DOI: 10.1111/jpn.12245
ORIGINAL ARTICLE
The fate of glycerol entering the rumen of dairy cows and sheep A. Werner Omazic1, C. Kronqvist1, L. Zhongyan2, H. Martens2 and K. Holtenius1 1 Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, Uppsala, Sweden, and 2 Department of Veterinary Physiology, Free University of Berlin, Berlin, Germany
Summary This study investigated the fate of glycerol entering the rumen, in particular whether glycerol could be absorbed across the rumen epithelium. Three non-lactating rumen-fistulated cows were used to calculate the overall disappearance rate of glycerol in vivo and evaluate the rate of ruminal glycerol absorption. Rumen epithelial tissues isolated from sheep were used to characterise glycerol transport properties. The rate of rumen microbial degradation of glycerol was then studied in an in vitro system under anaerobic and thermo-regulated conditions. The results showed that glycerol can be absorbed from the rumen in significant amounts. The fractional rate of absorption of glycerol was not affected by variations in glycerol concentration in the buffer solution in the in vivo study. The glycerol absorption apparently occurred largely by passive diffusion and was probably not facilitated by carriers. Glycerol also disappeared via microbial digestion and outflow from the rumen through the omasal orifice. Keywords glycerine, rumen metabolism, rumen epithelium, absorption, disappearance rate Correspondence Anna Werner Omazic, Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, Box 7024, 750 07 Uppsala, Sweden. Tel: + 46-18672948; E-mail: [email protected] Received: 2 September 2014; accepted: 7 August 2014
Introduction Glycerol (synonym: glycerine) is a by-product of biofuel production that is used in cattle feed, with mature cattle being capable of consuming up to 1 kg glycerol per day (S€ udekum, 2008). The energy value of glycerol is estimated to be 16.2 MJ metabolisable energy (ME) of DM for ruminants (Mach et al., 2009). Consumed glycerol may be fermented in the rumen, absorbed across the rumen epithelium or escape the rumen by outflow through the omasal orifice. Glycerol escaping the rumen may be absorbed from the intestine of cattle as it is in monogastric species (Otha et al., 2006). In humans, ingested glycerol is rapidly absorbed from the gastrointestinal tract (Massicotte et al., 2006). In young calves too, orally supplemented glycerol is rapidly absorbed from the gastrointestinal tract, presumably in the small intestine (Werner Omazic et al., 2013). Absorption of glycerol in the colon appears to be negligible even at high luminal glycerol concentrations and it does not cross the colonic membrane by passive diffusion or by paracellular routes in rats (Yuasa et al., 2003). However, it appears that glycerol can be absorbed from the stomach of rats by passive diffusion (Herting et al., 1956). 258
Absorbed glycerol entering the liver is channelled to triose phosphate and then further to glucose via gluconeogenesis, or it is catabolised via glycolysis (Lin, 1977; Danfaer et al., 1995). In cattle, it is generally assumed that glycerol is mainly fermented in the rumen and that the rate of absorption from the gastrointestinal tract is low (Kristensen and Raun, 2007; S€ udekum, 2008). However, the rate of fermentation of glycerol by rumen bacteria in vitro is only approximately 6% per hour (Trabue et al., 2007). The proportion of propionate and butyrate generally increases at the expense of acetate when diets are supplemented with glycerol (Linke et al., 2004; Kristensen and Raun, 2007; Carvalho et al., 2011). The aim of this study was to determine the fate of glycerol entering the rumen. A subject of particular interest was whether glycerol could be absorbed across the rumen epithelium. Materials and methods Animals Dairy cows
Rumen-fistulated cows of the Swedish Red Breed, not pregnant or lactating, with body weight (BW)
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ranging from 670–800 kg were used. The cows were housed indoors in individual tie stalls on rubber mats with chopped straw and sawdust as bedding and with free access to water and salt lick. The feed intake was 3.7 kg hay per animal and day (92% DM) and 1.6 kg concentrate per animal and day (88% DM). The hay contained per kg DM: 81 g crude protein (CP), 631 g neutral detergent fibre (NDF), 58 g ash and 9.8 MJ ME and the concentrate contained per kg DM 202 g CP, 268 g NDF, 68 g ash and 13.2 MJ ME. The feed ratio was fed twice daily, at 08:00 h and 16:00 h, and there were no feed refusals. All animal procedures were approved by the Uppsala Local Ethics Committee, Sweden (C: 17/8). Sheep
Tissues used for rumen epithelium isolation originated from sheep of the German dairy breed, aged 9–10 months, and were obtained at a local slaughterhouse. The animals were fed a pure hay diet ad libitum over a period of 6 weeks prior to slaughter to ensure adaptation of the rumen epithelium to a low-energy roughage diet. Hay was offered twice daily, at 07:00 h and 15:00 h. Feed intake was 1000 g per animal and day (88% DM) and contained per kg DM: 144 g CP, 28 g fat, 277 g crude fibre, 89 g ash, 29 g potassium, 2.2 g sodium and 8.5 MJ ME. One week before the experiment, the sheep were kept individually in pens on straw to control feed intake. All experiments were conducted in accordance with German laws on the care and use of experimental animals.
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each side with 16 ml buffer solution using a gas lift system and gassed with O2/CO2 (95:5) at 38 °C. The standard experimental buffer contained (mmol/l): 90 NaCl, 25 NaHCO3 25 Na-acetate, 10 Na-propionate, 5 Na-butyrate, 1.2 CaCl2, 1.2 MgCl2, 0.4 NaH2PO4, 2.4 Na2HPO4, 2.5 glutamine, 5 KCl, 5 glucose, 8 MOPS [3-(N-morpholino) propane sulphonic acid (C7H15NO4S)] and 1, 2 or 5 glycerol (purity ≥99%, Sigma, Sigma Aldrich Chemie Gmbh, Munich, Germany) according to the experimental design, adjusted to pH 7.4. Samples of epithelial tissues were obtained from each animal to be used in 24 different Ussing chambers in parallel. In the results, ‘N’ refers to the number of experimental animals, whereas ‘n’ refers to the number of epithelial tissues per treatment group. Electrical measurements and measurements of flux rates
The experiment was essentially performed as described previously by Abdoun et al. (2010). In brief, after stunning and exsanguination, the forestomachs were removed from the abdominal cavity within 2–3 min. A 250 cm2 piece of rumen wall was taken from the ventral sac, repeatedly cleaned in a buffer solution and stripped from the muscle layer. The tissues were transported to the laboratory in a buffer solution containing (mmol/l): 115 NaCl, 25 NaHCO3, 0.4 NaH2PO4, 2.4 Na2HPO4, 5 KCl, 5 glucose, 1.2 CaCl2, 1.2 MgCl2; pH 7.4 at 38.0 °C, adjusted to 300 mosmol/l with mannitol. The solution was gassed with O2/CO2 (95:5). Epithelium samples (3 9 3 cm) were mounted between the two halves of an Ussing chamber to give an exposed area of 3.14 cm2. The mounted tissue was bathed on
Electrical measurements were continuously obtained from a computer-controlled voltage-clamp device (Mussler Scientific Instruments, Aachen, Germany). Modified tips filled with KCl-Agar were positioned ~3 mm from each surface of the tissue and connected to Ag–AgCl electrodes for measurement of the transepithelial potential difference (PDt). Similar tips were inserted ~2 cm from the surface of the tissue for application of a short-circuit current (Isc). Transepithelial conductance (Gt) was calculated by measuring the displacement in potential difference (DPD) caused by application of a bipolar pulse of 100 lA and 1 s duration. All measurements were performed under classical short-circuit conditions (unless otherwise stated). Unidirectional mucosal-to-serosal (ms) and serosalto-mucosal (sm) fluxes (Jms, Jsm) of glycerol were determined on paired tissues from the same rumen under short-circuit conditions. Tissues were paired so that the tissue conductance (Gt) of each tissue in a pair did not differ by more than 25%. Net transepithelial flux was calculated as the difference between unidirectional fluxes in opposite directions (Jnet = Jms Jsm). Experiments commenced after the electrical parameters had stabilised in the open-circuit mode (generally 30–40 min after mounting). At this time, 14C-labelled glycerol (46.25 kBq) was added to the mucosal side of one tissue of each pair and to the serosal side of the other (‘hot side’). The first samples (1 ml) were taken from the unlabelled (‘cold’) side after 20 min of equilibration and then at 20 min intervals for three flux periods. Each sample was replaced by an equal volume of fresh corresponding buffer solution, and the data were corrected for this dilution. Samples (100 ll) from the labelled
