLA M700 Digital Thermometer).

Blood Analysis
Blood samples were obtained via coccygeal venipuncture from Donor cows at 0,8,16,24,30,36,48,72,and 96 h RAI,and at 0,8,16,24,48,72,and 96 h RFI for HF and AF cows.Plasma samples were collected in K 2 EDTA tubes (BD) and were subsequently centrifuged at 1,500 × g for 15 min at 4°C before being aliquoted into microcentrifuge tubes for storage at −20°C until analysis.An additional sample was collected in Donor cows in a tube containing lithium heparin (BD) for blood pH and gas analysis using an iSTAT handheld machine and cartridge (CG8+; Abbott Point of Care) at the previously discussed time points.

Fecal Analysis
All fecal samples (approximately 250 g wet basis) were manually collected via rectal grab throughout the experiment.After an initial −24 h sample, fecal pH was obtained every other hour from 0 to 36 h RAI, and again at 48, 60, 72, 84, and 96 h RAI in Donor cows.In HF/AF cows, fecal samples were obtained at −24 h, every other hour from 0 to 20 h RFI, and again at 24, 30, 36, 48, 72, and 96 h RFI.Fecal pH was measured using a 1:1 dilution method as previously described (Branstad et al., 2017).In brief, 50 g of fecal matter was mixed with 50 mL of double-distilled H 2 O and homogenized for 30 s using a stomacher (Lab-blender Stomacher 80, Seward Ltd.) and pH was immediately determined.Fecal scores were obtained before pH determination using a 1-to-5 manure scoring system (Zaaijer et al., 2001).

Statistical Analysis
All data were analyzed using the MIXED procedure of SAS version 9.4 (SAS Institute Inc.).Each animal's respective parameter was analyzed using repeated measures (represented as h or d) with an autoregressive covariance structure for DMI, milk yield, milk variables, ECM, thermal indices, and HF/AF inflammatory biomarkers (SAA and LBP), and spatial power for fecal and rumen pH, fecal score, blood gas parameters, blood pH, ionized calcium, hematocrit, metabolites, insulin, and Donor inflammatory biomarkers.For Donor cows, effects of time (h or d) were evaluated with lactation stage (dry vs. lactating) included as a fixed effect.Period 1 and P2 were analyzed separately, but to compare with baseline, an average of P1 data was included in an additional analysis with P2.For HF and AF cows, an average of each animal's respective P1 parameter was used as a covariate for analysis during P2.Effects of treatment, time (h or d), and treatment × time were assessed.To compare with baseline, an average of P1 data was included in an additional analysis with P2.All data were reported as least squares means and considered significant if P ≤ 0.05 and a tendency if 0.05 < P ≤ 0.10.

Donor Cows
Finely ground corn administration (approximately 25 kg/cow) steadily decreased Donor rumen pH from 1 to 8 h with the nadir occurring at 8 h RAI (pH = 5.64 vs. 6.82 at baseline; Figure 2A).However, unexpectedly, pH did not approach the desired threshold of 5.2, despite remaining decreased throughout the rest of P2 relative to baseline (0.79 units; P < 0.01).Interestingly, fecal pH responded to the corn challenge with strikingly more severity and began decreasing at 8 h RAI before reaching a low plateau from approximately 22 to 36 h RAI (4.75 vs. 6.97 during P1; 4.65 at the 36-h RAI nadir) and increasing again from 48 to 96 h (P < 0.01; Figure 2A).Notably, fecal pH of the Donor cows remained <5 for approximately 16 h during the plateau.Donor cow fecal score increased from 4 to 10 h RAI relative to baseline (0.7 units; P < 0.01; Figure 2B) before it began decreasing at 14 h RAI and plateaued from 18 to 36 h RAI (1.3 units relative to P1; P < 0.01).Like pH, fecal scores returned to baseline by 60 h RAI.
