J. Anim. Sci. 2003. 81:537-544
© 2003 American Society of Animal Science
Diet and procedures used to detach particle-associated microbes from ruminal digesta influence chemical composition of microbes and estimation of microbial growth in Rusitec fermenters1
M. J. Ranilla2 and
M. D. Carro
Departamento de Producción Animal I, Universidad de León, 24071 León, Spain
2 Correspondence:
Phone: +34 987 291234; fax: +34 987 291311; E-mail:
dp1mrg{at}unileon.es.
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Abstract
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Four different detachment methods were evaluated for their ability to remove particle-associated microorganisms (PAM) from ruminal digesta in semicontinuous fermenters fed two diets differing in their forage:concentrate ratio (80:20 [C20] and 20:80 [C80]). In the methylcellulose method, ruminal digesta was incubated at 38°C for 15 min with saline solution containing 0.1% methylcellulose before being stored at 4°C for 24 h. In the other procedures, samples were incubated with 0.1% methylcellulose before storage for 24 h at 4°C in different solutions (pH = 2): 1) saline solution with 0.1% Tween 80; 2) saline solution with 0.1% Tween 80 and 1% tertiary butanol; and 3) saline solution with 0.1% Tween 80, 1% methanol, and 1% tertiary butanol. Common to all treatments was subsequent homogenization, followed by filtration and resuspension of the residue five times in the treatment solutions. Microbial removal was estimated indirectly by measuring removal of 15N. There were no differences (P > 0.05) among detachment procedures, neither in the detaching efficiency (mean values of 79.7 and 88.1% for C20 and C80 diets, respectively) nor in the total recovery of PAM (54.9 and 34.9% for C20 and C80, respectively). There were no differences (P > 0.05) among PAM pellets obtained by the different detachment procedures in their N content, purine bases (PB) concentration, or PB:N ratio. For the C80 diet, 15N enrichment was greater (P < 0.05) in PAM pellets obtained with methylcellulose than in those obtained by the other methods. However, there were no differences (P > 0.05) due to the detachment procedure in the values of daily microbial growth estimated using as reference the different PAM pellets. The PAM pellets for diet C20 presented greater (P < 0.01) N content and lower (P < 0.01) PB concentration than those for diet C80 (mean values of 74.3 vs 49.1 mg of N/g of dry matter, and 22.8 vs 26.0 µmol PB/mg of dry matter, respectively). Daily microbial growth was greater (P < 0.05) for the C80 diet than for the C20 diet (121 vs 114 mg of microbial N, respectively). Results suggest that the treatment of ruminal digesta with a saline solution with 0.1% methylcellulose at 38°C for 15 min combined with homogenizing and chilling at 4°C for 24 h removed a major proportion of PAM, although further research is needed to decrease microbial losses during the isolation process.
Key Words: Adhesion • Fermentation • Microbial Proteins • Nitrogen • Purines • Rumen
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Introduction
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Methods of estimating ruminal microbial protein synthesis rely on marker techniques in which a particular microbial constituent is related to the microbial N content. Although differences between liquid-associated (LAM) and particle-associated microorganisms (PAM) in marker:N ratio are widely demonstrated (Merry and McAllan, 1983; Carro and Miller, 2002), in most of the studies, marker:N values have generally been established in LAM, and it has been assumed that the same relationship holds in the total population leaving the rumen (Merry and McAllan, 1983). Some studies have used a homogenate of total digesta to obtain a more representative microbial preparation (Cecava et al., 1990; Carro and Miller, 2002), but procedures commonly used to detach PAM have an efficiency lower than 50% (Legay-Carmier and Bauchart, 1989). Consequently, a pure PAM preparation may not be representative of the total PAM, and the validity of using a crude homogenate is also in doubt. Whitehouse et al. (1994) reported higher removal efficiencies (up to 82%), but recovery of microbial population was not reported. More recently, Martín-Orúe et al. (1998) showed a low recovery of microbes (from 19 to 22%) after detachment from ruminal digesta from sheep. Our hypothesis was that diet and/or procedure used to detach microbes from ruminal digesta could affect the characteristics of the microbial pellet obtained and therefore, the estimation of ruminal microbial protein synthesis. The aim of this work was to investigate the efficiency of recovery of PAM from ruminal digesta using four different detachment procedures, and to compare the estimates of microbial synthesis obtained with the microbial pellets isolated after each procedure. The study was conducted with semi-continuous fermenters fed two diets differing in their forage:concentrate ratio, as this factor can affect the relative proportion of LAM and PAM in the rumen.