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Experiment 1: Glycerol transport across sheep rumen epithelium Rumen epithelium isolation and incubation
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bathing solution (‘hot side’) were taken before the first and after the last flux period for calculation of specific radioactivity. 14C-labelled glycerol (46.25 kBq) was assayed in scintillation liquid (Rotiszint, Roth– Karlsruhe, Germany) using a b-counter (LKB Wal€ lace-Perkin-Elmer; Uberlingen, Germany). The inhibitors [phloretin (1 mmol/l) or HgCl2 (0.01 mmol/l)] was added to the mucosal side 30 min before the first sample was taken from the unlabelled side. The calculation of permeability (P) for glycerol was calculated according P = Jms in leq/cm2/h divided by concentration of glycerol (lmol/cm3). The calculation was performed with the concentration of 1 mmol/l glycerol. Experiment 2: In vivo rumen metabolism of glycerol in cows
To calculate the overall disappearance rate of glycerol from the rumen in vivo, three rumen-fistulated cows were given a bolus dose of 500 g glycerol (purity >99.5%, AkoGly 100; Aarhus Karlshamn, Karlshamn, Sweden) and 8 g CoLi-EDTA (as a fluid marker), dissolved in 1000 ml water. The solution was administered through the fistula at 10:00 h, 2 h after morning feeding. Approximately 100 ml rumen fluid was collected through a PVC tube inserted into the ventral sac of the rumen before the glycerol load, and at 15, 30, 45, 60, 90, 120, 180, 240, 300 and 360 min after the load. The samples were kept on ice until centrifuged at 1800 9 g for 5 min at 20 °C. The supernatant was divided into aliquots and stored at 20 °C until analysis. The cobalt concentration in samples was determined by means of atomic absorption spectrophotometry (AAnalyst 100; Perking Elmer, Norwalk, CT, USA). The fractional outflow of CoLi-EDTA through the omasal orifice was calculated assuming negligible net fluid transport across the rumen epithelium and uniform distribution of CoLi-EDTA in rumen fluid. Thus the fractional outflow rate was calculated according to first-order kinetics. The glycerol concentration in rumen fluid was analysed using an enzymatic colorimetric test (Glycerol; R-BIOPHARM AG, Darmstadt, Germany).
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system maintaining a temperature of 39 °C. The fractional rate of disappearance was assumed to reflect microbial degradation, as no absorption or outflow could occur from the tubes. The rumen of two rumen-fistulated cows was manually emptied completely. Every sixth handful taken from the rumen, 12 and 16 kg digesta, respectively, was transferred to each tube. The residual rumen content was returned to the rumen through the fistula. Six litres of McDougal’s buffer (Tilley and Terry, 1963) and 140 g glycerol (purity >99.5%, AkoGly 100, Aarhus Karlshamn) dissolved in 280 ml water were added to each tube. The initial concentration of glycerol was similar to that in the rumen of cows in Experiment 2 (in vivo study). The content was continuously gassed with CO2 and thoroughly mixed mechanically for 20 s every 5 min during the first 1 h and thereafter every 10 min for 3 h. Approximately 10 ml fluid was collected by suction 5 min before and 15, 30, 45, 60, 90, 120, 150, 180, 210 and 240 min after the glycerol solution was added to the tubes. The pH was recorded immediately after sampling. The samples were then centrifuged at 1800 9 g for 5 min at 20 °C. The supernatant was divided into aliquots and stored at 20 °C until analysis. Glycerol was analysed as described above. Experiment 4: In vivo rumen absorption
Microbial degradation of glycerol was studied in an in vitro system developed by Ud en (2011). Two polyethylene drainage pipes, 700 mm (height) and 250 mm (in diameter.), were used. The pipes were kept in a thermally insulated box with a warming
To study the rate of in vivo ruminal glycerol absorption, the rumen of the three rumen-fistulated cows was completely emptied. The digesta was stored in containers kept in water baths with a temperature of approximately 40 °C during the experiment. The rumen was then washed twice with body-temperature saline solution and thereafter with buffer solution. After this, 10 litres of three different experimental solutions with 15, 30 and 45 mmol/l glycerol, respectively, were introduced to the rumen and left for 60 min. The chemical composition of the buffer solution and the experimental solutions are shown in Table 1. Subsamples of the solution, approximately 40 ml, were collected before it was introduced to the rumen, and 10, 20, 30, 40, 50 and 60 min after introduction. Immediately, thereafter, the fluid was removed and its volume determined. The digesta was then brought back into the rumen. The experimental set-up was a 3 9 3 Latin square design and 1 week was allowed between experiments. The pH was determined in the samples immediately after collection and then samples were stored at 20 °C until analysis. After thawing, the glycerol concentration in the fluid was determined as
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Experiment 3: In vitro rumen metabolism of glycerol in cows
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Table 1 Chemical composition of the buffer solution and the experimental solutions (mmol/l unless otherwise stated). Each treatment solution contained 500 mg CoLi-EDTA
Item
Buffer solution
NaCl NaHCO3 Na2HPO4 KCl KHCO3 Na-acetate Propionate Butyrate MgCl CaCl Glucose Glycerol
45 45 2 5 20 25 10 5 2.5 2 10 –
to MIXED procedure of 2008) using the model:
SAS
(SAS 9.2; SAS Institute,
Yijk ¼ Treatmenti þ Periodj þ Cowk þ eijk
Treatment* GLY15
GLY30
GLY45
30 45 2 5 20 25 10 5 2.5 2 10 15
15 45 2 5 20 25 10 5 2.5 2 10 30
0 45 2 5 20 25 10 5 2.5 2 10 45
*Treatments: GLY15/GLY30/GLY45 = Experimental solution containing 15/30/45 mmol/l glycerol (purity >99.5%).
described above. The cobalt concentration in the samples was determined using the technique described above. The glycerol concentration was divided by the corresponding cobalt concentrations to correct for fluid movements other than across the rumen epithelium. The fractional rate of absorption (FRA) was calculated as Ct = C0ekt, assuming first-order kinetics, where C is the concentration of glycerol, k = FRA and t is the time relative to introduction of the experimental solution into the rumen. Statistical analysis
In total tissues from six sheep were used two for control (Table 2), two for the inhibitors and two for the reduced temperature. The data were calculated by SIGMA PLOT 10.0 for WINDOWS (SPSS, Chicago, IL, USA). The comparison between the groups was carried out in the form of a Student’s t-test or paired t-test. In Experiment 4 (in vivo rumen absorption), predicted values of glycerol FRA (n = 9) were subjected Table 2 Unidirectional transport rates of glycerol in vitro in sheep rumen epithelium Glycerol (mmol/l)
Jms
Jsm
Jnet
Isc
Gt
N/n
1 2 5
36.7 8.4 71.9 10.9 184.3 35.3
35.1 4.0 61.6 11.7 170.3 44.1
1.60 10.3 14.0
1.12 1.26 1.34
3.09 3.13 2.93
2/15 2/16 2/16
Jms (nmol/cm2/h) = transport in the mucosal serosal direction; Jsm (nmol/cm2/h) = transport in the serosal - mucosal direction; Jnet = Jms Jsm (nmol/cm2/h); ISc = leq/cm2/h; Gt = mS/cm2; N = number of sheep; n = number of tissues.
