3966

Experimental acute rumen acidosis in sheep: Consequences on clinical, rumen, and gastrointestinal permeability conditions and blood chemistry1

A. Minuti,*† S. Ahmed,*‡ E. Trevisi,*† F. Piccioli-Cappelli,* G. Bertoni,* N. Jahan,* and P. Bani*2

*Istituto di Zootecnica, Facoltà di Agraria, Università Cattolica del Sacro Cuore, via Emilia Parmense 84, 29122 Piacenza, Italy; †PRONUTRIGEN- Centro di Ricerca sulla Proteomica e Nutrigenomica, Università

Cattolica del Sacro Cuore, Piacenza, Italy; and ‡Bangladesh Livestock Research Institute, Savar, Dhaka 1341, Bangladesh

ABSTRACT: Acute acidosis was induced in sheep, and gastrointestinal permeability was assessed by using lactulose as a permeability marker. Metabolism was evaluated by monitoring blood metabolites. Four rams (72.5 ± 4.6 kg BW) were used in a 2 × 2 changeover design experiment. The experimental period lasted 96 h from –24 to 72 h. After 24 h of fasting (from –24 to 0 h) for both controls and acidosis-induced rams (ACID), 0.5 kg of wheat flour was orally dosed at 0 and 12 h of the experimental period to ACID, while the basal diet (grass hay, ad libitum) was restored to control. At 24 h, a lactulose solution (30 g of lactulose in 200 mL of water) was orally administered. Blood samples were collected at –24, 0, 24, 48, and 72 h of the experimental periods for the analysis of metabolic profiles and during the 10 h after lactulose dosage to monitor lactulose changes in blood. In addition, rumen and fecal samples were col-lected at 24 h of the experimental period. The acidotic challenge markedly reduced (P < 0.01) rumen pH and VFA but increased rumen d- and l-lactic acid (P < 0.01). Concurrently, a decrease of fecal pH and VFA occurred in ACID (P < 0.01), together with an abrupt increase (P < 0.01) of lactate and fecal alkaline phosphatase.

Blood lactulose was significantly increased in ACID peaking 2 h after lactulose dosage. Blood glucose, β-hydroxybutyrate, Ca, K, Mg, and alkaline phospha-tase showed a significant reduction (P < 0.05) at 24 h, whereas urea and NEFA declined (P < 0.05) from 48 to 72 h. A strong inflammatory acute phase response with oxidative stress in ACID group was observed from 24 to 72 h; higher values of haptoglobin (P < 0.01) were measured from 24 to 72 h and of ceruloplasmin from 48 (P < 0.05) to 72 h (P < 0.01). Among the negative acute phase reactants, plasma albumin, cholesterol, paraoxonase, and Zn concentration also decreased (P < 0.05) in ACID at different time points between 24 and 72 h after acidotic challenge start. A rise (P < 0.05) of reactive oxygen metabolites and a drop of vitamin E (P < 0.01) between 24 and 72 h were indicative of oxi-dative stress in ACID. The perturbation of these blood metabolites suggests that acute acidosis was effectively induced by our model. The increase of lactulose in blood in ACID indicates that gastrointestinal permeability for the marker increased and the large increment after 2 h from dosage suggests that most of the passage occurred through the rumen or abomasal walls.

Key words: acute ruminal acidosis, gastrointestinal permeability, lactulose test, sheep

© 2014 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2014.92:3966–3977 doi:10.2527/jas2014-7594

INTRODUCTION

During acute ruminal acidosis, the increase of pro-ton concentration in the rumen challenges the integrity of the ruminal epithelial cells with the appearance of microlesions and parakeratosis (Mohamed Nour et al.,

1998; Kleen et al., 2003; Ismail et al., 2010) that can affect animal metabolism. Furthermore, the acidic en-vironment, the high osmotic pressure, and the high concentration of lactate in the rumen can cause death and lysis of the gram-negative bacteria leading to an in-crease in cell-free lipopolysaccharide (LPS) in the fore-stomachs (Gozho et al., 2007; Zebeli and Ametaj, 2009). An impairment of the barrier function of the rumen epi-thelium resulting from feeding high grain-based diets is a possible mechanism of LPS translocation (Kleen et al., 2003). Translocation of LPS from the gastrointesti-

1This manuscript is based on research supported by the “Fondazione Romeo ed Enrica Invernizzi,” Milan, Italy.

2Corresponding author: paolo.bani@unicatt.itReceived January 14, 2014.Accepted June 14, 2014.

Published December 12, 2014

Acidosis and gastrointestinal permeability 3967

nal (GI) tract to the bloodstream causes an inflammatory response with possible relevant metabolic disorders such as laminitis (Bertoni et al., 1989; Nocek, 1997), displace-ment of the abomasum (Zebeli et al., 2011), fatty liver, and sudden death syndrome (Ametaj et al., 2005). Liver rapidly detoxifies the circulating LPS, and attempts to quantify LPS in peripheral blood circulation have faced many difficulties (Andersen et al., 1994; Gozho et al., 2007). The LPS has only occasionally been detected in ruminants’ peripheral circulation after an experimental induction of subacute ruminal acidosis by high grain diets (Khafipour et al., 2009a). In our previous studies (Ahmed et al., 2013; Minuti et al., 2013), we proved that lactulose can be used as a marker to assess GI permeability in adult ruminants. We hypothesized that the changes of GI epi-thelium integrity during rumen acidosis condition can be evaluated by the lactulose test.