Blood pH progressively decreased (P < 0.01; Figure 3A) after the corn challenge, to the extent that the threshold for metabolic acidosis (blood pH <7.35; Berne and Levy, 1998) was met by 24 h RAI.At nadir (30 h RAI), blood pH began increasing from 7.28 until 96 h RAI, but pH values persistently stayed below P1 even at 96 h RAI (7.35 vs. 7.46).Blood base excess, bicarbonate, and total carbon dioxide concentrations followed very similar patterns in that they began decreasing at 8 h RAI and reached a plateau from 24 to 48 h RAI, with the nadirs occurring at 48 h RAI (15.6 mmol/L, 42%, and 41% relative to P1, respectively; P < 0.01; Figure 3B-D).Further, partial pressure of carbon dioxide decreased from 16 to 24 h RAI and reached a nadir at 48 h RAI (18% relative to P1; P = 0.02; Figure 3E), before gradually increasing through 96 h RAI.Ionized Ca increased following corn administration until it apexed at 30 h RAI (12% relative to P1; P < 0.01; Figure 3F).There were no alterations in Cows were challenged with finely ground corn (2.75% of BW) via manual insertion to the rumen following 16 h of feed restriction to 25% of ad libitum feed intake.An additional 0.5% BW of finely ground corn was added to the rumen at 12 h relative to acidosis induction (RAI), and cows were fasted through 36 h RAI.Rumen fluid collections (approximately 7 L every 2 h) began at 12 h RAI and continued through 28 h RAI.Data were analyzed using PROC MIXED, and effects time (h) were evaluated.Period (P) 1 represents an average of the −24 and 0 h RAI samples and was analyzed separately.Results are expressed as LSM ± SEM and considered significant if P ≤ 0.05 and a tendency if 0.05 < P ≤ 0.10.oxygen saturation (P > 0.24; Figure 3G); however, the partial pressure of oxygen was sharply and transiently increased (2.4-fold; P = 0.02; Figure 3H) at 48 h RAI relative to baseline.Blood hematocrit mildly increased from 8 to 24 h RAI before markedly increasing at 30 h RAI (16% relative to P1; P < 0.01; Figure 3I).After peak, hematocrit slowly returned to baseline concentrations by 72 h RAI., (E) partial pressure of carbon dioxide (pCO 2 ), (F) ionized Ca (iCa), (G) saturated oxygen (sO 2 ), (H) partial pressure of oxygen (pO 2 ), and (I) hematocrit concentrations following a corn challenge in donor dairy cows acclimated to a high-fiber diet (NDF: 46% of DM).Cows were challenged with finely ground corn (2.75% of BW) via manual insertion to the rumen following 16 h of feed restriction to 25% of ad libitum feed intake.An additional 0.5% BW of finely ground corn was added to the rumen at 12 h relative to acidosis induction (RAI), and cows remained fasted through 36 h RAI.Rumen fluid collections (approximately 7 L every 2 h) began at 12 h RAI and continued through 28 h RAI.Data were analyzed using PROC MIXED, and effects of time (h) were evaluated.Period (P) 1 represents the 0 h RAI sample and was analyzed separately.Results are expressed as LSM ± SEM and considered significant if P ≤ 0.05 and a tendency if 0.05 < P ≤ 0.10.By design, DMI was decreased (75% relative to d 1 to 4 of P1; P < 0.01; Figure 4) on d 5 of P1 for Donor cows, and it remained decreased throughout d 3 to 4 of P2 (36% relative to baseline; P < 0.01) despite returning feed at 1300 h on d 2. Milk yield also progressively decreased in the lactating Donor cows from d 1 to 3 (47% on d 3 relative to P1; P = 0.03; Figure 4) before starting to return toward baseline on d 4. Interestingly, circulating acute phase proteins SAA and LBP were markedly increased by 48 h RAI (30-and 3-fold relative to P1, respectively; P < 0.01; Figure 5A and B), and LBP began increasing by 24 h RAI, which was temporally aligned with the fecal pH plateau at 22 h RAI but not the rumen pH nadir at 8 h.

HF/AF Cows
Abomasal infusion of acidotic rumen fluid decreased fecal pH in AF cows starting at 8 h, and it reached nadir by 12 h RFI (fecal pH = 6.33 vs. 7.07 in HF; P < 0.01; Figure 6A), after which it returned to HF levels by 24 h.Abomasal rumen fluid infusions decreased fecal score for both AF and HF cows from 8 to 12 h RFI (1.8 units; P < 0.01; Figure 6B), before progressively increasing to baseline levels at 30 h RFI.Further, there was a treatment-by-time interaction for HF/AF cows such that it was increased at 8 h RFI (1.0 units) and decreased at 14 and 30 h RFI (both 0.9 units) in AF relative to HF cows (P = 0.05).However, abomasal infusions of acidotic rumen fluid had no impact on DMI or milk yield in AF cows (P > 0.77; Table 2).Similarly, ECM, milk fat, protein, and lactose contents, their yields, MUN, SCS, and rectal temperature were unaf- .Dry matter intake and milk yield following a corn challenge in donor dairy cows acclimated to a high-fiber diet (NDF: 46% of DM).Cows were challenged with finely ground corn (2.75% of BW) via manual insertion to the rumen following 16 h of feed restriction to 25% of ad libitum feed intake on period (P) 1 d 5 (analyzed separately).An additional 0.5% BW ground corn was added to the rumen at 12 h relative to acidosis induction (RAI), and cows were fasted through 36 h RAI.Rumen fluid collections (approximately 7 L every 2 h) began at 12 h RAI and continued through 28 h RAI.Data were analyzed using PROC MIXED, and effects of time (d) were evaluated.Period 1 represents an average of d 1 to 4 of P1 and was analyzed separately.Results are expressed as LSM ± SEM and considered significant if P ≤ 0.05 and a tendency if 0.05 < P ≤ 0.10.