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Treatment (i = 3); Period (j = 3) and Cow (k = 3) are fixed effect and eijk is the random error term. The pH was analysed in MIXED procedure in SAS using the model: Yijkl ¼ Treatmenti þ Periodj þ Timek þ Cowl þ eijkl Treatment (i = 3); Period (j = 3); Time (k = 7) and Cow (l = 3) are fixed effect and eijk is the random error term. Results from the statistical analyses are presented as least squares means (LSM) and associated standard error of the means (SEM). Differences were considered significant at p < 0.05. Results In vitro study, sheep
The glycerol transport properties are shown in Table 2. Glycerol transport increased linearly with increasing glycerol concentration. Jms flux was slightly larger than Jsm, but no significant net transport was observed (p = 0.158 for 1 mmol/l, N = 2, n = 15; p = 0.059 for 2 mmol/l, N = 2, n = 16; p = 0.104 for 5 mmol/l, N = 2, n = 16. A weak correlation (r = 0.4) was found between tissue conductance and glycerol transport. Neither mucosal phloretin (1 mmol/l) nor HgCl2 (0.01 mmol/l) inhibited glycerol (1 mmol/l) transport across the epithelium. Glycerol (Jms) was transported in the control group at a rate of 27.0 6.37 nmol/cm2/h (N = 2; n = 8). Phloretin (N = 2; n = 8; 1 mmol/l lumen side) and HgCl2 (N = 2; n = 7; 0.01 mmol/l lumen side) did not change the flux rate, 26.7 6.64 and 24.8 6.54 nmol/cm2/h respectively. After equilibration and before addition of radioactivity, the incubation temperature was reduced to 28 °C in two sets, 12 of 24 Ussing chambers. This low temperature (38 vs. 28 °C) reduced glycerol transport from Jms 38.3 5.35 to 26.6 2.90 nmol/cm2/h (1 mmol/l, N = 2, n = 8) and from 70.6 5.49 to 55.2 9.55 nmol/cm2/h (2 mmol/l, N = 2, n = 8). In vivo rumen metabolism of glycerol, cows
Glycerol disappearance from the rumen was described by first-order kinetics and R2 ranged from 0.97–0.99 in the cows (n = 3). The disappearance rate ranged
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from 43 to 54% per h, reflecting the sum of microbial digestion, absorption and outflow through the omasal orifice (n = 3) (Fig. 1). The fractional outflow of cobalt ranged from 7.2 to 8.4% per h (n = 3). In vitro rumen metabolism of glycerol, cows
The decrease in glycerol concentration with time is shown in Fig. 2. The disappearance was described by first-order kinetics. The fractional disappearance rate was 6.6% and 10.8% per hour in the two tubes respectively.
Fig. 2 Disappearance of glycerol in a closed in vitro system (n = 2). The curve fit is exponential.
In vivo rumen absorption
The fluid volume increased during the experimental period. After 60 min, the volume ranged from 10.3 to 17.4 l (mean 12.3 l, SD = 2.5). The recovery of cobalt in the collected experimental solution ranged from 83% to 109% (mean 92%, SD = 10). The pH in the initial experimental solutions ranged from 7.1 to 7.7 (mean 7.5, SD = 0.21) and was neither affected by time of sampling (p = 0.10) nor by treatment (p = 0.17). The FRA of glycerol was not affected by variations in glycerol concentration in the buffer solution (p = 0.90). The FRA of glycerol for the different glycerol concentrations was 14, 14 and 11% per hour respectively (SEM = 6.1%).
Fig. 1 Concentration of glycerol in the rumen of cows (n = 3). The curve fit is exponential. Five hundred grams glycerol was added to the rumen compartment at time zero.
increased linearly with increasing glycerol concentration. The transport did not correlate with tissue conductance. Hence, paracellular transport is probably negligible and glycerol passes through the rumen epithelium predominantly via the cellular pathway. Calculation of the permeability of glycerol with the data obtained led to a permeability within the range of 9–10∙106 cm/s, which is within the range of permeability reported for artificial membranes (5.4∙106; Orbach and Finkelstein, 1980) and supports the assumption of lipophilic diffusion. The flux rates were slightly asymmetrical, with Jsm being numerically larger than Jms (Table 2). It is suggested that the tissue beneath the epithelium represents an unstirred layer and may disturb the pathway of glycerol in the serosal–mucosal direction. The transport rate of glycerol was only slightly influenced by temperature. According to the Q10 value, the velocity of a reaction or transport rate is enhanced twofold to fourfold by a change of 10 °C (28 °C vs. 38 °C). The reduction in glycerol flux observed at 28 °C was probably caused by an effect on the diffusion coefficient. The diffusion coefficient (D) was slightly reduced, because it is related to the absolute temperature: D = kT/6p∙r∙g, where k is the Boltzmann constant, T the absolute temperature (°K), r the radius and g the viscosity of the medium. The viscosity was probably increased by the decrease in temperature. The observed ratios of fluxes (38 °C vs. 28 °C) were 1.46 (1 mmol/l) and 1.27 (2 mmol/l) and within the range of 1.32 which has been observed by non-ionic diffusion of acetate in sheep omasum (Ali et al., 2006). It appears unlikely that a transport protein is involved. The flux rate increased from 36.7 to 184.3 nmol/ cm2/h with increasing glycerol concentration from 1 to 5 mmol/l. Assuming a further linear increase in
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Discussion This study showed that glycerol was transported across the rumen epithelium, very likely by passive diffusion. The studies with isolated sheets of ovine rumen epithelium exhibited no net transport under classical Ussing chamber conditions and transfer
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glycerol transport, extrapolation of the glycerol transport rate at a glycerol concentration. Twenty mmol/l led to a flux rate of 0.8 lmol/cm2/h, which is in the range of bovine rumen short-chain fatty acids (SCFA) transport rate at a glycerol concentration of 20 mmol/l (Sehested et al., 1999). Despite the assumption of lipophilic diffusion across the membranes of the epithelium, possible carrier-mediated transport of glycerol should be mentioned. Aquaporins are a family of small proteins that act as carriers of water and glycerol across cell membranes in numerous mammalian tissues, including the gastrointestinal tract (Ishibashi et al., 2009). Aquaporins have been demonstrated in the rumen epithelium of cows (AQP3, AQP7, AQP8 and AQP10; Rojen et al., 2011) and AQP3 and AQP7 are permeable to glycerol (Papadopoulos and Verkman, 2013). However, aquaporins probably did not facilitate ruminal transport of glycerol, because it was not reduced by phloretin or HgCl2, which are suggested to act as blockers of the aquaporin family (Ishibashi et al., 2009). The results from our in vivo studies with the washed rumen of cows support the results generated in the in vitro system based on isolated ovine epithelia. The fractional absorption rate of glycerol did not appear to be reduced when the glycerol concentration increased from 15 mmol/l up to 45 mmol/l in the experimental solution. Similarly, studies in rats have shown that glycerol is transported passively across the intestine when the glycerol concentration in the lumen is high (Otha et al., 2006). However, the present results must be viewed with caution, as the relationship is based on data from only three levels of glycerol and a limited range of glycerol concentrations. In the present study, the pH of the buffer experimental solutions was approximately 7.5, which is above rumen fluid pH of fed cows. It is well documented that the rate of absorption of SCFA is negatively correlated to rumen pH and even at pH levels above neutral, there was a substantial absorption of SCFA (Dijkstra et al., 1993; Melo et al., 2013). Glycerol is an alcohol which does not dissociate as SCFA do and it can be assumed that the glycerol absorption rate is less affected by rumen pH than SCFA. The rumen fluid pH in the present study reflects that of cattle subjected to an approximately 24-h period without feed (Galyean et al., 1981), but it cannot be excluded that the slightly alkalotic pH affected the permeability of the ruminal epithelium.
