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* Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, Scotland, U.K.;
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Wageningen Institute of Animal Sciences, Animal Nutrition Group, Wageningen Agricultural University, Marijkeweg 40, 6709 PG Wageningen, The Netherlands; and
and
Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Absorption • Infusion • Nutrient Uptake • Osmotic Pressure • Rumen • Volatile Fatty Acids
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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, 1992; Dijkstra et al., 1993), with the remainder passing with the liquid outflow to the omasum. Simple diffusion and facilitated transport are the main mechanisms of VFA absorption across the reticuloruminal wall (Gäbel and Sehested, 1997).
There is a close relationship between VFA concentration and osmotic pressure (OP) in the rumen, which affects the net epithelial water flux, changing indirectly the liquid outflow from the rumen, which, in turn, may have an effect on particulate matter passage, degradation rate, and fill in the rumen. Normally, OP is lower in the rumen than in plasma and water is absorbed. If ruminal OP exceeds that of plasma, there will be a net flux of water into the rumen. Our interest in water flux (López et al., 1994) was to enable the estimation of saliva flow, because this will be liquid outflow less water intake corrected for flux across the ruminal wall. The absorption of VFA in the rumen may contribute to keeping OP within physiological limits (López et al., 1994) and is affected by VFA concentration and pH (Dijkstra, 1994), which in turn are determined by the amount of VFA produced by substrate fermentation.
The intragastric nutrition of ruminants (Ørskov et al., 1979) provides a method whereby inputs of VFA and water to the rumen can be controlled very precisely. The objective of the work reported here was to study the effect of ruminal VFA concentration on VFA absorption, apparent water absorption, and ruminal liquid outflow. Changes in ruminal VFA concentration were induced by changing VFA infusion rate in sheep nourished by intragastric infusions.
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Materials and Methods |
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Three Finn Dorset x Suffolk female lambs (8 to 10 mo old and 35 [SE = 0.9] kg BW) were used in a Latin square design (three animals and three experimental periods), in which the animals were randomly allocated to experimental treatments (three different VFA infusion rates). The sheep were fitted with a ruminal cannula and an abomasal catheter. Each experimental period consisted of 1 d. The animals then rested for at least 1 wk before starting the next experimental period. The use of animals in this experiment was approved by the animal care and use committee of the Home Office (Project License No. PPL 60/00854).
Intragastric Infusion of Nutrients
Details of the management of the intragastric infusions were described by Hovell et al. (1987). After recovery from surgery, intragastric infusion of nutrients was introduced progressively during a 10- to 12-d adaptation period, and solid food was withdrawn in the second half of this period. The lambs were kept in metabolism cages and nourished by an intraruminal infusion of a solution of VFA and macrominerals, a separate intraruminal infusion of a solution of McDougall buffer, and an intraabomasal infusion of a casein solution, sufficient to provide about 600 to 650 kJ as VFA and 600 to 700 mg casein-N/kg BW0.75 daily. The VFA solution as infused contained approximately 2.09 M total VFA (650, 250, and 100 mmol/mol total VFA as acetate, propionate, and butyrate, respectively), with 40 mM CaCO3, 12 mM Ca(H2PO4)2•H2O and 7.5 mM MgCl2•6H2O to supply macrominerals, and the buffer solution about 170 mM NaHCO3, 75 mM KHCO3, and 25 mM NaCl. The VFA solution was infused at 132 (SE 2.1) mL/h, and the buffer solution at 245 (SE 3.4) mL/h, giving an overall infusion rate of 377 (SE 4.7) mL/h of liquid (i.e., approximately 0.63 L/kg BW0.75 daily). Vitamins were mixed into the casein solution, and microminerals were dosed per abomasum daily. At the end of the experiment, lambs were inoculated with ruminal fluid from a normally fed animal, and then reintroduced to solid food, and fed normally.
Experimental Procedures
On experimental days, the casein infusion was withdrawn, and access to drinking water was not allowed during the course of the experiment. On that particular day, animals received the basal infusion rate of total VFA (271 [SE 7.1] mmol/h) during the first 2 h, and then each animal received VFA at a different rate for the next 7.5 h. The total VFA supply was modified by changing the concentration of the infusate (0.5, 1.4, and 1.8 x the concentration of the solution described previously), giving infusion rates of 135 (SE 6.0), 394 (SE 5.5), and 511 (SE 15.2) mmol/h, respectively. The amount of liquid infused and the ratio between total VFA and buffer concentrations were kept constant. Samples were taken from the rumen at the time when the VFA infusion rate was changed, and thereafter every 1.5 h (at 1.5, 3, 4.5, 6, and 7.5). The infusion was then stopped, free access to drinking water was allowed, and the ruminal contents were emptied and replaced with warm water. The VFA and buffer infusions were resumed at low levels after 5 to 10 h. During the next 2 d, and depending on the recovery of each animal, the VFA and buffer infusions were increased up to the basal level of infusion.