The aim of this study was to evaluate, during an ex-perimentally induced acute ruminal acidosis in rams, the GI epithelium integrity through lactulose permeability test and the related consequences on animal metabolism and inflammatory status.

MATERIALS AND METHODS

All of the procedures involving animals were con-ducted in accordance with Italian laws on animal ex-perimentation and ethics (DL number 116, 27/01/1992), protocol number ZN2013002.

Animals and Feeding System

Four rams of the local population “Bardigiana” (72.5 ± 4.6 kg of initial BW) were used in this experiment. Animals were housed in separate cages with wooden floors and free access to water, in an environmentally controlled room (temperature 22°C and relative humidity 70%). Before and between the experimental periods the animals were fed twice daily (0700 and 1900 h) grass hay (11.4% CP and 8.11 MJ/kg ME, on a DM basis) for ad libitum intake, to ensure between 3 and 6% orts, and a vitamin–mineral sup-plement. During the experimental periods animals were fed according to the experimental design.

Experimental Design

The experiment was performed according to a changeover design (2 × 2), with two 4-d experimental periods (1 and 2) and a 28-d interval between periods. Figure 1 shows the time schedule of each experimental period. All the animals were fasted for 24 h (from –24 to 0 h of the experimental period). In the control rams (CTR), the previous grass hay diet was then restored. In the acidosis-induced rams (ACID), animals were then

orally dosed with wheat flour to induce acute acido-sis. Total amount of wheat flour was 1.0 kg/ram (cor-responding to 12.8 ± 0.9 SD g/kg BW), administered in 2 equivalent portions of 0.5 kg at 0 and 12 h of the experimental period. In the ACID group the grass hay diet availability was restored after 24 h.

Blood samples were collected (before morning feed delivery) at –24, 0, 24, 48, and 72 h during experimental periods. Rumen fluid through stomach tube and grab fe-cal samples were collected at 24 h. To assess GI perme-ability, at 24 h (lactulose dosage time [LDT]), a solution containing 30 g of lactulose (150 g/L) was orally admin-istered to every ram, and blood samples were collected at 0, 2, 4, 6, 8, and 10 h after LDT (0LDT, 2LDT, 4LDT, 6LDT, 8LDT, and 10LDT).

Feed Intake and Health Problems

During the trial rectal temperature was measured at –24, 24, 36, 48, 60, and 72 h of the experimental pe-riod (Fig. 1). At the same times animals were observed by general inspection to record development of clinical symptoms. In addition, feed intake and fecal features were monitored.

Blood Sampling and Analysis

Blood was collected from the jugular vein into evacuated tubes containing lithium heparin (Vacutainer; Becton Dickinson, Plymouth, UK) and immediately cooled in ice water. A small amount of blood was used for packed cell volume determination (Centrifugette 4203; ALC International Srl, Cologno Monzese, Italy); the remainder was centrifuged at 3,500 × g for 15 min at 6°C, and the plasma was frozen (–20°C) until analyses.

Blood samples were analyzed to assess the follow-ing profiles: metabolic (glucose, total cholesterol, urea, aspartate aminotransferase, γ-glutamyl transpeptidase, alkaline phosphatase [ALP], NEFA, β-hydroxybutyrate [BHBA], and d- and l-lactate), mineral (Ca, P, Mg, Na, K, Cl, and Zn), and oxidative–inflammatory (total protein, albumin, globulin, total bilirubin, haptoglobin, ceruloplasmin, vitamins A, vitamin E, reactive oxygen metabolites [ROM], thiol groups, total nitric oxide me-tabolites, and paraoxonase).

Figure 1. Experimental design. ▲ = 0.5 kg wheat flour administration; ● = blood samples; ↓ = lactulose dosage time; × = rumen and fecal samples collec-tion; ■ = rectal temperature; grey box = gastrointestinal permeability assessment.

Minuti et al.3968

Blood metabolites were analyzed by an automated biochemistry analyzer (ILAB 600; Instrumentation Laboratory, Lexington, MA). Total protein, albumin, total cholesterol, total bilirubin, triglycerides, urea, Ca, P, Mg, aspartate aminotransferase, γ-glutamyl transpep-tidase, and ALP were determined using kits purchased from Instrumentation Laboratory (Instrumentation Laboratory SpA, Milano, Italy). Globulin was calcu-lated as difference between total protein and albumin. Ions (Na, K, and Cl) were measured by the potentiomet-ric method (ion-selective electrode connected to ILAB 600). Zinc and NEFA were measured by commercial kits (Wako Chemicals GmbH, Neuss, Germany). The BHBA was determined by commercial kits Ranbut (Randox Laboratories Ltd., Crumlin, UK). Haptoglobin and ceru-loplasmin were analyzed using the methods described by Skinner et al. (1991) and Sunderman and Nomoto (1970), respectively, adapted to the ILAB 600 conditions. The ROM and thiol groups were measured by commercial kits (Diacron International s.r.l., Grosseto, Italy). The total ni-tric oxide metabolites were measured using the Griess test according to Gilliam et al. (1993) and Bouchard et al. (1999). Paraoxonase was measured by the method of Ferré et al. (2002) adapted to the ILAB 600 as previously described by Bionaz et al. (2007). Plasma vitamins A and E were extracted with hexane and analyzed by reverse-phase HPLC using an HPLC (Varian; ProStar, Walnut Creek, CA), equipped with an Allsphere ODS-2 3 μm, 150 by 4.6 mm column (Alltech, Deerfield, IL), and an UV detector set at 325 nm for vitamin A and 290 nm for vitamin E; a solution of 80:20 methanol:tetrahydrofurane was used as mobile phase. d- and l-lactate were analyzed using Megazyme L and D Lactate assay Kit (Megazyme International Ltd., Wicklow, Ireland).