Figure 5.
Circulating (A) serum amyloid A (SAA) and (B) LPSbinding protein (LBP) in corn-challenged donor cows acclimated to a high-fiber diet (NDF: 46% of DM).Cows were challenged with finely ground corn (2.75% of BW) via manual insertion to the rumen following 16 h of feed restriction to 25% of ad libitum feed intake.An additional 0.5% BW ground corn was added to the rumen at 12 h relative to acidosis induction (RAI), and cows were fasted through 36 h RAI.Rumen fluid collections (approximately 7 L every 2 h) began at 12 h RAI and continued through 28 h RAI.Data were analyzed using PROC MIXED, and effects of time (h) were evaluated.Period (P) 1 represents the 0 h RAI sample and was analyzed separately.Results are expressed as LSM ± SEM and considered significant if P ≤ 0.05 and a tendency if 0.05 < P ≤ 0.10.Values with differing lowercase letters denote differences (a-c = P ≤ 0.05) among days.
fected in AF relative to HF cows throughout P2 (P > 0.19; Table 2).There was a treatment-by-time interaction for respiration rate, which increased (5 breaths per minute; P = 0.02; Table 2) in AF cows on d 2 relative to HF cows but remained similar between treatments throughout the rest of the experiment.
There were no overall differences in circulating glucose for HF/AF cows (P > 0.25; Figure 7A); however, post hoc analysis revealed a tendency for glucose to be increased (9%; P = 0.06; Figure 7A) at 8 h RFI in AF relative to HF cows.Similarly, circulating insulin was increased at 8 h RAI in AF relative to HF cows (69%; P = 0.03; Figure 7B).Regardless of treatment, abomasal infusion of rumen fluid decreased circulating NEFA concentrations at 8 h RFI (46% relative to baseline; P = 0.05; Figure 7C).However, AF infusions decreased circulating NEFA throughout P2 relative to healthy fluid-infused cows (34%; P < 0.01; Figure 7C).Initially, circulating BHB was increased in both HF and AF cows at 8 h RFI (29% relative to baseline; P < 0.01; Figure 7D), after which concentrations were increased for AF cows throughout P2 relative to HF cows (15% increase; P = 0.01).There were no overall treatment differences in circulating BUN (P > 0.28; Figure 7E) throughout P2.Similarly, neither SAA nor LBP was altered for HF and AF cows due to treatment or time (P > 0.12; Figure 8A and B).

DISCUSSION
Inflammation is a reflection of suboptimal well-being and is detrimental to productivity; thus, it is important to better characterize potential sources of immune activation in dairy cows.Postruminal GIT barrier function appears to be compromised during rumen acidosis despite evidence that this damage does not emanate from moderate and isolated HGA (Mainardi et al., 2011;van Gastelen et al., 2021a,b;Abeyta et al., 2023a,b,c,d;Piantoni et al., 2022).Acidotic ruminal fluid contains many potentially toxic substances (e.g., endotoxin, ethanol, and bioactive amines; Slyter, 1976;Saleem et al., 2012), which have previously been associated with a variety of pathologies, including intestinal barrier dysfunction (Hietbrink et al., 2009;Bala et al., 2014).Thus, the deleterious impacts of acidotic ruminal fluid may weaken or damage the small and large intestinal barriers, a scenario that would contribute to the negative consequences associated with rumen acidosis (i.e., inflammation, decreased production; Kleen et al., 2003;Plaizier et al., 2022).However, it is difficult to alienate the regional sources of inflammation using traditional in vivo acidosis models.Identifying the GIT segments that are vulnerable to hyperpermeability is seemingly a prerequisite in developing strategies aimed at fortifying epithelial barrier defenses.Thus, objectives of the present study were to evaluate the effects of abomasal infusion of acidotic rumen fluid from corn-challenged donor cows on systemic inflammation, metabolism, and production in healthy lactating Holstein cows that did not have rumen acidosis.We hypothesized that acidotic rumen fluid would compromise the postruminal intestinal barrier and elicit a systemic immune response, alter metabolism, and create an immune-activated phenotype characterized by decreased milk production and increased rectal temperature in abomasally infused healthy recipients.