Markers for the liquid phase were dosed, and samples were collected at different intervals. Polyethylene glycol (PEG) was included in both solutions infused into the rumen at a concentration of 1 g/L, initiating its continuous infusion 3 d before the experimental day to have a stable level of PEG in the ruminal contents. The marker used to estimate ruminal volume was the Cr complex of EDTA (Cr-EDTA), prepared as described by Downes and McDonald (1964) and injected directly in the rumen via the cannula as pulse doses at each sampling time. The ruminal contents were mixed thoroughly after dosing the marker by repetitively removing and returning some liquid using a 60-mL syringe. Samples of ruminal fluid were taken before and after dosing and mixing the marker into the ruminal contents. They were then centrifuged at 10,000 x g for 15 min, and 4 mL of the supernatant was acidified with 1 mL of a 20 mM solution of ethyl-butyric acid in 25% metaphosphoric acid and stored at 4°C for VFA analysis. The remaining supernatant was frozen and retained for PEG and Cr analyses.
Analytical Methods
Analytical determinations of pH, osmolality, PEG, Cr, and VFA concentrations were described by López et al. (1994). The pH was measured directly with a pH-meter, and osmolality was determined with an osmometer based on the depression in the critical freezing point. Concentration of PEG was determined by the turbidity method, using a spectrophotometer to measure optical density at 650 nm. Concentration of Cr in the ruminal samples was analyzed by atomic absorption spectrophotometry at a wavelength of 357.9 nm using an air-acetylene flame. Total VFA concentrations and molar proportions were determined by gas-liquid chromatography with flame ionization detector, using ethyl-butyric acid as the internal standard.
Calculations
The calculations used to estimate parameters of liquid and VFA kinetics in the rumen were as described by López et al. (1994). The model assumes that the ruminal pools of PEG and of VFA are in steady state at the time of sampling and has been described in detail by Warner and Stacy (1968) and France et al. (1991). Ruminal volume (V, L) was estimated as
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where D (milligrams of Cr) is the pulse dose at time t and C- and C+ (both milligrams of Cr/L) are the ruminal concentrations of chromium at time t immediately before and after dosing, respectively. Because ruminal contents are liquid, it was assumed that the marker was completely mixed when the second sample was taken at each time.
Fractional passage rate (k, h-1) was calculated as
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where IPEG (milligrams per hour) is the rate of infusion of PEG and PEGt (milligrams per liter) is its ruminal concentration at time t. For the average passage rate (F, milliliters per hour) between two consecutive sampling times, the following relation was used (Warner and Stacy, 1968):
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where PEGa is the average PEG concentration (milligrams per milliliter) in two consecutive sampling times.
Let I (milliliters per hour) be the rate of water infusion into the rumen, and then the apparent water absorption through the ruminal wall (W, milliliters per hour) was estimated as
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If W is positive, then there is a net absorption of water into the plasma. Negative values of W indicate that there is a net inflow of water into the rumen coming either from the saliva flow or from the movement of water across the ruminal wall. It is not possible to derive the contribution of saliva production to the estimated overall net inflow of liquid, but it can be assumed that this is a minor amount in animals receiving a continuous infusion of nutrients.
Assuming the VFA concentration of the liquid leaving the rumen is equal to the mean VFA concentration of the ruminal contents, the VFA absorption rate (AVFA, millimoles per hour) can be estimated as
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where IVFA (millimoles per hour) is the VFA infusion rate, F (milliliters per hour) is the liquid passage rate and CVFA (millimoles per milliliter) is the average VFA concentration between both sampling times.