Lactulose Analysis

The lactulose concentration in plasma samples col-lected at 0LDT, 2LDT, 4LDT, 6LDT, 8LDT, and 10LDT was analyzed through GLC adapting the methodology used for urinary sugar detection by Farhadi et al. (2003). Plasma samples were prepared for the analysis as follows: 250 μL of sample was added with 250 μL of solution con-taining internal standard (0.2 g/L of myo-inositol in 7% wt/vol trichloroacetic acid). Samples were mixed by vor-texing and centrifuged (15,000 × g for 10 min at 20°C). After that, 400 μL of the supernatant were transferred to a new tube and evaporated to dryness at 70°C under a stream of nitrogen. The dried residues were then taken up in 200 μL of anhydrous pyridine containing 25 mg/mL of hydroxylamine chlorhydrate, mixed by vortexing, heated at 70°C for 1 h, and centrifuged (15,000 × g for 10 min at 20°C). An aliquot (100 μL) of the supernatant was transferred to a new tube, and sugar oximes were

silylated with 100 μL of N-trimethylsilylimidazole for 30 min at 70°C. An aliquot of the silylated derivatives was sealed in a vial for analysis.

The lactulose was quantified using a gas chromato-graph (model 7820A GC; Agilent Technologies, Santa Clara, CA) equipped with a DB-1 capillary column (15 m by 530 μm by 1.5 μm; Agilent J&W GC column) and flame-ionization detection. The oven temperature was 220°C held for 5 min and then increased by 10°C/min for 2 min, 5°C/min for 4 min, and 3.5°C/min for 4 min to a final temperature of 274°C, held for 9 min. The analysis time was approximately 24 min. The injec-tor temperature was 250°C and the detector temperature was 280°C. Glass wool packed liners were used to pre-vent contamination of GC with dirty particles from the sample. The samples were injected by auto sampler, and 1 μL at 25:1 split ratio was injected. Hydrogen and air were used for flame ionization detection. Helium at a flow rate of 10 mL/min was used as carrier gas.

Rumen and Feces Sampling and Analysis

Rumen fluid was collected through a stomach tube (rubber tube of approximately 150 cm of length for 1.5 cm of diameter) at 24 h, before morning feeding. The pH of the fluid was measured immediately after it was withdrawn from the rumen by a pH meter (GLP 21; Crison Instruments SA, Alella, Barcelona, Spain). An aliquot of the remaining fluid was immediately cooled in ice water and centrifuged at 3,000 × g for 10 min at 10°C; 2-mL aliquots of the supernatant were transferred into tubes with 1 mL of 0.12 M oxalic acid and frozen at –20°C for later analysis of VFA and lactate. Fecal sam-ples were collected manually at 24 h directly from the rectum immediately after rumen sampling, diluted with distilled water at 1:3 ratio, homogenized in a Stomacher 400 (Tekmar, Cincinnati, OH) for 3 min, and centrifuged as for the rumen fluid. The pH measure and storing of the extract were performed as for the rumen samples.

The l- and d-lactate were analyzed in rumen liquor and fecal extract by the same method used for lactate in blood. Alkaline phosphatase was analyzed in the fe-cal extract using a kit from Instrumentation Laboratory (Instrumentation Laboratory, Lexington, MA). The VFA concentration in rumen fluid and fecal extract was ana-lyzed by a gas chromatograph (model 7820A; Agilent Technologies, Santa Clara, CA) equipped with a capillary column (30 m × 250 μm × 0.25 μm; DB-FFAP, Agilent J&W GC column) and a flame-ionization detector. The oven temperature was 60°C held for 5 min and then in-creased by 5°C/min to 140°C. The injector temperature was 250°C and the detector temperature was 300°C. The injector was equipped with a glass liner of glass wool to separate particles of dirt from the sample. The samples

Acidosis and gastrointestinal permeability 3969

were dosed by auto sampler at an injection size of 1 μL us-ing the split method and a 25:1 splitting ratio. Hydrogen and air were used for flame ionization detection. The car-rier gas was nitrogen, with constant flow of 1.78 mL/min. Pivalic acid was used as an internal standard.

Statistical Analysis

Data of each parameter were previously tested for nor-mal distribution using the Shapiro test (SAS Inst. Inc., Cary, NC) and normalized by natural log transformation, when necessary. Data were analyzed using mixed model proce-dures of SAS (release 8.0) for repeated measures. The sta-tistical model used treatment time (from trial start or from LDT) and interaction of treatment × time as fixed factors and the individual ram were random effects. Each param-eter was subjected to 4 covariance structures—autore-gressive order, compound symmetry, spatial power, and toeplitz—and the best covariance structure was retained (Littell et al., 2006). The effect of the period (1 and 2) was initially included in the model and then removed, being not significant for any parameter. All values are presented as the means and SEM. The effect of the period (1 and 2) was initially included in the model and then removed, being not significant for any parameter. Statistical significance was established by using a conventional P-value of 0.05 or 0.01.