Despite abruptly administrating approximately 25 kg of finely ground corn, rumen pH in the Donor cows never reached the intended threshold for acute rumen acidosis (pH <5.2; Owens et al., 1998).Based on previous literature (Nagaraja et al., 1981) and a preliminary experiment (Abeyta et al., 2021), we anticipated ruminal pH would decrease to <5.2 by approximately 10 h RAI.However, none of the Donor cows' ruminal fluid reached a pH of <5.2 in the first 24 h, and only 1 of the 8 cows' ruminal fluid did so by 30 h RAI.In fact, although there are several defined thresholds, the pH nadir remained above 5.6, indicating our Donor cows did not even develop SARA (Krause and Oetzel, 2006;Plaizier et al., 2018).We anticipated some level of variation in response to the corn challenge, as others have reported substantial variation in individual cow response to grain challenges (Nagaraja and Titgemeyer, 2007;Penner et al., 2009;Petri et al., 2013).Reasons why we did not observe an acute pH response to corn administration are unclear.However, we did not add water to the ground corn (i.e., we did not create a "mash") before administering it, which is seemingly in contrast with methods from Nagaraja and colleagues (1981;personal communication, T. G. Nagaraja).Regardless, our ineffectiveness in creating a true ruminal acidotic environment limited our ability to thoroughly test the original hypothesis (that noxious compounds created during rumen acidosis cause damage to the postruminal epithelial barrier).
Despite adjusting the pH of the abomasally infused acidotic rumen fluid to between 5.0 and 5.2, there were no negative impacts of infusions on production, body temperature, or inflammatory metrics in the AF cows.This provides further evidence that low pH associated with SARA is likely not the causative driver in postruminal barrier damage, and that other characteristics (e.g., ethanol, endotoxin, and osmolarity) of rumen fluid may be more important mediators to consider.In addition to the lack of rumen acidosis in our Donor cows, another limitation in the current experiment was the shortened duration of abomasal infusions (16 h) from our original goal (36 h).We hypothesized that 24 to 36 h of abomasal infusions would be sufficient to detect an immune-activated phenotype based on findings from Danscher et al. (2011), who reported substantial increases in SAA and haptoglobin starting at approximately 24 h following acute acidosis induction by intraruminal oligofructose administration.In their study, a very mild increase in SAA was detected as early as 6 h RAI, but this effect became substantially more pronounced past 24 h RAI.Unfortunately, Donor cows were unable to supply the full quantity of rumen fluid required to continue with abomasal infusions past 16 h of collections.In addition to the limited availability of cannulated cows, it is likely that the lack of rumen acidosis developed contributed to the insufficient quantity of fluid for infusions.Acute ruminal acidosis causes an influx of fluids to the rumen pool Trt = treatment; HF = healthy rumen fluid infused (5 L/h abomasal infusions of healthy rumen fluid collected from cows eating a high-fiber diet; n = 5); AF = acidotic fluid infused (5 L/h abomasal infusions of acidotic rumen fluid collected from corn-challenged donor cows; n = 5); bpm = breaths per minute.Cows were abomasally infused with their respective treatments for 16 h relative to the first infusion.Cows were abomasally infused with acidotic rumen fluid collected from corn-challenged donor cows (AF; 5 L/h; n = 5) or healthy rumen fluid from cows fed a high-fiber diet (HF; 5 L/h; n = 5) until 16 h relative to the first infusion (RFI).Data were analyzed using PROC MIXED and included fixed effects of treatment (Trt), time (h), and their interaction, and each animal's respective period (P) 1 value served as a covariate for P2.Results are expressed as LSM ± SEM and considered significant if P ≤ 0.05 and a tendency if 0.05 < P ≤ 0.10.*Denotes a significant (P ≤ 0.05) difference between HF and AF cows.†Indicates that post hoc analysis revealed a tendency for a difference (P = 0.06; standard error for 8 h RAI: 2 mg/dL) between HF and AF cows at 8 h RFI.
due to increased osmolarity (Owens et al., 1998).We had therefore hypothesized that the pool of available rumen fluid would increase to potentially support rumen fluid collections for up to 36 h.Additionally, Penner et al. (2009) reported that, regardless of diet (low vs. high concentrate), the liquid fraction of the rumen digesta was approximately 76 L in Holstein cows weighing about 815 kg, which was slightly heavier than the Donor cows used herein (760 ± 70 kg SD).By the end of the 16 h, approximately 56 L of fluid had been removed from each Donor cow and there was little to no ruminal fluid remaining.This is likely explained by the absorbent capacity of the finely ground dry corn delivered into the rumen.Regardless, in addition to not creating a "noxious rumen fluid," the short duration time of abomasal infusions could also be a potential cause for the lack of inflammatory perturbations in our recipient cows.