The volume of fluid in the rumen increased during the measurement period. This probably mainly reflected saliva inflow. A significant net inflow of fluid across the rumen epithelium is less probable, given that the experimental fluid was designed to have similar osmolality to blood plasma and that net movements of fluid are largely driven by osmotic differences (Warner and Stacy, 1972). If the in vitro system used in the present study accurately reflected microbial degradation of glycerol in vivo, the observed rapid rate of glycerol disappearance from the rumen could scarcely be explained by rumen microbial digestion. The determined rate of in vitro microbial digestion in the present study was in agreement with observations made by Trabue et al. (2007). Approximately 25% of the glycerol entering the rumen was estimated to be rumen fermented while approximately 45% was absorbed from the rumen according to the equation described by McDonald (1981). Provided that the observed fractional outflow rate of CoLi-EDTA through the omasal orifice in Experiment 2 accurately reflected the glycerol fractional outflow rate, approximately 30% of the glycerol escaped through the omasal orifice. This fraction was most likely absorbed from the small intestine. Taken together, the results indicated that approximately 75% of the glycerol escaped rumen fermentation and may have been an available gluconeogenic substrate. This finding is favourable especially for high-yielding dairy cows, as the absorbed glycerol can be efficiently converted to glucose via gluconeogenesis in the liver and might in the end improve the milk yield.
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Conclusions This study showed that glycerol is absorbed from the rumen in significant amounts. The absorption appears to occur largely via passive diffusion and is probably not facilitated by carriers. Such a mechanism would be beneficial, especially for high-yielding dairy cows, as the absorbed glycerol can be efficiently converted to glucose via gluconeogenesis in the liver. Glycerol also disappeared via microbial digestion and outflow from the rumen through the omasal orifice. Acknowledgements Aarhus Karlshamn, Sweden, is gratefully acknowledged for providing the glycerol supplement used in the study. The authors thank H akan Wallin and Camilla Andersson for technical assistance and for performing the analysis.
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References Abdoun, K.; Stumpff, F.; Rabbani, I.; Martens, H., 2010: Modulation of urea transport across sheep rumen epithelium in vitro by SCFA and CO2. American Journal of Physiology-Gastrointestinal and Liver Physiology 298, G190–G202. Ali, O.; Shen, Z.; Tietjen, U.; Martens, H., 2006: Transport of acetate and sodium in sheep omasum: mutual, but asymmetric interactions. Journal of Comparative Physiology B, Biochemical Systemic and Environmental Physiology 176, 477–487. Carvalho, E. R.; Schmelz-Roberts, N. S.; White, H. M.; Doane, P. H.; Donkin, S. S., 2011: Replacing corn with glycerol in diets for transition dairy cows. Journal of dairy science 94, 908–916. Danfaer, A.; Tetens, V.; Agergaard, N., 1995: Review and an experimentalstudy on the physiological and quantitative aspects of gluconeogenesis in lactating ruminants. Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology 111, 201–210. Dijkstra, J.; Boer, H.; Vanbruchem, J.; Bruining, M.; Tamminga, S., 1993: Absorption of volatile fatty acids from the rumen of lactating dairy cows as influenced by volatile fatty acid concentration, pH and rumen liquid volume. British Journal of Nutrition 69, 385–396. Galyean, M. L.; Lee, R. W.; Hubbert, M. E., 1981: Influence of fasting and transit on ruminal and blood metabolites in beef steers. Journal of Animal Science 53, 7–18. Herting, D. C.; Embree, N. D.; Harris, P. L., 1956: Absorption of acetic acid and glycerol from the rat stomach. American Journal of Physiology – Legacy Content 187, 224–226. Ishibashi, K.; Hara, S.; Kondo, S., 2009: Aquaporin water channels in mammals. Clinical and Experimental Nephrology 13, 107–117. Kristensen, N. B.; Raun, B. M. L., 2007: Ruminal fermentation, portal absorption and hepatic metabolism of glycerol infused into the rumen of lactating cows. In: Energy and protein metabolism and nutrition. Proc. 2nd International symposium on energy and protein
264
metabolism and nutrition. Wageningen, the Netherlands. Paper No 124. Lin, E. C. C., 1977: Glycerol utilization and its regulation in mammals. Annual Review of Biochemistry 46, 765–795. Linke, P. L.; DeFrain, J. M.; Hippen, A. R.; Jardon, P. W., 2004: Ruminal and plasma responses in dairy cows to drenching or feeding glycerol. Journal of Dairy Science 82 (Suppl. 1), 343. Mach, N.; Bach, A.; Devant, M., 2009: Effects of crude glycerin supplementation on performance and meat quality of Holstein bulls fed high-concentrate diets. Journal of Animal Science 87, 632– 638. Massicotte, D.; Scotto, A.; Peronnet, F.; M’Kaouar, H.; Milot, M.; Lavoie, C., 2006: Metabolic fate of a large amount of C-13-glycerol ingested during prolonged exercise. European Journal of Applied Physiology 96, 322–329. McDonald, E., 1981: A revised model for the estimation of protein degradability in the rumen. The Journal of Agricultural Science 96, 251–252. Melo, L. Q.; Costa, S. F.; Lopes, F.; Guerreio, M. C.; Armentano, L. E.; Pereira, M. N., 2013: Rumen morphometrics and the effect of digesta pH and volume on volatile fatty acid absorption. Journal of Animal Science 91, 1775–1783. Orbach, E.; Finkelstein, A., 1980: The nonelectrolyte permeability of planar lipid bilayer membranes. Journal of General Physiology 75, 427–436. Otha, K.; Inoue, K.; Hayashi, Y.; Yuasa, H., 2006: Carrier-mediated transport of glycerol in the perfused rat small intestine. Biological and Pharmaceutical Bulletin 29, 785–789. Papadopoulos, M. C.; Verkman, A. S., 2013: Aquaporin water channels in the nervous system. Nature Reviews Neuroscience 14, 265–277. Rojen, B. A.; Poulsen, S. B.; Theil, P. K.; Fenton, R. A.; Kristensen, N. B., 2011: Short communication: effects of dietary nitrogen concentration on messenger RNA expression and protein abundance of urea transporter-B and aquaporins in
ruminal papillae from lactating Holstein cows. Journal of Dairy Science 94, 2587– 2591. SAS Institute, 2008: SAS 9.2. SAS Institute Inc., Cary, USA. Sehested, J.; Diernaes, L.; Moller, P. D.; Skadhauge, E., 1999: Ruminal transport and metabolism of short-chain fatty acids (SCFA) in vitro: effect of SCFA chain length and pH. Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology 123, 359–368. S€ udekum, K.-H., 2008: Co- products from biodiesel production. In: P. C. Garnsworthy, J. Weisman (eds), Recent Advances in Animal Nutrition – 2007. Nottingham University Press, Nottingham, UK, pp. 201–219. Tilley, J. M. A.; Terry, R. A., 1963: A twostage technique for the in vitro digestion of forage crops. Journal of the British Grassland Society 18, 104–111. Trabue, S.; Scoggin, K.; Tjandrakusuma, S.; Rasmussen, M. A.; Reilly, P. J., 2007: Ruminal fermentation of propylene glycol and glycerol. Journal of Agricultural and Food Chemistry 55, 7043–7051. Ud en, P., 2011: Using a novel macro in vitro technique to estimate differences in absorption rates of volatile fatty acids in the rumen. Journal of Animal Physiology and Animal Nutrition 95, 27–33. Warner, A. C. I.; Stacy, B. D., 1972: Water, sodium and potassium movements across rumen wall of sheep. Quarterly Journal of Experimental Physiology and Cognate Medical Sciences 57, 103–119. Werner Omazic, A.; Tr av en, M.; Roos, S.; Mellgren, E.; Holtenius, K., 2013: Oral rehydration solution with glycerol to dairy calves: effects on fluid balance, metabolism, and intestinal microbiota. Acta Agriculturae Scandinavica, Section A – Animal Science 63, 47–56. Yuasa, Y.; Hamamoto, K.; Dogu, S.; Marutani, T.; Nakajima, A.; Kato, T.; Hayashi, Y.; Inoue, K.; Watanabe, J., 2003: Saturable absorption of glycerol in the rat intestine. Biological and Pharmaceutical Bulletin 26, 1633–1636.
