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Effect of the magnitude of the decrease of rumen pH on rumen fermentation in a dual-flow continuous culture system¹

Grup de Recerca en Nutrició, Maneig i Benestar Animal, Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain

	Abstract

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The negative effect of pH on rumen microbial fermentation has been associated with the total amount of time that pH is below a certain threshold. However, not only the time, but also the magnitude of the pH reduction, is important. Eight 1,325-mL dual-flow continuous culture fermenters were used in 2 replicated periods to determine the effect of the magnitude of pH reduction (5.6 vs. 5.1) during 4 h/d on rumen microbial fermentation. Fermenters were maintained at a constant temperature (38.5°C) and fed 97 g/d of a 60:40 forage:concentrate diet (19.2% CP, 29.0% NDF, and 18.2% ADF, DM basis), and the solid and liquid dilution rates were controlled at 5.0 and 10.0%/h, respectively. Treatments were a constant pH 6.4 (H), 4 h/d at pH 5.6 (L), 4 h/d at pH 5.1 (VL), and 2 h/d at pH 5.1 and 2 h/d at pH 7.1 (HL). Relative to H, L did not affect OM and NDF digestion, the VFA profile, NH₃-N concentration, CP degradation, or the flow of dietary N. In contrast, VL tended (P < 0.10) to reduce true OM digestion, reduce the NDF digestibility and the acetate and branch-chained VFA proportions, and increase the propionate proportion. Compared with H, VL reduced the CP degradation and the flow of dietary N. Relative to H, treatment HL did not affect OM and NDF digestibility, the acetate proportion, CP degradation, or the flow of dietary N but increased the propionate proportion and decreased the branch-chained VFA proportion and NH₃-N concentration. There were no differences among treatments in the efficiency of microbial protein synthesis, the flow of bacterial N, or the flow of essential and nonessential AA. In summary, fermentation was not affected by either 4 h/d at pH 5.6 or fluctuating pH between 5.1 (2 h/d) and 7.1 (2 h/d), but when pH was at 5.1 for 4 h/d, rumen microbial fermentation was modified, suggesting that effects of low pH on rumen microbial fermentation are dependent on the magnitude of the pH decrease.

Key Words: acidosis • pH • rumen fermentation

	INTRODUCTION

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Recent studies indicate that the effect of low pH on rumen microbial fermentation is dependent not merely on the average pH but on the time that pH is suboptimal (Sauvant et al., 1999; de Veth and Kolver, 2001b; Cerrato et al., 2007a) and the extent of pH variations (de Veth and Kolver, 2001b; Wales et al., 2004). In fact, Sauvant et al. (1999) suggested that the time that pH is below 6.0 is a good indicator of the effects of pH on rumen microbial fermentation and that, up to 4 h/d, no negative effects are expected. The experiment reported here is part of a larger project that evaluates the effects of ruminal pH and its fluctuations on rumen microbial fermentation in vitro. Preliminary studies identified a ruminal pH of 6.4 and 5.5 as optimal and suboptimal, respectively (Cardozo et al., 2000). Cerrato et al. (2007a) reported that the effects of time at this suboptimal pH on rumen microbial fermentation began immediately after the reduction of pH. Furthermore, the effects of low pH are dependent on the total amount of time that pH is suboptimal and are not reduced by splitting it into various cycles (Cerrato et al., 2007b). There is also increasing evidence that large variations in ruminal pH affect rumen microbial fermentation to a greater extent than a low, but less variable, pH (de Veth and Kolver, 2001b; Wales et al., 2004). However, what is uncertain is the extent to which the magnitude of the decrease in ruminal pH affects ruminal fermentation, and it would be important to determine if bacteria would resist transitory reductions to a lower pH (5.1). Therefore, the objective of the present experiment was to determine the effect of the magnitude of pH decrease (5.6 vs. 5.1) during 4 h/d on rumen microbial fermentation.

	MATERIALS AND METHODS

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The research protocol was approved and supervised by the Laboratory Animal Care Committee of the Universsitat Autonoma de Barcelona (Spain).

Apparatus, Experimental Design, and Diets

Eight 1,325-mL dual-flow continuous culture fermenters (Hoover et al., 1976) were used in 2 replicated periods of 8 d each (5 d of adaptation, 3 d of sampling). Fermenters were inoculated with ruminal fluid obtained from a rumen-fistulated lactating dairy cow fed a 60% forage and 40% concentrate diet. Temperature (38.5°C) and liquid (10%/h) and solid (5%/h) dilution rates were maintained constant and were monitored using a personal computer and the LabView Software (FieldPoint, National Instruments, Austin, TX). Anaerobic conditions were maintained by the infusion of N₂ at a rate of 40 mL/min. Artificial saliva (Weller and Pilgrim, 1974) was continuously infused into flasks and contained 0.4 g/L of urea to simulate recycled N. Fermenters were fed 97 g of DM/d in 3 equal portions, at 0800, 1600, and 2400.