Mild alterations in circulating metabolites and insulin suggest rumen acidotic fluid from Donor cows contained more energetic nutrients [e.g., soluble carbohydrates, short-chain fatty acids (SCFA)] than the abomasally infused healthy fluid.Despite the lack of ruminal acidosis in Donor cows, corn administration still decreased Donor rumen pH approximately 1.2 units at nadir, suggesting there was a moderate increase in SCFA concentration, as would be expected with increased grain fermentation (Khafipour et al., 2009b;Steele et al., 2011).Because both glucose and SCFA (mainly propionate) are insulin secretagogues (Manns and Boda, 1967;Harmon, 1992), it is likely the rise in circulating glucose and insulin at 8 h RFI can be explained by a relative increase in SCFA, residual sugars, or both within the abomasally infused acidotic rumen fluid.Further, the transient increase in circulating insulin likely explains the initial decrease in circulating NEFA because insulin is intensely antilipolytic (Brockman and Laarveld, 1986).Interestingly, fecal pH decreased in AF cows from 8 to 12 h RFI (0.9 units from baseline), and this may indicate an increase in hindgut fermentation of residual carbohydrates from acidotic fluid infusions.Notably, we manually altered the pH of acidotic rumen fluid with HCl before abomasal infusions, and thus the effects of this on fecal pH cannot be distinguished from that of hindgut fermentation.However, there was a numerical (albeit insignificant) decrease in circulating BUN at 8 h RFI that temporally aligns with the initial decrease in fecal pH, which may be explained by elevated microbial ammonia sequestration with increased hindgut fermentation (Røjen et al., 2012), as has been repeatedly shown in isolated HGA experiments (van Gastelen et al., 2021a,b;Abeyta et al., 2023a,b,c,d).Additionally, acidic chyme leaving the abomasum is typically neutralized by pancreatic bicarbonate secretion (pH = approximately 8; Pierzynowski et al., 1988;Mills et al., 2017), suggesting the reduction in fecal pH most likely stemmed from cecal/colonic carbohydrate fermentation.Regardless, fecal pH transiently decreased to levels below the arbitrary threshold for HGA conditions (fecal pH < 6.6; Plaizier et al., 2018), providing further evidence that a moderate reduction in pH in the hindgut lumen alone does not cause an immune-activated phenotype or systemic inflammation in lactating dairy cows.
In stark contrast to the recipients, the corn challenge initiated a delayed inflammatory response in Donor cows, as indicated by the striking increase in SAA and LBP at 48 h RAI.Interestingly, these acute phase proteins began mildly increasing around 24 h, which temporally aligns with the plateau in fecal pH (pH = approximately 4.75) from 22 to 36 h and development of metabolic acidosis (blood pH < 7.35; Berne and Levy, 1998) by 24 h RAI.In contrast, rumen pH nadir (pH = 5.64) occurred at 8 h RAI before increasing again, suggesting the inflammatory pattern was mediated by postrumen pathology.Interestingly, the marked alterations in blood gas parameters (i.e., decreased blood pH, bicarbonate, CO 2 , and base excess) strongly resemble those reported following acute ruminal lactic acidosis induction in cows and sheep (Nagaraja et al., 1981;Thoefner et al., 2004;Reis et al., 2018).Because rumen pH remained relatively stable in the current study, these findings further support the postulation that distal segments of the GIT contribute to the pathophysiology of acute ruminal acidosis, as has been previously suggested by others (Huber, 1976;Zust et al., 2000).Interestingly, Huber (1976) reported preliminary data from a steer that was abruptly switched from a hay to a high-concentrate (80%) diet immediately following ruminal inoculation with 1 L of pure lactic acid utilizing bacteria.Apparently due to the inoculant, ruminal conditions remained largely unaltered (pH > 5.6, no lactic acid accumulation), but by d 5 the steer had developed profuse diarrhea, went off-feed, and had reduced ruminal motility (Huber, 1976).Similarly, Tremere et al. (1968) reported that grain-challenged dairy heifers still went off-feed despite rumen conditions (i.e., pH and lactic acid accumulation) being controlled by intraruminal buffer administration.In the present study, feed intake remained depressed in Donor cows even after full feed had been returned at 36 h RAI, which may be explained by the increase in acute phase proteins starting at 24 h because hypophagia is a well-characterized consequence of immune activation (Kvidera et al., 2017;Kuhla, 2020).Considering these similarities, we speculated that reports from Tremere et al. (1968) and Huber (1976) may be partially explained by immune activation attributed to lactic acidosis and subsequent hyperpermeability in the hindgut, particularly because in addition to causing anorexia, endotoxemia decreases ruminal motility (Lohuis et al., 1988).However, control over ruminant forestomach motility is complex and altered motility may be explained by multiple factors (e.g., acid receptors, osmoreceptors, or central nervous system ;Huber, 1976;Gregory, 1987;Carter and Grovum, 1990) during ruminal acidosis or HGA.