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ORIGINAL ARTICLE
The fate of glycerol entering the rumen of dairy cows and sheep A. Werner Omazic1, C. Kronqvist1, L. Zhongyan2, H. Martens2 and K. Holtenius1 1 Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, Uppsala, Sweden, and 2 Department of Veterinary Physiology, Free University of Berlin, Berlin, Germany
Summary This study investigated the fate of glycerol entering the rumen, in particular whether glycerol could be absorbed across the rumen epithelium. Three non-lactating rumen-fistulated cows were used to calculate the overall disappearance rate of glycerol in vivo and evaluate the rate of ruminal glycerol absorption. Rumen epithelial tissues isolated from sheep were used to characterise glycerol transport properties. The rate of rumen microbial degradation of glycerol was then studied in an in vitro system under anaerobic and thermo-regulated conditions. The results showed that glycerol can be absorbed from the rumen in significant amounts. The fractional rate of absorption of glycerol was not affected by variations in glycerol concentration in the buffer solution in the in vivo study. The glycerol absorption apparently occurred largely by passive diffusion and was probably not facilitated by carriers. Glycerol also disappeared via microbial digestion and outflow from the rumen through the omasal orifice. Keywords glycerine, rumen metabolism, rumen epithelium, absorption, disappearance rate Correspondence Anna Werner Omazic, Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, Box 7024, 750 07 Uppsala, Sweden. Tel: + 46-18672948; E-mail: [email protected] Received: 2 September 2014; accepted: 7 August 2014
Introduction Glycerol (synonym: glycerine) is a by-product of biofuel production that is used in cattle feed, with mature cattle being capable of consuming up to 1 kg glycerol per day (S€ udekum, 2008). The energy value of glycerol is estimated to be 16.2 MJ metabolisable energy (ME) of DM for ruminants (Mach et al., 2009). Consumed glycerol may be fermented in the rumen, absorbed across the rumen epithelium or escape the rumen by outflow through the omasal orifice. Glycerol escaping the rumen may be absorbed from the intestine of cattle as it is in monogastric species (Otha et al., 2006). In humans, ingested glycerol is rapidly absorbed from the gastrointestinal tract (Massicotte et al., 2006). In young calves too, orally supplemented glycerol is rapidly absorbed from the gastrointestinal tract, presumably in the small intestine (Werner Omazic et al., 2013). Absorption of glycerol in the colon appears to be negligible even at high luminal glycerol concentrations and it does not cross the colonic membrane by passive diffusion or by paracellular routes in rats (Yuasa et al., 2003). However, it appears that glycerol can be absorbed from the stomach of rats by passive diffusion (Herting et al., 1956). 258
Absorbed glycerol entering the liver is channelled to triose phosphate and then further to glucose via gluconeogenesis, or it is catabolised via glycolysis (Lin, 1977; Danfaer et al., 1995). In cattle, it is generally assumed that glycerol is mainly fermented in the rumen and that the rate of absorption from the gastrointestinal tract is low (Kristensen and Raun, 2007; S€ udekum, 2008). However, the rate of fermentation of glycerol by rumen bacteria in vitro is only approximately 6% per hour (Trabue et al., 2007). The proportion of propionate and butyrate generally increases at the expense of acetate when diets are supplemented with glycerol (Linke et al., 2004; Kristensen and Raun, 2007; Carvalho et al., 2011). The aim of this study was to determine the fate of glycerol entering the rumen. A subject of particular interest was whether glycerol could be absorbed across the rumen epithelium. Materials and methods Animals Dairy cows
Rumen-fistulated cows of the Swedish Red Breed, not pregnant or lactating, with body weight (BW)
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ranging from 670–800 kg were used. The cows were housed indoors in individual tie stalls on rubber mats with chopped straw and sawdust as bedding and with free access to water and salt lick. The feed intake was 3.7 kg hay per animal and day (92% DM) and 1.6 kg concentrate per animal and day (88% DM). The hay contained per kg DM: 81 g crude protein (CP), 631 g neutral detergent fibre (NDF), 58 g ash and 9.8 MJ ME and the concentrate contained per kg DM 202 g CP, 268 g NDF, 68 g ash and 13.2 MJ ME. The feed ratio was fed twice daily, at 08:00 h and 16:00 h, and there were no feed refusals. All animal procedures were approved by the Uppsala Local Ethics Committee, Sweden (C: 17/8). Sheep
Tissues used for rumen epithelium isolation originated from sheep of the German dairy breed, aged 9–10 months, and were obtained at a local slaughterhouse. The animals were fed a pure hay diet ad libitum over a period of 6 weeks prior to slaughter to ensure adaptation of the rumen epithelium to a low-energy roughage diet. Hay was offered twice daily, at 07:00 h and 15:00 h. Feed intake was 1000 g per animal and day (88% DM) and contained per kg DM: 144 g CP, 28 g fat, 277 g crude fibre, 89 g ash, 29 g potassium, 2.2 g sodium and 8.5 MJ ME. One week before the experiment, the sheep were kept individually in pens on straw to control feed intake. All experiments were conducted in accordance with German laws on the care and use of experimental animals.
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each side with 16 ml buffer solution using a gas lift system and gassed with O2/CO2 (95:5) at 38 °C. The standard experimental buffer contained (mmol/l): 90 NaCl, 25 NaHCO3 25 Na-acetate, 10 Na-propionate, 5 Na-butyrate, 1.2 CaCl2, 1.2 MgCl2, 0.4 NaH2PO4, 2.4 Na2HPO4, 2.5 glutamine, 5 KCl, 5 glucose, 8 MOPS [3-(N-morpholino) propane sulphonic acid (C7H15NO4S)] and 1, 2 or 5 glycerol (purity ≥99%, Sigma, Sigma Aldrich Chemie Gmbh, Munich, Germany) according to the experimental design, adjusted to pH 7.4. Samples of epithelial tissues were obtained from each animal to be used in 24 different Ussing chambers in parallel. In the results, ‘N’ refers to the number of experimental animals, whereas ‘n’ refers to the number of epithelial tissues per treatment group. Electrical measurements and measurements of flux rates
The experiment was essentially performed as described previously by Abdoun et al. (2010). In brief, after stunning and exsanguination, the forestomachs were removed from the abdominal cavity within 2–3 min. A 250 cm2 piece of rumen wall was taken from the ventral sac, repeatedly cleaned in a buffer solution and stripped from the muscle layer. The tissues were transported to the laboratory in a buffer solution containing (mmol/l): 115 NaCl, 25 NaHCO3, 0.4 NaH2PO4, 2.4 Na2HPO4, 5 KCl, 5 glucose, 1.2 CaCl2, 1.2 MgCl2; pH 7.4 at 38.0 °C, adjusted to 300 mosmol/l with mannitol. The solution was gassed with O2/CO2 (95:5). Epithelium samples (3 9 3 cm) were mounted between the two halves of an Ussing chamber to give an exposed area of 3.14 cm2. The mounted tissue was bathed on
Electrical measurements were continuously obtained from a computer-controlled voltage-clamp device (Mussler Scientific Instruments, Aachen, Germany). Modified tips filled with KCl-Agar were positioned ~3 mm from each surface of the tissue and connected to Ag–AgCl electrodes for measurement of the transepithelial potential difference (PDt). Similar tips were inserted ~2 cm from the surface of the tissue for application of a short-circuit current (Isc). Transepithelial conductance (Gt) was calculated by measuring the displacement in potential difference (DPD) caused by application of a bipolar pulse of 100 lA and 1 s duration. All measurements were performed under classical short-circuit conditions (unless otherwise stated). Unidirectional mucosal-to-serosal (ms) and serosalto-mucosal (sm) fluxes (Jms, Jsm) of glycerol were determined on paired tissues from the same rumen under short-circuit conditions. Tissues were paired so that the tissue conductance (Gt) of each tissue in a pair did not differ by more than 25%. Net transepithelial flux was calculated as the difference between unidirectional fluxes in opposite directions (Jnet = Jms Jsm). Experiments commenced after the electrical parameters had stabilised in the open-circuit mode (generally 30–40 min after mounting). At this time, 14C-labelled glycerol (46.25 kBq) was added to the mucosal side of one tissue of each pair and to the serosal side of the other (‘hot side’). The first samples (1 ml) were taken from the unlabelled (‘cold’) side after 20 min of equilibration and then at 20 min intervals for three flux periods. Each sample was replaced by an equal volume of fresh corresponding buffer solution, and the data were corrected for this dilution. Samples (100 ll) from the labelled