The diet consisted of (DM basis) 38.0% alfalfa hay, 20.4% ground corn grain, 17.5% ground corn silage, 14.6% soybean meal, 8.8% ground barley grain, 0.22% NaCl, and 0.48% of a vitamin and mineral mix. The vitamin and mineral mix contained (per kg) the following: 7 mg of Co, 167 mg of Cu, 33 mg of I, 2,660 mg of Mn, 27 mg of Se, 660 mg of Zn, 1,000 kIU of vitamin A, 200 kIU of vitamin D₃, 1,330 mg of vitamin E, 2.67 g of urea, 67 g of NaCl, 33 g of sulfur, and 300 g of MgO. The diet was designed to meet or exceed current nutrient recommendations for a Holstein cow (650 kg of BW) producing 30 kg/d of milk (NRC, 2001) and contained (DM basis) 19.2% CP, 29.0% NDF, 18.2% ADF, 2.79% ether extract, and 6.3% ash.

Treatments were a constant pH 6.4 (H), 4 h/d at pH 5.6 (L), 4 h/d at pH 5.1 (VL), or 2 h/d at pH 5.1 followed by 2 h/d at pH 7.1 (HL). For the rest of the day, fermenters were maintained at pH 6.4. The period of suboptimal pH began at 0800. Ruminal pH was controlled from the beginning of each period at either optimal pH (6.4 ± 0.05) or suboptimal pH (5.6 ± 0.05 or 5.1 ± 0.05) by automatic infusion of 3 M HCl or 5 M NaOH. The change in pH from optimal to suboptimal or vice versa due to the programmed fluctuations was achieved within 15 min.

Sample Collection and Processing

Each experimental period consisted of 5 d for adaptation and 3 d for sampling. During the 3 d for sampling of each period, 8 mL of filtered fermenter fluid was taken at 0800 (after 20 h of pH 6.4 and before beginning the period of suboptimal pH) and at 1200 (after 4 h of suboptimal pH) to determine VFA and NH₃-N concentration. In addition, during sampling days, liquid and solid effluent collection vessels were maintained at 4°C to impede microbial action, their contents were mixed and homogenized for 1 min, and a 500-mL sample was removed via aspiration. Upon completion of each period, effluents from the 3 d of sampling were composited and mixed within fermenter and homogenized for 2 min. Samples were taken for total N, NH₃-N, and VFA analyses. The remainder of the sample was lyophilized. Dry samples were analyzed for DM, ash, NDF, and purine contents.

Bacterial cells were isolated from fermenter flasks on the last day of each period by a combination of several procedures selected to obtain the maximum detachment without affecting microbial cell integrity (Whitehouse et al., 1994). One hundred milliliters of a 0.2% (wt/vol) of methylcellulose buffer solution and small marbles (30 of 2 mm and 15 of 4 mm in diameter) were added to each fermenter, incubated in the same fermenter flask at 39°C for 1 h to remove attached bacteria, and refrigerated at 4°C for 24 h. After refrigeration, fermenter contents were agitated for 1 h to dislodge loosely attached bacteria. The content was filtered through cheesecloth and washed with saline solution. Bacterial cells were isolated by differential centrifugation at 1,000 x g for 15 min to eliminate feed particles and at 20,000 x g for 20 min to isolate the bacterial pellet. Pellets were rinsed twice with saline solution (0.9% NaCl) and recentrifuged at 20,000 x g for 20 min. The last rinse was done with distilled water to prevent contamination of bacteria with ash. Bacterial cells were lyophilized and analyzed for DM, ash, N, and purine contents. Digestibility of OM, NDF, and CP and flows of total, nonammonia, microbial, and dietary N were calculated as described by Stern and Hoover (1990).