Evidence from this and other experiments suggests that hindgut lactic acidosis may cause tissue damage and increase intestinal hyperpermeability, potentially contributing to the malaise phenotype associated with ruminal lactic acidosis (e.g., off-feed, metabolic acidosis, and ruminal stasis; Huber, 1976;Nagaraja and Titgemeyer, 2007).In support, Clayton and Jones (2001) reported a simultaneous increase in fecal lactic acid (fecal pH = 5.00) and fecal TNF-α concentrations following acute ruminal lactic acidosis in sheep, indicating these hindgut conditions may have caused a postruminal inflammatory response.Additionally, Bissell and Hall (2010) described gut tissue damage (excretion of tissue and mucin casts) and severe morbidity in 2 cows that had developed acute HGA following abomasal infusion of 4 kg/d of corn starch (fecal pH = 4.6; personal communication with Mary Beth Hall, USDA-ARS, Madison, WI).Lactic acid is a much stronger acid (pKa = 3.9) than acetate, propionate, or butyrate (pKa = approximately 4.8), thus its accumulation is implicated in the reduction of ruminal pH to <5 during acute ruminal acidosis (see reviews by Mackenzie, 1967;Aschenbach et al., 2011).Hence, the particularly low fecal pH in Bissell and Hall (2010), Clayton and Jones (2001), and the current experiment (4.6, 5.0, and 4.8, respectively) indicate the predominant driver in hindgut acidity was likely lactic acid.In support, Zust et al. (2000) indicated that abomasal infusion of 4 kg/d of a 65% corn feed mixture markedly increased hindgut and circulating lactic acid concentrations and caused metabolic acidosis (blood pH = 7.18), severe inappetence, ruminal stasis, and general weakness, a phenotype that is strikingly similar to that of immune activation (Horst et al., 2021).
The aforementioned lactic acid discussion is in contrast with recent reports indicating that moderate reductions in hindgut pH do not cause intestinal barrier dysfunction in dairy cows (van Gastelen et al., 2021a,b;Abeyta et al., 2023a,b,c,d), steers (Mainardi et al., 2011), or pigs (Mayorga et al., 2021).Akin to the comparison between subacute and acute ruminal acidosis, discrepancies between HGA experiments may be attributed to the relative severity of the acidosis, primarily whether the pH depression is explained by an accumulation of SCFA or lactic acid.For instance, fecal pH in our previous cattle HGA experiments averaged approximately 5.7 (Abeyta et al., 2023a,b,c,d), and in other reports it varied between 5.15 and 6.26 (van Gastelen et al., 2021a,b;Piantoni et al., 2022).Despite successfully inducing HGA, no indication of immune activation, reduced production, or other negative impacts associated with HGA were observed, contrasting with findings from the literature where lactic acid appeared to predominate (Zust et al., 2000;Bissell and Hall, 2010).Interestingly, lactic acid accumulation is thought to be one of the drivers in causing increased intestinal permeability, which precedes carbohydrate overload-induced laminitis in horses (Bailey et al., 2004), a concept that is reinforced by the prevention of laminitis through antibiotic inhibition of lactic acidproducing bacteria (Rowe et al., 1994).Additionally, ethanol increases in the rumen (Allison et al., 1964) and hindgut (Sato and Kurosawa, 2011) with increased fermentation, and it can damage the intestinal mucous layer (Qin and Deitch, 2015), potentially exposing the underlying epithelium.Although the effects of lactic acid and ethanol cannot be singled out, Weiss et al. (2000) further demonstrated the link between excessive hindgut fermentation and intestinal hyperpermeability when they reported that exposing equine colonic tissue to cecal digesta (which had been incubated with corn starch, resulting in decreased pH and increased lactic acid) caused hyperpermeability.Interestingly, there appears to be a similar relationship in rodents, as Genda et al. (2018) reported that altered fermentation dynamics between different basal diets (semipurified vs. nonpurified) plus fructo-oligosaccharide supplementation (a prebiotic) resulted in either the accumulation of lactic acid, abolition of the mucosal layer, and bacterial translocation (semipurified diet) or an increase in SCFA with no evidence of bacterial translocation or inflammation (nonpurified diet).As aforementioned, there are a plethora of potential toxins produced during excessive microbial fermentation, which may contribute to the hyperpermeable phenotype associated with rumen or hindgut acidosis, not just lactic acid (Slyter, 1976;Saleem et al., 2012).However, the presence of lactic acid within the hindgut digesta may be a useful indicator of whether increased hindgut fermentation tilts over the threshold of becoming detrimental to the epithelial barrier.Thus, there may be benefits to better identifying the effects of large intestine lactic acid accumulation and hindgut fermentation characteristics.