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Experiment 1: Glycerol transport across sheep rumen epithelium Rumen epithelium isolation and incubation
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bathing solution (‘hot side’) were taken before the first and after the last flux period for calculation of specific radioactivity. 14C-labelled glycerol (46.25 kBq) was assayed in scintillation liquid (Rotiszint, Roth– Karlsruhe, Germany) using a b-counter (LKB Wal€ lace-Perkin-Elmer; Uberlingen, Germany). The inhibitors [phloretin (1 mmol/l) or HgCl2 (0.01 mmol/l)] was added to the mucosal side 30 min before the first sample was taken from the unlabelled side. The calculation of permeability (P) for glycerol was calculated according P = Jms in leq/cm2/h divided by concentration of glycerol (lmol/cm3). The calculation was performed with the concentration of 1 mmol/l glycerol. Experiment 2: In vivo rumen metabolism of glycerol in cows
To calculate the overall disappearance rate of glycerol from the rumen in vivo, three rumen-fistulated cows were given a bolus dose of 500 g glycerol (purity >99.5%, AkoGly 100; Aarhus Karlshamn, Karlshamn, Sweden) and 8 g CoLi-EDTA (as a fluid marker), dissolved in 1000 ml water. The solution was administered through the fistula at 10:00 h, 2 h after morning feeding. Approximately 100 ml rumen fluid was collected through a PVC tube inserted into the ventral sac of the rumen before the glycerol load, and at 15, 30, 45, 60, 90, 120, 180, 240, 300 and 360 min after the load. The samples were kept on ice until centrifuged at 1800 9 g for 5 min at 20 °C. The supernatant was divided into aliquots and stored at 20 °C until analysis. The cobalt concentration in samples was determined by means of atomic absorption spectrophotometry (AAnalyst 100; Perking Elmer, Norwalk, CT, USA). The fractional outflow of CoLi-EDTA through the omasal orifice was calculated assuming negligible net fluid transport across the rumen epithelium and uniform distribution of CoLi-EDTA in rumen fluid. Thus the fractional outflow rate was calculated according to first-order kinetics. The glycerol concentration in rumen fluid was analysed using an enzymatic colorimetric test (Glycerol; R-BIOPHARM AG, Darmstadt, Germany).
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system maintaining a temperature of 39 °C. The fractional rate of disappearance was assumed to reflect microbial degradation, as no absorption or outflow could occur from the tubes. The rumen of two rumen-fistulated cows was manually emptied completely. Every sixth handful taken from the rumen, 12 and 16 kg digesta, respectively, was transferred to each tube. The residual rumen content was returned to the rumen through the fistula. Six litres of McDougal’s buffer (Tilley and Terry, 1963) and 140 g glycerol (purity >99.5%, AkoGly 100, Aarhus Karlshamn) dissolved in 280 ml water were added to each tube. The initial concentration of glycerol was similar to that in the rumen of cows in Experiment 2 (in vivo study). The content was continuously gassed with CO2 and thoroughly mixed mechanically for 20 s every 5 min during the first 1 h and thereafter every 10 min for 3 h. Approximately 10 ml fluid was collected by suction 5 min before and 15, 30, 45, 60, 90, 120, 150, 180, 210 and 240 min after the glycerol solution was added to the tubes. The pH was recorded immediately after sampling. The samples were then centrifuged at 1800 9 g for 5 min at 20 °C. The supernatant was divided into aliquots and stored at 20 °C until analysis. Glycerol was analysed as described above. Experiment 4: In vivo rumen absorption
Microbial degradation of glycerol was studied in an in vitro system developed by Ud en (2011). Two polyethylene drainage pipes, 700 mm (height) and 250 mm (in diameter.), were used. The pipes were kept in a thermally insulated box with a warming
To study the rate of in vivo ruminal glycerol absorption, the rumen of the three rumen-fistulated cows was completely emptied. The digesta was stored in containers kept in water baths with a temperature of approximately 40 °C during the experiment. The rumen was then washed twice with body-temperature saline solution and thereafter with buffer solution. After this, 10 litres of three different experimental solutions with 15, 30 and 45 mmol/l glycerol, respectively, were introduced to the rumen and left for 60 min. The chemical composition of the buffer solution and the experimental solutions are shown in Table 1. Subsamples of the solution, approximately 40 ml, were collected before it was introduced to the rumen, and 10, 20, 30, 40, 50 and 60 min after introduction. Immediately, thereafter, the fluid was removed and its volume determined. The digesta was then brought back into the rumen. The experimental set-up was a 3 9 3 Latin square design and 1 week was allowed between experiments. The pH was determined in the samples immediately after collection and then samples were stored at 20 °C until analysis. After thawing, the glycerol concentration in the fluid was determined as
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Experiment 3: In vitro rumen metabolism of glycerol in cows
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Table 1 Chemical composition of the buffer solution and the experimental solutions (mmol/l unless otherwise stated). Each treatment solution contained 500 mg CoLi-EDTA
Item
Buffer solution
NaCl NaHCO3 Na2HPO4 KCl KHCO3 Na-acetate Propionate Butyrate MgCl CaCl Glucose Glycerol
45 45 2 5 20 25 10 5 2.5 2 10 –
to MIXED procedure of 2008) using the model:
SAS
(SAS 9.2; SAS Institute,
Yijk ¼ Treatmenti þ Periodj þ Cowk þ eijk
Treatment* GLY15
GLY30
GLY45
30 45 2 5 20 25 10 5 2.5 2 10 15
15 45 2 5 20 25 10 5 2.5 2 10 30
0 45 2 5 20 25 10 5 2.5 2 10 45
*Treatments: GLY15/GLY30/GLY45 = Experimental solution containing 15/30/45 mmol/l glycerol (purity >99.5%).
described above. The cobalt concentration in the samples was determined using the technique described above. The glycerol concentration was divided by the corresponding cobalt concentrations to correct for fluid movements other than across the rumen epithelium. The fractional rate of absorption (FRA) was calculated as Ct = C0ekt, assuming first-order kinetics, where C is the concentration of glycerol, k = FRA and t is the time relative to introduction of the experimental solution into the rumen. Statistical analysis
In total tissues from six sheep were used two for control (Table 2), two for the inhibitors and two for the reduced temperature. The data were calculated by SIGMA PLOT 10.0 for WINDOWS (SPSS, Chicago, IL, USA). The comparison between the groups was carried out in the form of a Student’s t-test or paired t-test. In Experiment 4 (in vivo rumen absorption), predicted values of glycerol FRA (n = 9) were subjected Table 2 Unidirectional transport rates of glycerol in vitro in sheep rumen epithelium Glycerol (mmol/l)
Jms
Jsm
Jnet
Isc
Gt
N/n
1 2 5
36.7 8.4 71.9 10.9 184.3 35.3
35.1 4.0 61.6 11.7 170.3 44.1
1.60 10.3 14.0
1.12 1.26 1.34
3.09 3.13 2.93
2/15 2/16 2/16
Jms (nmol/cm2/h) = transport in the mucosal serosal direction; Jsm (nmol/cm2/h) = transport in the serosal - mucosal direction; Jnet = Jms Jsm (nmol/cm2/h); ISc = leq/cm2/h; Gt = mS/cm2; N = number of sheep; n = number of tissues.