RESULTS

Health Status of the AnimalsAt 24 h after induction of acidosis, all ACID showed

nervous depression and exhibited periods of trembling. All could stand up, but mostly with head hung low and dull eyes. Parallel to this, ACID showed acute symp-toms including watery, yellowish, and acidic smelling diarrhea. Cessation of feed intake occurred in all ACID, and no appetite was evident until 72 h when a little in-terest in food and water appeared. All ACID showed more or less severe symptoms of foot pain and difficulty walking. Meanwhile, no clinical signs could be observed in CTR. At the start of the experimental period, rectal temperature was 38.9 ± 0.23°C in both groups, but 24 h after the induction of acidosis it increased (P < 0.01) in ACID, peaked (40.7°C) at 36 h, and then declined but remained higher than CTR until 72 h after the acidotic challenge (39.0 in CTR vs. 39.7°C in ACID; P < 0.01).

Ruminal and Fecal Parameters

The mean pH and VFA concentrations for rumen samples collected at 24 h are presented in Table 1. Rumen pH was lower (P < 0.01) in ACID compared to CTR, and the pH for ACID indicated an acute acidosis condition

(threshold value ≤ 5.0; Nagaraja and Town, 1990). Total VFA concentration and acid molar proportions differed between groups. In ACID compared with CTR, a marked decrease of total VFA concentration (P < 0.01) and higher concentrations of l- and d-lactate (P < 0.01) were ob-served. Fatty acid molar proportions also differed between groups. Compared to CTR, ACID showed higher acetate (P < 0.05) and lower propionate (P < 0.05) proportions.

Characteristics of fecal samples are shown in Table 2. Similar to the rumen, the fecal pH was lower in ACID compared to CTR (P < 0.01). In ACID a lower total fecal VFA concentration (P < 0.01) was also found, whereas l- and d-lactate concentrations were higher in ACID than in CTR (P < 0.01). The feces of ACID had a higher molar proportion of acetate (P < 0.01) and a lower proportion of propionate (P < 0.01) than those of CTR. In feces from ACID, isobutyrate, valerate, and isovalerate could not be detected and ALP was higher compared to CTR (1,543 vs. 30 units/100 g fresh matter; P < 0.01).

Blood Parameters

At –24 and 0 h (prechallenge period), no differences for any blood parameters were detected between ACID and CTR.

Metabolic Blood Parameters

The results of metabolic blood parameters are pre-sented in Fig. 2 and Table 3. Before the acidotic chal-lenge the concentrations of d- and l-lactate in blood of ACID were quite similar, but their behavior after acidotic challenge was different. In ACID there was a rapid rise of d-lactate (Fig. 2A), with a maximum reached at 24 h (2.68 vs. 0.07 mmol/L in ACID and CTR; P < 0.01) and

Table 1. Effects of induction of acute ruminal acidosis in rams on ruminal pH, l-lactate, d-lactate, total volatile fatty acids, and their molar proportions after 24 h from induc-tion of acidosis (mean ± SEM). The groups are control rams (CTR) or acidosis-induced rams (ACID)

Item

Treatment SEM

P-valueCTR ACID

pH 6.89 4.94 0.13 <0.001l-lactate, mmol/L 1.1 56.6 3.7 <0.001d-lactate, mmol/L 1.1 50.4 2.0 <0.001Total VFA, mmol/L 87.6 11.9 5.0 <0.001VFA, mol/100 mol

Acetate 66.4 75.9 2.6 0.04Propionate 21.1 14.0 1.8 0.03Butyrate 10.5 9.0 1.2 0.40Isobutyrate 0.45 0.32 0.062 0.19Valerate 0.95 0.34 0.099 0.005Isovalerate 0.48 0.43 0.11 0.75

Minuti et al.3970

by 72 h d-lactate was restored to prechallenge values. The rise of l-lactate was more gradual (Fig. 2B); it was higher in ACID from 24 to 72 h (P < 0.01), but the peak was reached at 48 h (2.96 vs. 0.72 mmol/L in ACID vs. CTR). Glucose (Fig. 2C) and BHBA (Fig. 2D) showed a decrease, starting from 24 h, as a consequence of induc-tion of acidosis. Plasma concentrations of urea, NEFA, and bilirubin all increased in ACID (Table 3) and differed from CTR starting from 24 (P < 0.05) to 48 (bilirubin) or 72 h (urea and NEFA). On the contrary, the concentra-tions of Ca, K, Mg, and ALP (Table 3) were lower in the ACID compared to CTR at the different time points (P < 0.05). No changes were observed for aspartate amino-transferase and γ-glutamyl transpeptidase.