Isolating the segments of intestinal hyperpermeability in in vivo acidosis experiments is challenging.Thus, we aimed to isolate the postruminal effects of SARA by abomasally infusing acidotic rumen fluid from a subset of acidotic donor cows into a healthy group of recipients, with the hypothesis that abomasal infusions would result in increased inflammation and an immuneactivated phenotype due to altered barrier integrity.Unfortunately, our original hypothesis was unable to be fully evaluated, as Donor cows did not develop rumen acidosis following finely ground corn administration.However, despite the lack of rumen acidosis, Donor cows developed profound HGA, as indicated by a se-vere decrease in fecal pH, and this temporally aligned with a marked increase in inflammatory biomarkers, indicating inflammation may be the result of pathology associated with hindgut barrier damage.If true, these findings contrast with a series of HGA experiments in cows and pigs that indicated a moderate reduction in hindgut pH does not elicit an inflammatory response or malaise phenotype.It may be that these discrepancies can be explained by the relative severity of HGA developed, primarily whether the pH depression is characterized by the accumulation of SCFA or lactic acid.We speculate that lactic acid accumulation may be a sine qua non for intestinal barrier dysfunction caused by excessive hindgut fermentation.Given the relevance of immune activation on animal well-being and farm profitability, a better characterization of lactic acid's role in hindgut barrier function is needed.

Figure 1 .
Figure1.Experimental schematic.Cows destined to be donors (Donor; n = 8), healthy rumen fluid infused (HF; n = 5), and acidotic rumen fluid infused (AF; n = 5) were acclimated to a high-fiber (NDF: 46% of DM) diet for an 11-d prefeeding period (PF) before period (P) 1. Baseline data were collected during P1, and on d 5 Donor cows were given a corn challenge (Acidosis Day) during which they were feed restricted to 25% of their ad libitum feed intake for 16 h before manual insertion of 2.75% BW finely ground corn at 0000 h on P2 d 1.After their initial feed allotment was consumed, Donor cows were fasted until 36 h and data were collected until 96 h relative to acidosis induction (RAI).At 12 h RAI, an additional 0.5% BW finely ground corn was manually inserted into the rumen, and rumen fluid collections (approximately 7 L every other hour) began to facilitate abomasal infusions for AF cows.Starting at 1330 h on d 1 of P2, HF and AF cows were abomasally infused with their respective treatments for 16 h and data were collected through 96 h relative to the first infusion (RFI).*Indicates when blood samples were obtained for respective groups.
Figure 2. (A)Rumen and fecal pH and (B) fecal score following a corn challenge in donor dairy cows acclimated to a high-fiber diet (NDF: 46% of DM).Cows were challenged with finely ground corn (2.75% of BW) via manual insertion to the rumen following 16 h of feed restriction to 25% of ad libitum feed intake.An additional 0.5% BW of finely ground corn was added to the rumen at 12 h relative to acidosis induction (RAI), and cows were fasted through 36 h RAI.Rumen fluid collections (approximately 7 L every 2 h) began at 12 h RAI and continued through 28 h RAI.Data were analyzed using PROC MIXED, and effects time (h) were evaluated.Period (P) 1 represents an average of the −24 and 0 h RAI samples and was analyzed separately.Results are expressed as LSM ± SEM and considered significant if P ≤ 0.05 and a tendency if 0.05 < P ≤ 0.10.