Statistical Analysis
Data were examined by ANOVA using a repeated measures design in which the experimental units (each sheep within each experimental period) were arranged in a Latin square, and the sequential samples during the day were considered as repeated observations on each experimental unit. Linear and quadratic effects of the level of VFA infusion were separated by orthogonal polynomials. Differences between means for each level of VFA infusion were evaluated by Bonferroni t-tests. To test statistical significance of treatment (level of VFA infusion) effects, polynomial contrasts, and multiple comparisons among treatment means, the error term used was the residual variance among experimental units (df = 2). The effect of the sampling time was tested using the Greenhouse-Geisser adjustment with the within units residual variance as the error term (df = 12) and, when significant, it was examined by analysis of orthogonal contrasts (Helmert) over time (Rowell and Walters, 1976). Statistical dependence between variables was established by simple linear correlation and regression analyses. Procedures GLM and REG of SAS (SAS Inst. Inc., Cary, NC) were used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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), due to the increase in total VFA concentration (Figure 2). With the lowest infusion rate (135 mmol/h), ruminal pH remained stable, whereas osmolality and total VFA concentration tended to decrease with time. Ruminal fluid volumes (data not shown) and liquid outflow (Figure 2) remained fairly stable during the course of the experiment, as the effect of the sampling time was not significant statistically. The Helmert contrast (orthogonal single-degree-of-freedom contrast) was used (procedure GLM of SAS) to compare values observed at each sampling time with the mean of subsequent times to detect when stable levels of the variable were achieved. This analysis indicated that, in all treatments, stable levels of the variables were reached 4.5 h after initiating the change in VFA infusion rate. The variation from the mean across the last three sampling times within each experimental treatment was smaller than 6% for ruminal pH, osmolality, liquid outflow, and VFA concentrations and absorption rates, which was a variation considered acceptable for a steady-state situation. Therefore, only the values recorded over the last three sampling times were used in the statistical analyses to evaluate the treatment effects.
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. There was no significant effect of VFA infusion rate on ruminal volume, liquid outflow (expressed either as fractional or as absolute outflow rates), or on the apparent water absorption in the rumen.
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. The ruminal total VFA concentration increased linearly (P < 0.05) with VFA infusion rate. There was also a linear increase (P < 0.05) in ruminal osmolality and a linear decline (P < 0.05) in ruminal pH.
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However, for total VFA concentrations in the rumen higher than 80.1 mM, liquid outflow remained stable at an average rate of 407 mL/h.
The effect of VFA infusion rate on the molar proportions of VFA was significant (P < 0.001), with higher proportions of acetic acid and lower proportions of propionic acid as VFA infusion rate was increased (Table 2
). Proportions of butyrate were unaffected (P > 0.10) by the level of VFA infusion rate.
The calculated VFA absorption rates (expressed either as absolute or as fractional rates) are presented in Table 3. There was a significant effect of VFA infusion rate on the VFA absorption rate, with both a linear and a quadratic response to the level of VFA supply. Fractional absorption rates and the proportion of the VFA supply that was absorbed in the rumen tended to be higher as the VFA infusion rate was increased, but differences did not reach the level of statistical significance (P > 0.05). The proportion of total VFA supplied that was absorbed in the rumen was calculated as the slope of the linear relationship between VFA infused (IVFA) and VFA absorbed (AVFA). The regression equation obtained was
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The absorption rates of acetate, propionate, and butyrate are also shown in Table 3. Absolute absorption rates of each acid were significantly affected by the VFA infusion rate in a similar trend for total VFA. Using regression analysis, the proportion of acid infused that was absorbed was 0.822 (SE 0.021), 0.877 (SE 0.017), and 0.910 (SE 0.015) for acetate, propionate, and butyrate, respectively. At the same infusion rate, the fractional rate of absorption and the proportion of acid infused that was absorbed were greatest for butyrate and smallest for acetate.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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The partition of water loss from the rumen between absorption across the ruminal wall and passage to the omasum and abomasum may have important effects on ruminal fermentation patterns, on the absorption of nutrients from the rumen, and on the extent and composition of daily flows of digesta from the reticulorumen. The main factor defining the transepithelial movement of water seems to be the osmotic gradient between the ruminal contents and the plasma, which determines the extent and direction of the diffusion of water (Dobson et al., 1976). In animals fed normally, the addition of PEG (Harrison et al., 1975) or mineral salts (Thomson et al., 1978; Rogers and Davis, 1982; Hart and Polan, 1984) increased both the OP and the dilution rate in the rumen. This significant relationship between ruminal osmolality and liquid outflow from the rumen was confirmed in sheep nourished by the intragastric infusion of nutrients (López et al., 1994), in which OP was changed by infusing various amounts of NaCl into the rumen. The infusion of different amounts of VFA also induced significant changes in ruminal OP, but, in contrast with experiments using mineral salts, liquid passage rate was not affected by the increase in OP or in total VFA concentration in the rumen. The lack of an observable