Positive and Negative Acute Phase Proteins and Oxidative Stress Reactants

Haptoglobin (Fig. 3A) in the ACID started to increase after 24 h from the induction of acidosis (P < 0.01) and continued until 72 h (P < 0.01). Similarly, ceruloplasmin (Fig. 3B) started to rise in ACID from 24 h but differences were found at 48 (P < 0.05) and 72 h (P < 0.01). Among negative acute phase proteins, plasma albumin (Table 3) declined in ACID compared to CTR from 48 h with sig-nificant differences at 72 h (P < 0.01). Major changes were found in the plasma concentration of Zn and paraoxonase (Fig. 3C and 3D), with values significantly lower in the ACID compared to the CTR from 24 to 72 h for Zn (P < 0.01) and from 48 (P < 0.05) to 72 h (P < 0.01) for paraox-onase. Total cholesterol and vitamin A had a common trend (Table 3), decreasing in ACID after acidotic challenge, but

differences with respect to CTR became significant from 24 h (P < 0.05) for cholesterol and from 48 h (P < 0.05) for vitamin A. Both parameters continued to decline in ACID until 72 h (P < 0.01). Among the parameters related to oxi-dative stress, ROM started to rise in ACID after 24 h with differences from 48 (P < 0.05) to 72 h (P < 0.01; Fig. 3E). Vitamin E showed lower concentration in ACID (Fig. 3F) from 24 to 72 h (P < 0.01). No changes were observed for thiol groups and total nitric oxide metabolites.

Lactulose Concentration in Blood

The lactulose concentrations in blood plasma at dif-ferent time points after oral dosing are presented in Fig. 4. No detectable concentration of lactulose was found at 0LDT for either CTR or ACID. After the oral dosage, the plasma concentration of lactulose in CTR slightly in-creased at 2LDT and remained steady until 10LDT, with an average concentration of 3 μg/mL. In contrast, plasma concentration of lactulose rose rapidly in the ACID group after 2 h from lactulose dosage (P < 0.01) and remained higher compared to the CTR until 6LDT (P < 0.05). The highest value (39 μg/mL) was recorded 2 h after LDT whereas predosage values were restored at about 8LDT.

DISCUSSION

The high energy demand for increasing productive performance often leads to feeding ruminants with highly fermentable diets, largely based on concentrates and in particular on cereal grains. This can lead to the onset of ru-minal acidosis situations with varying degrees of intensity. As a consequence of the acute rumen acidosis, an impair-ment of the GI epithelium can occur, and several authors have hypothesized the translocation into the bloodstream of bacterial LPS and biogenic amines, which are more abun-dant in ruminal digesta during ruminal acidosis (Plaizier et al., 2008; Khafipour et al., 2009a,b; Saleem et al., 2012). When LPS translocation happens, the consequence on the animal can be an overt systemic inflammatory response with associated metabolic disorders such as compromised insulin sensitivity and stimulation of lipolysis.

In the present experiment the digestive epithelium integrity of the rams was challenged by inducing an acute experimental acidosis. After the induction of aci-dosis rams developed some typical clinical signs such as anorexia, abdominal pain, periods of trembling with problems in movement, and watery diarrhea with yel-lowish color and acidic odor. Similar clinical symptoms were also reported by Patra et al. (1996), Mohamed Nour et al. (1998), and Ismail et al. (2010) after acute acidosis induction in sheep and goats.

Our model for the induction of acidosis was based on 24 h fasting followed by administration of 1 kg of wheat

Table 2. Effects of induction of acute ruminal acidosis in rams on fecal pH, DM, alkaline phosphatase (ALP), l-lactate, d-lactate, total volatile fatty acids, and their molar proportions after 24 h from induction of acidosis (mean ± SEM). The groups are control rams (CTR) or acidosis-induced rams (ACID)

Item

Treatment SEM

P-valueCTR ACID

pH 8.35 4.81 0.10 <0.001DM, % 26.4 21.7 2.9 0.294ALP, units/100 g fresh matter 29 1,543 291 0.010l-lactate, mmol/100 g fresh matter 0.3 10.3 1.1 <0.001d-lactate, mmol/100 g fresh matter 0.29 3.50 0.25 <0.001Total VFA, mmol/100 g fresh matter 3.31 0.47 0.40 0.002VFA proportions, mol/100 mol

Acetate 80.9 96.6 1.8 <0.001Propionate 14.4 1.8 0.95 <0.001Butyrate 2.8 1.6 0.89 0.375Isobutyrate 0.75 0.02 0.039 <0.001Valerate 0.59 0.01 0.028 <0.001Isovalerate 0.44 0.01 0.057 <0.001

Acidosis and gastrointestinal permeability 3971

flour in 2 equal doses spaced by 12 h. In such conditions the mean value of rumen pH measured 24 h after the chal-lenge in ACID was 4.94. In some other comparable experi-ments, higher doses of flour or starchy concentrates were used. Patra et al. (1996) induced acute acidosis in adult sheep after 24 h fasting by feeding soaked wheat at 90 g per kg BW and after 24 h rumen pH was reduced to 4.70. Lettat et al. (2010) and Ismail et al. (2010) induced rumen acute acidosis in rumen-cannulated sheep and goats by intraru-minal supply of wheat at 12 g/kg BW in sheep and wheat flour at 50 g/kg BW in goats, and in these conditions ru-minal pH dropped to 4.85 and 4.23 after 24 h, respectively.