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Treatment (i = 3); Period (j = 3) and Cow (k = 3) are fixed effect and eijk is the random error term. The pH was analysed in MIXED procedure in SAS using the model: Yijkl ¼ Treatmenti þ Periodj þ Timek þ Cowl þ eijkl Treatment (i = 3); Period (j = 3); Time (k = 7) and Cow (l = 3) are fixed effect and eijk is the random error term. Results from the statistical analyses are presented as least squares means (LSM) and associated standard error of the means (SEM). Differences were considered significant at p < 0.05. Results In vitro study, sheep
The glycerol transport properties are shown in Table 2. Glycerol transport increased linearly with increasing glycerol concentration. Jms flux was slightly larger than Jsm, but no significant net transport was observed (p = 0.158 for 1 mmol/l, N = 2, n = 15; p = 0.059 for 2 mmol/l, N = 2, n = 16; p = 0.104 for 5 mmol/l, N = 2, n = 16. A weak correlation (r = 0.4) was found between tissue conductance and glycerol transport. Neither mucosal phloretin (1 mmol/l) nor HgCl2 (0.01 mmol/l) inhibited glycerol (1 mmol/l) transport across the epithelium. Glycerol (Jms) was transported in the control group at a rate of 27.0 6.37 nmol/cm2/h (N = 2; n = 8). Phloretin (N = 2; n = 8; 1 mmol/l lumen side) and HgCl2 (N = 2; n = 7; 0.01 mmol/l lumen side) did not change the flux rate, 26.7 6.64 and 24.8 6.54 nmol/cm2/h respectively. After equilibration and before addition of radioactivity, the incubation temperature was reduced to 28 °C in two sets, 12 of 24 Ussing chambers. This low temperature (38 vs. 28 °C) reduced glycerol transport from Jms 38.3 5.35 to 26.6 2.90 nmol/cm2/h (1 mmol/l, N = 2, n = 8) and from 70.6 5.49 to 55.2 9.55 nmol/cm2/h (2 mmol/l, N = 2, n = 8). In vivo rumen metabolism of glycerol, cows
Glycerol disappearance from the rumen was described by first-order kinetics and R2 ranged from 0.97–0.99 in the cows (n = 3). The disappearance rate ranged
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from 43 to 54% per h, reflecting the sum of microbial digestion, absorption and outflow through the omasal orifice (n = 3) (Fig. 1). The fractional outflow of cobalt ranged from 7.2 to 8.4% per h (n = 3). In vitro rumen metabolism of glycerol, cows
The decrease in glycerol concentration with time is shown in Fig. 2. The disappearance was described by first-order kinetics. The fractional disappearance rate was 6.6% and 10.8% per hour in the two tubes respectively.
Fig. 2 Disappearance of glycerol in a closed in vitro system (n = 2). The curve fit is exponential.
In vivo rumen absorption
The fluid volume increased during the experimental period. After 60 min, the volume ranged from 10.3 to 17.4 l (mean 12.3 l, SD = 2.5). The recovery of cobalt in the collected experimental solution ranged from 83% to 109% (mean 92%, SD = 10). The pH in the initial experimental solutions ranged from 7.1 to 7.7 (mean 7.5, SD = 0.21) and was neither affected by time of sampling (p = 0.10) nor by treatment (p = 0.17). The FRA of glycerol was not affected by variations in glycerol concentration in the buffer solution (p = 0.90). The FRA of glycerol for the different glycerol concentrations was 14, 14 and 11% per hour respectively (SEM = 6.1%).
Fig. 1 Concentration of glycerol in the rumen of cows (n = 3). The curve fit is exponential. Five hundred grams glycerol was added to the rumen compartment at time zero.
increased linearly with increasing glycerol concentration. The transport did not correlate with tissue conductance. Hence, paracellular transport is probably negligible and glycerol passes through the rumen epithelium predominantly via the cellular pathway. Calculation of the permeability of glycerol with the data obtained led to a permeability within the range of 9–10∙106 cm/s, which is within the range of permeability reported for artificial membranes (5.4∙106; Orbach and Finkelstein, 1980) and supports the assumption of lipophilic diffusion. The flux rates were slightly asymmetrical, with Jsm being numerically larger than Jms (Table 2). It is suggested that the tissue beneath the epithelium represents an unstirred layer and may disturb the pathway of glycerol in the serosal–mucosal direction. The transport rate of glycerol was only slightly influenced by temperature. According to the Q10 value, the velocity of a reaction or transport rate is enhanced twofold to fourfold by a change of 10 °C (28 °C vs. 38 °C). The reduction in glycerol flux observed at 28 °C was probably caused by an effect on the diffusion coefficient. The diffusion coefficient (D) was slightly reduced, because it is related to the absolute temperature: D = kT/6p∙r∙g, where k is the Boltzmann constant, T the absolute temperature (°K), r the radius and g the viscosity of the medium. The viscosity was probably increased by the decrease in temperature. The observed ratios of fluxes (38 °C vs. 28 °C) were 1.46 (1 mmol/l) and 1.27 (2 mmol/l) and within the range of 1.32 which has been observed by non-ionic diffusion of acetate in sheep omasum (Ali et al., 2006). It appears unlikely that a transport protein is involved. The flux rate increased from 36.7 to 184.3 nmol/ cm2/h with increasing glycerol concentration from 1 to 5 mmol/l. Assuming a further linear increase in
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Discussion This study showed that glycerol was transported across the rumen epithelium, very likely by passive diffusion. The studies with isolated sheets of ovine rumen epithelium exhibited no net transport under classical Ussing chamber conditions and transfer
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glycerol transport, extrapolation of the glycerol transport rate at a glycerol concentration. Twenty mmol/l led to a flux rate of 0.8 lmol/cm2/h, which is in the range of bovine rumen short-chain fatty acids (SCFA) transport rate at a glycerol concentration of 20 mmol/l (Sehested et al., 1999). Despite the assumption of lipophilic diffusion across the membranes of the epithelium, possible carrier-mediated transport of glycerol should be mentioned. Aquaporins are a family of small proteins that act as carriers of water and glycerol across cell membranes in numerous mammalian tissues, including the gastrointestinal tract (Ishibashi et al., 2009). Aquaporins have been demonstrated in the rumen epithelium of cows (AQP3, AQP7, AQP8 and AQP10; Rojen et al., 2011) and AQP3 and AQP7 are permeable to glycerol (Papadopoulos and Verkman, 2013). However, aquaporins probably did not facilitate ruminal transport of glycerol, because it was not reduced by phloretin or HgCl2, which are suggested to act as blockers of the aquaporin family (Ishibashi et al., 2009). The results from our in vivo studies with the washed rumen of cows support the results generated in the in vitro system based on isolated ovine epithelia. The fractional absorption rate of glycerol did not appear to be reduced when the glycerol concentration increased from 15 mmol/l up to 45 mmol/l in the experimental solution. Similarly, studies in rats have shown that glycerol is transported passively across the intestine when the glycerol concentration in the lumen is high (Otha et al., 2006). However, the present results must be viewed with caution, as the relationship is based on data from only three levels of glycerol and a limited range of glycerol concentrations. In the present study, the pH of the buffer experimental solutions was approximately 7.5, which is above rumen fluid pH of fed cows. It is well documented that the rate of absorption of SCFA is negatively correlated to rumen pH and even at pH levels above neutral, there was a substantial absorption of SCFA (Dijkstra et al., 1993; Melo et al., 2013). Glycerol is an alcohol which does not dissociate as SCFA do and it can be assumed that the glycerol absorption rate is less affected by rumen pH than SCFA. The rumen fluid pH in the present study reflects that of cattle subjected to an approximately 24-h period without feed (Galyean et al., 1981), but it cannot be excluded that the slightly alkalotic pH affected the permeability of the ruminal epithelium.