effect in the present experiment could have been due to a more limited range in OP because effects of OP on liquid outflow have been demonstrated in experiments having a wide range in OP (Peters et al., 1990, 1992; López et al., 1994). When the rumen becomes hypertonic because of the addition of mineral salts, there is an increased water secretion across the ruminal epithelium and, subsequently, a greater liquid outflow to the omasum. As OP is increased by the accumulation of VFA, the net water movement across the epithelium is more moderate, as the osmotic forces are offset by the absorption of water associated with the VFA absorption. Thus, for the higher ruminal VFA concentrations, a direct relationship between ruminal OP and liquid outflow rate was not observed. However, for total VFA concentrations lower than 80.1 mM, any increase in VFA concentration was associated with a greater water passage rate. If the relationship between liquid outflow and total VFA concentration is assumed to be curvilinear, then the data can be described by the negative exponential
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There is evidence that ruminal outflow may, at least in part, be controlled by the omasum because infusion of VFA solutions into the omasum increased ruminal outflow of normally fed animals and infusion of a high osmolality buffer decreased ruminal outflow (Afzalzadeh et al., 1997)
The effects of VFA infusion level on ruminal pH, osmolality, and total VFA concentration reflect the accumulation of VFA in the rumen as result of the balance between the rate of VFA infusion and the rate of VFA loss from the rumen, either by absorption or passage. The pool of total VFA in the rumen was increased with the level of VFA infusion, even though ruminal volume was unaffected and the VFA absorption rate was also increased.
The molar proportions of VFA absorbed were affected by the level of VFA infusion into the rumen, with the proportion of propionate decreasing and that of acetate increasing, suggesting that both acids have to be absorbed at different rates. When the absorptions of acetate, propionate, and butyrate were expressed either as fractional absorption rates or as the proportion of acid infused that is absorbed, it was confirmed that the ruminal absorption rate of short-chain fatty acids is directly related to the length of their carbon chain. This may have been pH related, because the accumulation of total VFA in the rumen tended to reduce ruminal pH, thus favoring the absorption of the longer-chain fatty acids (Danielli et al., 1945). Fractional absorption rates of acetic acid were not affected by VFA infusion rate, whereas absorption rates of propionic and butyric acids tended to increase (46% and 33%, respectively) from Level 1 to Level 3 of VFA infusion. The decrease in fractional absorption rates (moles of acid absorbed • mol-1 acid in the rumen • h-1) as pH was increased was quantified by regression analysis, with regression coefficients of -0.024 ± 0.0205, -0.094 ± 0.0224, and -0.106 ± 0.0353 for acetate, propionate, and butyrate, respectively, confirming the differential effect of pH on each VFA. As the infusion of VFA was elevated, the differences between propionic and acetic acid or between butyric and acetic acid in their fractional absorption rates were increased. In line with results reported by Dijkstra et al. (1993) in the washed rumen of dairy cattle, the fractional absorption rate of propionic and butyric acid is higher than that of acetic acid at lower pH values. Given the lower energetic content per mole of acetic acid, an isoenergetic production of acetic acid, compared with that of propionic or butyric acid, results in a higher accumulation of VFA in the rumen and a lower pH. In other words, for an isoenergetic amount of VFA, an increased rate of propionic acid production at the expense of acetic acid production when pH is reduced will help to reduce the accumulation of VFA in the rumen. Oshio and Tahata (1984) reported no differences between the absorption rates of acetic, propionic, and butyric acids from the rumen of sheep at a pH range of 6.8 to 8.0, much higher than those observed at the higher levels of VFA infusion in the present experiment. Stevens (1970) reviewed several in vitro and in vivo VFA absorption studies and concluded that the greater absorption rate of acids with longer chain length may be the result of increased lipid solubility.
Ruminal VFA absorption has been measured by various methods (in vitro, in the isolated rumen, or using ruminal pouches or resorption chambers), but direct applicability of the results obtained to the normal ruminant has been questioned (Stevens, 1970). In animals fed normally, it is difficult to estimate VFA absorption given the difficulty of measuring accurately the production rates of VFA in the rumen, and molar proportions of VFA in ruminal fluid do not necessarily represent proportions in which they are formed (Dijkstra, 1994). With the use of animals nourished by the intragastric infusion of nutrients, the rate of VFA entry into the rumen could be controlled and kept constant. It can be assumed that the different levels of VFA infusion used in this study would correspond with different rates of VFA production in animals fed normally, as it has been reported that the intraruminal infusion of 225 mmol VFA/h is associated with ruminal VFA concentrations similar to those observed after feeding (Matsunaga et al., 1999). An indication of VFA absorption in the rumen can be also obtained from the rate of appearance of VFA in venous blood draining the rumen. However, using this method, the recoveries of propionate (Seal et al., 1989; Kristensen et al., 2000b) and butyrate (Kristensen et al., 2000a; Noziere et al., 2000) were smaller than absorption rates observed in this study. Propionate and butyrate are extensively metabolized in the ruminal epithelial tissue (mainly to lactate and ß-hydroxybutyrate, respectively) in a dose-related manner (Baldwin and Jesse, 1996), and thus the acids measured in the mesenteric veins account for only a variable proportion of those actually absorbed from the luminal side of the ruminal wall (Kristensen et al., 2000a,b; Noziere et al., 2000).