The marked reduction in ruminal pH that occurred in the present study indicates that acute acidosis was effec-tively induced. This strong acidification was associated with the reduction of total VFA and the marked increase of lactic acid in the rumen (50-fold increase in ACID compared with CTR). Similar results were observed in previous studies of induction of acute acidosis (Muir et al., 1980; Ismail et al., 2010; Lettat et al., 2010). The de-cline of VFA seems caused by a marked impairment of the bacterial flora, whereas the marked accumulation of the lactic acid can be a consequence of its increased pro-duction but reduced utilization within the rumen (Slyter, 1976; Nagaraja and Titgemeyer, 2007).

Rumen concentration of both isomers of lactate in ACID was very similar, but in feces a 3:1 ratio was

found between l- and d-lactic acid, respectively. Acid-tolerant lactobacilli, mainly producing d-lactate isomer (Slyter and Rumsey, 1991), may proliferate in both acute and subacute acidosis (Slyter, 1976; Nagaraja and Miller, 1989; Goad et al., 1998) leading to an increase in produc-tion of total and in particular d-lactate. Microbial changes in the large intestine associated with experimentally in-duced acidosis have also been reported but they did not parallel those taking place in the forestomachs as in that site d-lactate potentially yielding lactobacilli did not pro-liferate (Metzler-Zebeli et al., 2013). Zust et al. (2000) measured a cecal concentration of l-lactate 8 times higher than d-lactate after having induced a mild intestinal aci-dosis but a 3-fold higher l-lactate when an acute acidosis was induced. Ewaschuk et al. (2004) measured a 3-fold higher level of fecal d-lactate compared with l-lactate on milk-fed diarrheic calves but lower and much similar con-centrations of the 2 isoforms in healthy calves.

The concentrations of d- and l-lactic acid in blood (Fig. 2) were markedly increased during the experiment, suggesting that our protocol likely led the animals to a condition of metabolic acidosis. This circumstance is typi-cally observed during acute acidosis experiments in sheep (Muir et al., 1980; Patra et al., 1996; Ismail et al., 2010). Lactic acid isomers reached similar maximum concentra-tions in blood but had different patterns: d-lactate peaked at 24 h after the start of the induction of the acidosis and

Figure 2. Effects of induction of acute ruminal acidosis on plasma concentration of (A) d-lactate, (B) l-lactate, (C) glucose, and (D) β-hydroxybutyrate (BHBA). Values are means ± SEM. The groups are control rams (CTR) or acidosis-induced rams (ACID). The triangle (▲) indicates the wheat flour administration during acute acidosis induction. Significance of differences between groups at each time point indicated by * for P < 0.05 and ** for P < 0.01.

Minuti et al.3972

then declined rapidly, whereas l-lactate increased more slowly, reaching the peak at 48 h. Although d-lactic acid can be metabolized in humans (Connor et al., 1983) and other mammals (Giesecke and von Wallenberg, 1985), l-lactic acid is reported to be more rapidly metabolized than the D- isomer (Prior, 1983; Harmon et al., 1984).

Lactic acid concentration in the rumen and feces was measured after 24 h from the onset of the acidosis induc-tion, whereby it is not possible to assess the relative contri-bution to blood pool of lactic acid from the different sites of absorption, that is, rumen or lower GI tract. Slyter (1976) and Patra et al. (1996) observed that diarrhea is often as-sociated with acute and with subacute acidosis. Diarrhea is mainly due to an increased osmolality in the intestine consequent to high lactic acid concentration in the digesta (Huber, 1976), which in turn drives the pH decrease in feces. High concentrations of lactic acid and diarrhea oc-curred in the present experiment. Similar to our results, a significant reduction of fecal pH was also reported in sheep

by Al Jassim et al. (2003) at 24 h after acute acidosis induc-tion by barley engorgement (900 g DM/animal).

Interestingly, fecal ALP markedly increased after the acidotic challenge. In fact, fecal ALP concentra-tions were 50-fold higher in ACID compared to CTR. The causes of this abrupt boost of ALP in feces of ACID are not clear. Data regarding ALP in feces of ruminants are lacking in the literature. However, Lehmann et al. (1981) proved that in rat the administration of toxic agents for the small intestinal mucosa (bleomycin or triparanol) were able to increase the release of the intes-tinal ALP into the feces after 1 d of treatment. For this reason several other studies (Davis, 2003; Lapré et al., 1993; Taché et al., 2006) used the intestinal ALP in feces as marker of in vivo epithelial cell loss during intestinal damage investigations. Thomas and Henton (1985) did not find changes in the excretion of intestinal ALP after drug treatments with bleomycin in rats, which is a mol-ecule able to cause the intestinal damage. Altogether this research supports ALP in feces as a marker of damage of the small intestinal epithelium in ruminants, but ad-ditional studies are advisable to confirm this hypothesis.

In our rams, the clinical symptoms of anorexia were confirmed from changes in metabolic blood parameters. The fall of glucose and the rise of NEFA in plasma in-dicated an energy deficit state due to the fall of VFA consequent to the interruption of the feed intake and also to the impairment of the microbial activity (as discussed above). Generally, in fasted animals NEFA and BHBA have a similar pattern of change and are interpreted as markers of adipose tissue mobilization during energy deficiency. The same pattern of changes and the same in-terpretation was reported by Odongo et al. (2009) during metabolic acidosis in sheep. In the present experiment, blood BHBA had an unusual decline in ACID, likely re-lated to the lower concentration of butyrate from rumen fermentation that is an important precursor of BHBA in healthy and nourished ruminants.