Chemical Analyses

Effluent DM was determined by lyophilizing 200-mL aliquots in triplicate, and a subsample was dried at 103°C in a forced-air oven for 24 h to determine the final DM content. The DM contents of diets and bacterial samples were determined by drying samples for 24 h in a 103°C forced-air oven according to the AOAC (1990). Dry samples were ashed overnight at 550°C in a muffle furnace. Fiber components of diets and effluents were analyzed by the detergent system (Van Soest et al., 1991) sequentially using a thermostable alpha-amylase and sodium sulfite. Total N in feed, effluents, and bacterial samples were determined by the Kjeldahl method (AOAC, 1990). Ammonia N was analyzed in a 4-mL subsample of filtered fluid that was acidified with 4 mL of 0.2 M HCl and frozen. Samples were centrifuged at 25,000 x g for 20 min, and the supernatant was analyzed for NH₃-N by colorimetry (Chaney and Marbach, 1962). Samples for VFA were prepared as described by Jouany (1982) using 4-methylvaleric acid as the internal standard. The analysis was performed by GLC (model 6890, Hewlett Packard, Palo Alto, CA) using a polyethylene glycol, nitroterephthalic acid-treated capillary column (BP21, SGE Europe Ltd., Buckinghamshire, UK). Dry effluent and mixed bacterial cells were analyzed for purine base content by HPLC (Beckman Instruments, Palo Alto, CA) according to the procedure of Balcells et al. (1992). Amino acids were analyzed by hydrolyzing samples (25 mg) with 1 mL of 6 M HCl containing 0.5 µL/mL of mercaptoethanol at 110°C for 22.75 h in sealed evacuated tubes. Derivatization was conducted using the AccQ-Tag Amino Acid Analysis Method (Waters Co., Milford, MA). This involved the derivatization of samples with 30 µL per tube of the Waters AccQFluor Reagent (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) at 55°C for 9.33 min. Amino acid analysis was performed by reversed-phase HPLC (HP1100 Agilent, AccQ-Tag Amino Acid Analysis Column 3.9 mm x 150 mm silica base bonded with C18) with UV-visible spectroscopy detection using modified procedures of Waters Amino Acid Analysis Method to ensure separation of derivatized AA. Norleucine was used as the internal standard.

Statistical Analysis

Results were analyzed using the MIXED procedure (SAS Inst. Inc., Cary, NC). Data for nutrient digestion, composite VFA concentrations, and N fractions were analyzed using the model Y_i = µ + pH_i + e_i, where Y_i = the dependent variable; µ = the population mean; pH_i = the mean effect of treatment; and e_i = the residual error. The period was considered a random effect. Treatment means (n = 4 for each treatment) are reported as least squares means, and differences were tested using the Tukey’s multiple comparison test and declared at P < 0.05 unless otherwise indicated.

Results of total and individual VFA and of NH₃-N concentrations of samples obtained from the fermenter flask at 0800 and 1200 during the 3 sampling days were analyzed using the same model with repeated measures (Littell et al., 1998). Data were subjected to 2 covariance structures: compound symmetric and unstructured. The covariance structure that yielded the smallest Schwarz’s Bayesian criterion was considered the most desirable analysis, and the least squares means for treatments are reported. Differences between treatments were declared at P < 0.05 using the Bonferroni comparison test.

	RESULTS AND DISCUSSION

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Because pH was different in each treatment, daily consumption of acid (9.5, 19.8, 31.9, and 23.2 mL/d for H, L, VL, and HL, respectively) and base (4.7, 14.2, 15.9, and 20.3 mL/d for H, L, VL, and HL, respectively) was different between treatments. When pH was reduced to 5.6 for 4 h/d, there were no differences in OM and NDF digestibility (Table 1) compared with a constant pH 6.4. The effects of pH on OM and NDF digestibility are well documented (Mould and Ørskov, 1983; Hoover et al., 1984), but few studies have evaluated the effects of short periods at suboptimal pH on rumen microbial fermentation. It has been suggested that ruminal bacteria are able to resist short periods at low pH without affecting rumen fermentation (Sauvant et al., 1999; de Veth and Kolver, 2001a; Calsamiglia et al., 2002). Although nonsignificant, the reduction in true OM (P < 0.57) and NDF (P < 0.13) digestibility in L compared with H was of similar magnitude to the reduction reported by de Veth and Kolver (2001b) and Cerrato et al. (2007a) for OM and NDF digestibility when pH was maintained suboptimal (pH 5.4 and 5.5, respectively) for 4 h/d.