The different mechanisms of VFA fluxes (absorption and secretion) through the ruminal wall have been extensively reviewed (Rechkemmer et al., 1995; Remond et al., 1996; Gäbel and Sehested, 1997). The VFA are absorbed across the ruminal wall both as anions and as undissociated acids (Kramer et al., 1996). Whereas it is clear that passive diffusion is the mechanism for absorption of acids in the nonionic form, much less is known about absorption of the acids as anions. Absorption of VFA is primarily affected by variations in their intraruminal concentrations and pH, although other factors may also be implicated (Remond et al., 1996). In our study, we used undissociated acids in the infusion solutions. However, at pH 5.3, some 76% of the VFA are present in the ionized form according to the Henderson-Hasselbalch equation; at pH 6.8, this is close to 99%. Ionized acids do not diffuse passively across the ruminal cell membrane (Bugaut, 1987). Yet, substantial fractional absorption rates have been observed. This is in line with findings of Dijkstra et al. (1993), who calculated from VFA absorption studies at various pH levels a half-maximal rate of VFA absorption at pH 6.0, when only 6% of the VFA are present in the undissociated form. It is obvious that protons have to be supplied to the dissociated VFA to establish such relatively high fractional absorption rates, probably in an acid microclimate near the ruminal wall (Bugaut, 1987).
In agreement with other authors using different experimental methods in sheep (Rechkemmer et al., 1995; Kristensen et al., 1996, 1998) or in cattle (Peters et al., 1990, 1992; Obitsu and Taniguchi, 1994), absorption of total VFA and of the individual acids was significantly greater with increasing the VFA infusion rate and VFA concentration in the rumen, even though this was accompanied by increases in the ruminal osmolality (Peters et al., 1990, 1992). When the osmotic pressure in the rumen is increased by the addition of NaCl, VFA absorption is reduced (Oshio and Tahata, 1984; López et al., 1994), probably due to the effect of the net influx of water across the ruminal epithelium with the higher OP, which may hinder VFA absorption. Our results indicate that the increase in ruminal osmolality associated with higher total VFA concentrations would not have any detrimental effect on VFA absorption. In contrast with our results, Oshio and Tahata (1984) reported that absorption rates of VFA in an almost dissociated state at neutral or high pH were not affected significantly by their concentrations. The absorption of VFA is reduced at higher pH values, and a significant interaction of pH with VFA concentration on VFA absorption has been reported (Dijkstra et al., 1993).
The ruminal epithelium can show, in its ability to absorb VFA, water, and electrolytes, a reversible adaptive response to different diets (Gäbel et al., 1987; Sehested et al., 2000) and to different levels of feed intake (Perrier et al., 1996; Doreau et al., 1997). The increase in the absorptive capacity due to high VFA concentrations in the ruminal fluid could be a medium-term response, but immediately after the VFA concentration in the rumen is increased, the concentration gradient between the luminal side of the rumen and the blood is greater, resulting in an increased flux of acids to the blood. This would support the idea that passive diffusion is the main driving force for the movement of short-chain fatty acids across the ruminal wall (Rechkemmer et al., 1995; Remond et al., 1996). This diffusion through lipid membranes would be favored by the lipid solubility of the acids, which is in agreement with higher absorption rates of VFA as the carbon chain is longer.
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Implications |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Footnotes |
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3 Present address: Dpto. Producción Animal, Universidad de León, E-24071 León, Spain.
4 Present address: Affrusk, Banchory, Kincardineshire AB31 6LD, Scotland, U.K.
2 Correspondence—phone: +34 987 291 291; fax: +34 987 291 311; E-mail: dp1slp{at}unileon.es.
Received for publication February 18, 2003. Accepted for publication May 26, 2003.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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