The rise of plasma urea in acidotic sheep, observed also by Patra et al. (1996), can be interpreted as an index of the increased catabolism of AA of muscle tissues, although an increase of ruminal ammonia (not monitored in this experi-ment) could have also happened. Moreover, in similar con-ditions, the urea increase in blood could be also due to a renal failure, as previously hypothesized by Dunlop (1972).

The increase of blood bilirubin concentration in ACID is in agreement with the results obtained by Nikolov (1998) in buffalo and by Nikolov (2003) in sheep, and it may signal the impairment of hepatic func-tion (i.e., reduced bilirubin clearance). A decrease of plasma concentration of Ca, K, and Mg comparable to our results has been reported after acute acidosis induc-tion in cows (Zebeli et al., 2010) and sheep (Irwin et al., 1979). The reduction of plasma Mg mainly reflects

Table 3. Effects of induction of acute ruminal acidosis in rams on blood parameters at different times after induc-tion of acidosis (mean ± SEM). The groups are control rams (CTR) or acidosis-induced rams (ACID)Item Time, h CTR ACID SEM P-valueUrea, mmol/L

24 2.78 3.26 0.62 0.4548 2.39 4.74 0.000972 2.53 4.76 0.0015

NEFA,1 mmol/L

24 0.24 0.41 0.12 0.6048 0.30 0.68 0.00472 0.18 0.50 0.01

Bilirubin, μmol/L

24 0.47 1.38 2.0 0.6548 0.59 5.86 0.0272 0.41 8.24 0.10

Ca, mmol/L

24 2.69 2.44 0.073 0.00348 2.69 2.32 <0.000172 2.76 2.21 <0.0001

K, mmol/L

24 5.15 4.15 0.49 0.0548 5.23 3.68 0.00472 5.34 2.70 <0.0001

Mg, mmol/L

24 0.92 0.64 1.22 0.00148 0.93 0.76 0.0272 0.96 0.72 0.006

Alkaline phosphatase, units/L

24 154 87 18 0.00148 150 50 <0.000172 171 40 <0.0001

Albumin, g/L

24 37.3 38.2 1.2 0.4848 37.7 36.8 0.4572 37.8 33.7 0.003

Cholesterol, mmol/L

24 1.20 0.86 0.13 0.0148 1.20 0.66 0.000372 1.13 0.60 0.0004

Vitamin A, μg/100 mL

24 48 34 8.3 0.1248 39 20 0.0372 44 25 0.02

Acidosis and gastrointestinal permeability 3973

the reduced absorption of this mineral by the rumen (Schweigel and Martens, 2000), whereas the low plas-ma K could be justified by the reduction of feed intake as well as the increased intestinal losses as consequence of diarrheic condition (Gennari, 1998), while the renal conservation of K takes several days to compensate the deficient potassium intake (Carlson, 1997). The low plasma Ca and Zn in ACID sheep can be interpreted as a consequence of the inflammatory condition (Bertoni and Trevisi, 2013) and it has been previously observed for lactating cows challenged with starch (Zebeli et al., 2010) and during intravenously infusion of LPS in cows (Waldron et al., 2003) and sheep (Bertoni et al., 1989). Besides this, a possible role of hyperaldosteronism dur-ing oxidative stress and consequent alteration of Ca and Mg metabolism as well as disturbance of electrolytes level could be supposed (Sun et al., 2006).

A significant rise of blood lactulose concentration in ACID after oral ingestion indicates its paracellular ab-

sorption into systemic circulation. This event happens only when disruption of tight junctions in epithelial bar-rier occurs (Bjarnason et al., 1995; Meddings et al., 1999; Melichar et al., 2005). In a previous study (Minuti et al., 2013) an indomethacin-induced enteropathy model was used in sheep and proved that the lactulose test was a suit-able, noninvasive, and sensitive method to evaluate GI permeability in adult ruminants. Minuti et al. (2013) re-corded the highest values of lactulose in blood at 6 h after oral dosage. In the present experiment, the blood lactu-lose peaked in ACID just after 2 h from LDT. Ruminal acidosis is associated to reticulorumen hypomotility (Kezar and Church, 1979) and reduction of rumen out-flow rate (Harmon et al., 1985). The early appearance of lactulose in blood might consequently suggest that its absorption mostly occurred before the small intestine. Therefore, our results indicate that acute ruminal acidosis impairs epithelial barrier integrity in the ruminoreticulum, the abomasum, or both, allowing lactulose passage. In

Figure 3. Effects of induction of acute ruminal acidosis on plasma concentration of (A) haptoglobin, (B) ceruloplasmin, (C) zinc, (D) paraoxonase, (E) reactive oxygen metabolites (ROM), and (F) vitamin E. Values are means ± SEM. The treatments are control or induction of acute ruminal acidosis. The triangle (▲) indicates the wheat flour administration during acute acidosis induction treatment. Significance of differences between groups at each time point indicated by * for P < 0.05 and ** for P < 0.01.