The volume of fluid in the rumen increased during the measurement period. This probably mainly reflected saliva inflow. A significant net inflow of fluid across the rumen epithelium is less probable, given that the experimental fluid was designed to have similar osmolality to blood plasma and that net movements of fluid are largely driven by osmotic differences (Warner and Stacy, 1972). If the in vitro system used in the present study accurately reflected microbial degradation of glycerol in vivo, the observed rapid rate of glycerol disappearance from the rumen could scarcely be explained by rumen microbial digestion. The determined rate of in vitro microbial digestion in the present study was in agreement with observations made by Trabue et al. (2007). Approximately 25% of the glycerol entering the rumen was estimated to be rumen fermented while approximately 45% was absorbed from the rumen according to the equation described by McDonald (1981). Provided that the observed fractional outflow rate of CoLi-EDTA through the omasal orifice in Experiment 2 accurately reflected the glycerol fractional outflow rate, approximately 30% of the glycerol escaped through the omasal orifice. This fraction was most likely absorbed from the small intestine. Taken together, the results indicated that approximately 75% of the glycerol escaped rumen fermentation and may have been an available gluconeogenic substrate. This finding is favourable especially for high-yielding dairy cows, as the absorbed glycerol can be efficiently converted to glucose via gluconeogenesis in the liver and might in the end improve the milk yield.
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Conclusions This study showed that glycerol is absorbed from the rumen in significant amounts. The absorption appears to occur largely via passive diffusion and is probably not facilitated by carriers. Such a mechanism would be beneficial, especially for high-yielding dairy cows, as the absorbed glycerol can be efficiently converted to glucose via gluconeogenesis in the liver. Glycerol also disappeared via microbial digestion and outflow from the rumen through the omasal orifice. Acknowledgements Aarhus Karlshamn, Sweden, is gratefully acknowledged for providing the glycerol supplement used in the study. The authors thank H akan Wallin and Camilla Andersson for technical assistance and for performing the analysis.
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References Abdoun, K.; Stumpff, F.; Rabbani, I.; Martens, H., 2010: Modulation of urea transport across sheep rumen epithelium in vitro by SCFA and CO2. American Journal of Physiology-Gastrointestinal and Liver Physiology 298, G190–G202. Ali, O.; Shen, Z.; Tietjen, U.; Martens, H., 2006: Transport of acetate and sodium in sheep omasum: mutual, but asymmetric interactions. Journal of Comparative Physiology B, Biochemical Systemic and Environmental Physiology 176, 477–487. Carvalho, E. R.; Schmelz-Roberts, N. S.; White, H. M.; Doane, P. H.; Donkin, S. S., 2011: Replacing corn with glycerol in diets for transition dairy cows. Journal of dairy science 94, 908–916. Danfaer, A.; Tetens, V.; Agergaard, N., 1995: Review and an experimentalstudy on the physiological and quantitative aspects of gluconeogenesis in lactating ruminants. Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology 111, 201–210. Dijkstra, J.; Boer, H.; Vanbruchem, J.; Bruining, M.; Tamminga, S., 1993: Absorption of volatile fatty acids from the rumen of lactating dairy cows as influenced by volatile fatty acid concentration, pH and rumen liquid volume. British Journal of Nutrition 69, 385–396. Galyean, M. L.; Lee, R. W.; Hubbert, M. E., 1981: Influence of fasting and transit on ruminal and blood metabolites in beef steers. Journal of Animal Science 53, 7–18. Herting, D. C.; Embree, N. D.; Harris, P. L., 1956: Absorption of acetic acid and glycerol from the rat stomach. American Journal of Physiology – Legacy Content 187, 224–226. Ishibashi, K.; Hara, S.; Kondo, S., 2009: Aquaporin water channels in mammals. Clinical and Experimental Nephrology 13, 107–117. Kristensen, N. B.; Raun, B. M. L., 2007: Ruminal fermentation, portal absorption and hepatic metabolism of glycerol infused into the rumen of lactating cows. In: Energy and protein metabolism and nutrition. Proc. 2nd International symposium on energy and protein
264
metabolism and nutrition. Wageningen, the Netherlands. Paper No 124. Lin, E. C. C., 1977: Glycerol utilization and its regulation in mammals. Annual Review of Biochemistry 46, 765–795. Linke, P. L.; DeFrain, J. M.; Hippen, A. R.; Jardon, P. W., 2004: Ruminal and plasma responses in dairy cows to drenching or feeding glycerol. Journal of Dairy Science 82 (Suppl. 1), 343. Mach, N.; Bach, A.; Devant, M., 2009: Effects of crude glycerin supplementation on performance and meat quality of Holstein bulls fed high-concentrate diets. Journal of Animal Science 87, 632– 638. Massicotte, D.; Scotto, A.; Peronnet, F.; M’Kaouar, H.; Milot, M.; Lavoie, C., 2006: Metabolic fate of a large amount of C-13-glycerol ingested during prolonged exercise. European Journal of Applied Physiology 96, 322–329. McDonald, E., 1981: A revised model for the estimation of protein degradability in the rumen. The Journal of Agricultural Science 96, 251–252. Melo, L. Q.; Costa, S. F.; Lopes, F.; Guerreio, M. C.; Armentano, L. E.; Pereira, M. N., 2013: Rumen morphometrics and the effect of digesta pH and volume on volatile fatty acid absorption. Journal of Animal Science 91, 1775–1783. Orbach, E.; Finkelstein, A., 1980: The nonelectrolyte permeability of planar lipid bilayer membranes. Journal of General Physiology 75, 427–436. Otha, K.; Inoue, K.; Hayashi, Y.; Yuasa, H., 2006: Carrier-mediated transport of glycerol in the perfused rat small intestine. Biological and Pharmaceutical Bulletin 29, 785–789. Papadopoulos, M. C.; Verkman, A. S., 2013: Aquaporin water channels in the nervous system. Nature Reviews Neuroscience 14, 265–277. Rojen, B. A.; Poulsen, S. B.; Theil, P. K.; Fenton, R. A.; Kristensen, N. B., 2011: Short communication: effects of dietary nitrogen concentration on messenger RNA expression and protein abundance of urea transporter-B and aquaporins in
ruminal papillae from lactating Holstein cows. Journal of Dairy Science 94, 2587– 2591. SAS Institute, 2008: SAS 9.2. SAS Institute Inc., Cary, USA. Sehested, J.; Diernaes, L.; Moller, P. D.; Skadhauge, E., 1999: Ruminal transport and metabolism of short-chain fatty acids (SCFA) in vitro: effect of SCFA chain length and pH. Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology 123, 359–368. S€ udekum, K.-H., 2008: Co- products from biodiesel production. In: P. C. Garnsworthy, J. Weisman (eds), Recent Advances in Animal Nutrition – 2007. Nottingham University Press, Nottingham, UK, pp. 201–219. Tilley, J. M. A.; Terry, R. A., 1963: A twostage technique for the in vitro digestion of forage crops. Journal of the British Grassland Society 18, 104–111. Trabue, S.; Scoggin, K.; Tjandrakusuma, S.; Rasmussen, M. A.; Reilly, P. J., 2007: Ruminal fermentation of propylene glycol and glycerol. Journal of Agricultural and Food Chemistry 55, 7043–7051. Ud en, P., 2011: Using a novel macro in vitro technique to estimate differences in absorption rates of volatile fatty acids in the rumen. Journal of Animal Physiology and Animal Nutrition 95, 27–33. Warner, A. C. I.; Stacy, B. D., 1972: Water, sodium and potassium movements across rumen wall of sheep. Quarterly Journal of Experimental Physiology and Cognate Medical Sciences 57, 103–119. Werner Omazic, A.; Tr av en, M.; Roos, S.; Mellgren, E.; Holtenius, K., 2013: Oral rehydration solution with glycerol to dairy calves: effects on fluid balance, metabolism, and intestinal microbiota. Acta Agriculturae Scandinavica, Section A – Animal Science 63, 47–56. Yuasa, Y.; Hamamoto, K.; Dogu, S.; Marutani, T.; Nakajima, A.; Kato, T.; Hayashi, Y.; Inoue, K.; Watanabe, J., 2003: Saturable absorption of glycerol in the rat intestine. Biological and Pharmaceutical Bulletin 26, 1633–1636.
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