Figure3.Blood (A) pH, (B) base excess, (C) bicarbonate (HCO 3 ), (D) total carbon dioxide (TCO 2 ), (E) partial pressure of carbon dioxide (pCO 2 ), (F) ionized Ca (iCa), (G) saturated oxygen (sO 2 ), (H) partial pressure of oxygen (pO 2 ), and (I) hematocrit concentrations following a corn challenge in donor dairy cows acclimated to a high-fiber diet (NDF: 46% of DM).Cows were challenged with finely ground corn (2.75% of BW) via manual insertion to the rumen following 16 h of feed restriction to 25% of ad libitum feed intake.An additional 0.5% BW of finely ground corn was added to the rumen at 12 h relative to acidosis induction (RAI), and cows remained fasted through 36 h RAI.Rumen fluid collections (approximately 7 L every 2 h) began at 12 h RAI and continued through 28 h RAI.Data were analyzed using PROC MIXED, and effects of time (h) were evaluated.Period (P) 1 represents the 0 h RAI sample and was analyzed separately.Results are expressed as LSM ± SEM and considered significant if P ≤ 0.05 and a tendency if 0.05 < P ≤ 0.10.
Figure 4. Dry matter intake and milk yield following a corn challenge in donor dairy cows acclimated to a high-fiber diet (NDF: 46% of DM).Cows were challenged with finely ground corn (2.75% of BW) via manual insertion to the rumen following 16 h of feed restriction to 25% of ad libitum feed intake on period (P) 1 d 5 (analyzed separately).An additional 0.5% BW ground corn was added to the rumen at 12 h relative to acidosis induction (RAI), and cows were fasted through 36 h RAI.Rumen fluid collections (approximately 7 L every 2 h) began at 12 h RAI and continued through 28 h RAI.Data were analyzed using PROC MIXED, and effects of time (d) were evaluated.Period 1 represents an average of d 1 to 4 of P1 and was analyzed separately.Results are expressed as LSM ± SEM and considered significant if P ≤ 0.05 and a tendency if 0.05 < P ≤ 0.10.

Figure 6 .
Figure 6.Effects of abomasal infusion of acidotic rumen fluid on (A) fecal pH and (B) fecal score.Cows were abomasally infused with acidotic rumen fluid collected from corn-challenged donor cows (AF; 5 L/h; n = 5) or healthy rumen fluid from cows fed a high-fiber diet (HF; 5 L/h; n = 5) until 16 h relative to the first infusion (RFI).Data were analyzed using PROC MIXED and included fixed effects of treatment (Trt), time (h), and their interaction, and each animal's respective period (P) 1 values served as a covariate for P2.Results are expressed as LSM ± SEM and considered significant if P ≤ 0.05 and a tendency if 0.05 < P ≤ 0.10.*Denotes a significant (P ≤ 0.05) difference between HF and AF cows.

Figure 7 .
Figure 7. Effects of abomasal infusion of acidotic rumen fluid on circulating (A) glucose, (B) insulin, (C) nonesterified fatty acids (NEFA), (D) BHB, and (E) BUN.Cows were abomasally infused with acidotic rumen fluid collected from corn-challenged donor cows (AF; 5 L/h; n = 5) or healthy rumen fluid from cows fed a high-fiber diet (HF; 5 L/h; n = 5) until 16 h relative to the first infusion (RFI).Data were analyzed using PROC MIXED and included fixed effects of treatment (Trt), time (h), and their interaction, and each animal's respective period (P) 1 value served as a covariate for P2.Results are expressed as LSM ± SEM and considered significant if P ≤ 0.05 and a tendency if 0.05 < P ≤ 0.10.*Denotes a significant (P ≤ 0.05) difference between HF and AF cows.†Indicates that post hoc analysis revealed a tendency for a difference (P = 0.06; standard error for 8 h RAI: 2 mg/dL) between HF and AF cows at 8 h RFI.

Figure 8 .
Figure 8. Effects of abomasal infusion of acidotic rumen fluid on circulating (A) serum amyloid A (SAA) and (B) LPS-binding protein (LBP).Cows were abomasally infused with acidotic rumen fluid collected from corn-challenged donor cows (AF; 5 L/h; n = 5) or healthy rumen fluid collected from cows fed a high-fiber diet (HF; 5 L/h; n = 5) until 16 h relative to the first infusion (RFI).Data were analyzed using PROC MIXED and included fixed effects of treatment (Trt), time (h), and their interaction, and each animal's respective period (P) 1 value served as a covariate during P2.Results are expressed as LSM ± SEM and considered significant if P ≤ 0.05 and a tendency if 0.05 < P ≤ 0.10.
Abeyta et al.: ACIDOSIS AND POSTRUMINAL BARRIER FUNCTION

Table 1 .
Abeyta et al.: ACIDOSIS AND POSTRUMINAL BARRIER FUNCTION Ingredients and composition of diet 1

Table 2 .
Abeyta et al.:ACIDOSIS AND POSTRUMINAL BARRIER FUNCTION Effects of abomasal infusion of acidotic rumen fluid on production metrics, milk variables, and thermal indices