Compared with a constant pH 6.4, treatment VL reduced the flow of NH₃-N and increased the flow of dietary N but did not affect the flow of bacterial N or the flow of total, essential, or nonessential AA (Tables 3 and 4). When samples were taken from the fermenter flask, NH₃-N concentration was lower in VL compared with H at 0800 and tended (P < 0.10) to be lower at 1200. Relative to a constant pH 6.4, VL also reduced CP degradation, which is supported by the decrease in BCVFA and NH₃-N concentration and flow (Mansfield et al., 1994). In general, results in treatment VL suggest that 4 h/d at pH 5.1 had detrimental effects on rumen microbial fermentation, which is contrary to the hypothesis that ruminal fermentation is not affected by short periods at suboptimal pH (Sauvant et al., 1999). Nevertheless, the present trial is the first study that evaluates short periods at pH 5.1. The studies cited above (de Veth and Kolver, 2001b; Cerrato et al., 2007a) evaluated the effects of 4 h/d at a greater pH value (pH 5.4 and 5.5), and therefore comparisons with treatment VL are not appropriate. Relative to H, treatment VL did not affect the EMPS. Studies on the effect of low pH on the EMPS are contradictory. Although some in vitro studies have shown no effect (Hoover and Miller, 1992; Cardozo et al., 2000; Calsamiglia et al., 2002), another reported a reduction in EMPS (Wales et al., 2004). Results in the present trial indicate that 4 h/d at pH 5.6 or 5.1 had no effect on EMPS.

Results from the current study and others (de Veth and Kolver, 2001b; Calsamiglia et al., 2002) suggest that the effects of pH on rumen microbial fermentation are dependent on the combined effects of the magnitude and time that pH is low. Furthermore, some authors (de Veth and Kovler, 2001b; Wales et al., 2004) also suggested that large variations in ruminal pH may be more critical for rumen fermentation than a low, but less variable, pH. In the present experiment, treatment HL was designed to test the effect of transitory pronounced variations of pH on rumen microbial fermentation. In samples taken from the daily composite, treatment HL had no effects on true OM and NDF digestibility (Table 1), the total VFA concentration, and the acetate and valerate proportions compared with H (Table 2). The propionate proportion was higher and the butyrate and BCVFA proportion was lower compared with H. Results were similar in samples taken from the fermenter flask, although acetate proportion was lower in HL compared with H (at 0800 and 1200), and total VFA concentration was higher in HL compared with H at 0800. Relative to H, treatment HL reduced the average NH3-N concentration and flow but did not affect the flow of dietary and bacterial N, CP degradation, the EMPS, and the flow of total, essential, and nonessential AA (Tables 3 and 4). When samples were taken from the fermenter flask, NH₃-N concentration was lower in HL compared with H at 0800 but was not different at 1200. Changes in the VFA profile and NH₃-N concentration in treatment HL may be explained by the 2 h/d at pH 5.1 but could also be due to fluctuations in pH between an extreme low pH (5.1) and a high pH (7.1). Wales et al. (2004) reported that fluctuations between 5.1 and 6.0 affect rumen bacteria more than the same pH (5.6) kept constant throughout the day and concluded that the inhibition of fermentation was not due only to the lower average pH but also to fluctuations in pH. They suggested that at constant suboptimal pH, there was a shift toward strains of bacteria that tolerate low pH, whereas large diurnal variations in pH may reduce the ability of microbial populations to cope with these changes. However, the experimental design in the present trial does not allow us to determine whether the differences are due to the lower pH alone or to the large fluctuations in ruminal pH.

Short periods (4 h/d) at pH 5.6 had small effects on rumen microbial fermentation compared with a constant optimal pH (6.4), but the effects were negative if pH fell at 5.1 for 4 h/d. When pH fluctuated between pH 5.1 (2 h/d) and 7.1 (2 h/d), effects were negligible. It appears that the effects of low pH on rumen microbial fermentation are due not only to the amount of time pH is below 6.0 (Sauvant et al., 1999) but to the magnitude of decrease of ruminal pH. This supports the hypothesis that the area below pH 5.6 or 5.8, which measures the time and extent below this threshold, may be the most appropriate measurement to assess rumen acidosis (Cooper et al., 1999; Beauchemin et al., 2003).

	Footnotes

 
¹ We express our gratitude to the Spanish Ministry of Education, Culture and Sport for its support through grant AP2002-3180 and the Spanish Interministerial Commission for Science and Technology for its support through CICYT project (AGL 2002 - 01642).

² Corresponding author: sergio.calsamiglia{at}uab.es

Received for publication March 22, 2007. Accepted for publication September 29, 2007.

	LITERATURE CITED

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This Article Abstract Full Text (PDF) All Versions of this Article: jas.2007-0180v1 86/2/378    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cerrato-Sánchez, M. Articles by Ferret, A. Search for Related Content PubMed PubMed Citation Articles by Cerrato-Sánchez, M. Articles by Ferret, A.