Minuti et al.3974

human and rats studies, the increase in intestinal perme-ability, measured using the lactulose test, has been found to be positively correlated with the presence of LPS in the systemic circulation (Schietroma et al., 2013; Liu et al., 2008) and also related with bacterial translocation (Yue et al., 2012). If this were confirmed, we can hyp-notize that LPS as well as other bacterial immunogens, some other large molecules, and even microbes could enter from the forestomachs into systemic circulation as well as interact with immune cells resident in the GI mucosa (macrophage and dendritic cell) leading to lo-cal and systemic inflammation. It was previously found that subacute rumen acidosis (SARA) and acute rumen acidosis increase LPS concentration in rumen and stimu-late systemic inflammatory response (Gozho et al., 2007; Nagaraja and Titgemeyer, 2007; Emmanuel et al., 2008; Khafipour et al., 2009a). Ismail et al. (2010) conducted a postmortem examination of rumen epithelium samples of goats after the induction of acute acidosis and ob-served easily detached ruminal papillae, patchy areas of sloughed ruminal papillae, and hemorrhage in different areas of ruminal and reticular wall sometimes associated with ruminal wall ulceration, and similar results were ob-served during acute acidosis also by Mohamed Nour et al. (1998). Rumen wall is lined by keratinized stratified squamous epithelium but is not protected by mucus, as it occurs with abomasal epithelium, and can be vulner-able to the chemical damage by acids. Low ruminal pH has been demonstrated to lead to rumenitis and eventually

to parakeratosis, erosion, and ulceration of the ruminal epithelium (Garry, 2002). Moreover, acute and repeated episodes of ruminal acidosis are associated with promi-nent histological alterations in rumen epithelium, which strongly suggest an impaired barrier function (Steele et al., 2009) and provide the explanation for the transloca-tion of toxins and bacteria during this disorder (Plaizier et al., 2008). In a recent paper (Plaizier et al., 2012), re-sults from 2 models of induction of SARA (alfalfa pellet vs. grain; Khafipour et al., 2009a,b) were reviewed and led to the suggestion that during grain based SARA the translocation of LPS mainly occurs from the large intes-tine. In our study, the maximum concentrations of plasma lactulose were reached just 2 h after LDT, which suggests an increased permeability of the forestomachs barrier, be-coming a site for LPS translocation during acute acidosis.

In this study the consequences of acute ruminal aci-dosis on systemic inflammatory response were evident. Rectal temperature increased significantly in the ACID and this increment may be due to translocation of LPS into systemic circulation. Naylor and Kronfeld (1986) and Bertoni et al. (1989) found similar temperature in-crements after intravenous infusion of LPS in sheep. Haptoglobin and ceruloplasmin, both positive acute phase proteins, strongly increased in blood after 24 and 48 h, respectively, from the induction of acidosis. Negative markers of acute phase reaction, such as Zn, paraoxonase, retinol, and albumin, decreased in blood 24, 48, 48, and 72 h, respectively, after the start of the acidosis challenge.

Figure 4. Effect of induction of acute ruminal acidosis on plasma concentration of lactulose after its oral administration. The groups are control rams (CTR) or acidosis-induced rams (ACID).. Significance of differences between groups at each time point indicated by * for P < 0.05 and ** for P < 0.01.

Acidosis and gastrointestinal permeability 3975

The inflammatory responses during ruminal acido-sis have been reported in the literature. Serum amyloid A and haptoglobin increased during SARA conditions (Khafipour et al., 2009a; Gozho et al., 2006, 2007). In agreement with our observations, Danscher et al. (2011), inducing acute ruminal acidosis in cattle by oligofructose overload and demonstrated that haptoglobin concentra-tion in blood increases after 18 to 36 h from the challenge.

In our study the intense oxidative stress, highlighted by blood markers changes (i.e., strong fall of antioxidant vitamin E and a rise of the concentration of ROM) af-ter 48 h from the onset of the rumen acidosis induction, could support the damage of tissues by acidotic insult. Altogether, these hematic changes appear to be consistent with an inflammatory response mediated by immune cell resident in the GI tract as well as translocation of LPS into bloodstream as consequence of the increased permeabil-ity of the GI epithelia. In fact, LPS is a potent inducer of the acute phase response (Bertoni et al., 1989; Jacobsen et al., 2004) and its concentrations increases dramatically into the ruminal fluid during acidosis events (Nagaraja et al., 1978; Zebeli and Ametaj, 2009).

In conclusion, the adopted model successfully in-duced acute rumen acidosis in sheep as indicated by severe clinical signs, the significant changes in rumen fluid parameters, the metabolic variations, and the in-flammatory condition. The large increase of lactulose concentration in blood after acidosis induction indicat-ed a disruption of the GI epithelium integrity and the consequent increase of its permeability to lactulose and likely also to large molecules. The earlier appearance of the lactulose in blood compared to a previous work us-ing drugs known to damage intestinal epithelium sug-gests also that the permeability of rumen epithelium was likely altered by acidotic conditions. The results of this experiment suggest that the rumen represents a vulner-able area for the translocation of LPS, toxic biogenic amines, and perhaps different microbes during acute aci-dosis conditions. Results also established that lactulose can be a valuable probe to study rumen permeability in ruminants and that best times for collection of blood are from 2 to 6 h after oral dosage of the sugar. Finally, the present study also indicated the measurement of ALP in feces as potential marker of GI damage in ruminants.

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