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Materials and Methods
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Apparatus, Diets, and Experimental Procedure
One 15-d incubation trial was carried out using semi-continuous fermenters (Rusitec). The Rusitec unit consisted of eight vessels with an effective volume of 700 mL each, and the general incubation procedure was as described by Czerkawski and Breckenridge (1977). The dietary treatments consisted of two complete diets composed of chopped alfalfa hay (about 2-cm pieces) and concentrate in the proportions of 80:20 (C20) and 20:80 (C80; g/100 g; air-dry basis). Composition of experimental diets is shown in Table 1
. Dietary treatments were assigned randomly, so that each diet was fed to four vessels. Each vessel received 15.6 g of DM of the corresponding diet fed into nylon bags (100-µm pore size) daily. Solid and liquid fermentation inocula were collected from four ruminally fistulated sheep immediately before feeding in the morning and transferred to the in vitro system within 30 min. Two sheep were fed diet C20 and the other two received diet C80 for 15 d before commencing the trial. Sheep were managed according to the protocols approved by the León University Institutional Animal Care and Use Committee.
A continuous infusion of artificial saliva (pH = 8.4; 9.8 g of NaHCO3, 3.72 g of Na2HPO4, 0.47 g of NaCl, 0.57 g of KCl, 0.053 g of CaCl2 • 2H2O, and 0.128 g of MgCl2 • 6H2O per liter) at a rate of 500 mL/d was maintained through the four vessels receiving diet C20. The composition of the artificial saliva was modified (pH = 7.0; 6.5 g of NaHCO3, 1.86 g of Na2HPO4, 0.47 g of NaCl, 0.57 g of KCl, 0.053 g of CaCl2 • 2H2O, and 0.128 g of MgCl2 • 6H2O per liter) for the vessels fed the C80 diet in order to achieve a pH value similar to that found before feeding in the rumen of sheep fed this diet. On d 9, a dose of 1.85 mg of 15N (95% enriched SO4 [15NH4]2; Sigma Chemical, Madrid, Spain) was added into each vessel to instantaneously label the ammonia N pool. Then a solution of SO4 (15NH4)2 was added to the artificial saliva at a rate of 3.70 mg of 15N per liter. On d 9, 10, and 11, the pH of vessel fluid was determined immediately before feeding, and the following samples were collected. Liquid effluent was collected daily in overflow flasks containing a solution of H2SO4 (20%, vol/vol) to maintain pH values below 2. One milliliter of effluent was added to 1 mL of deproteinizing solution (10% of metaphosphoric acid and 0.06% crotonic acid, wt/vol) for VFA determination, and 5 mL of effluent were stored at -20°C for ammonia N determination. One nylon bag from each vessel was collected daily, washed twice with 40 mL of non-15N-enriched artificial saliva, and then washed in the cold rinse cycle (20 min) of a washing machine. Disappearance of DM after 48 h of incubation was calculated from the loss in weight after oven drying at 60°C for 48 h and the residues were analyzed for ash to determine the apparent disappearance of organic matter.
On d 12, 13, 14 and 15, 5 mL of saturated HgCl2 was added to the overflow flasks, which were held at 4°C by a cold-water bath to impede microbial growth. During these days, the residues of the nylon bags collected daily from the vessels were subjected to four different detachment procedures to obtain PAM. Each day, the four vessels receiving diet C20 or diet C80 were allocated randomly to one of the detachment procedures with the only restriction that each detachment procedure was applied to each vessel only once. The nylon bags were washed twice with 40 mL of artificial saliva, and then their content was emptied and weighed. One sample (about 5 g of fresh matter) was taken, frozen, and lyophilized to determine DM, N, and 15N enrichment. The rest of the residue was subjected to one of the following treatments (see Figure 1): 1) the residue was incubated with saline solution (0.85% NaCl) containing 0.1% methylcellulose at 38°C for 15 min with continuous shaking; the residue was filtered through two layers of nylon cloth (40-µm pore size), resuspended in cold (4°C) saline solution containing 0.1% methylcellulose, and chilled at 4°C for 24 h; 2) after incubating the residue with saline solution containing 0.1% methylcellulose at 38°C for 15 min with continuous shaking, the residue was filtered through two layers of nylon cloth (40-µm pore size), resuspended in saline solution containing 0.1% Tween 80 (pH = 2), and chilled at 4°C for 24 h; 3) as in 2, except that 1% tertiary butanol was included in the treatment solution; and 4) as in 3 except that 1% butanol was included in the treatment solution. All solutions were added at a rate of 3 mL/g of residue. The filtrate obtained in each treatment after the 15-min incubation with methylcellulose was stored at 4°C and mixed with that obtained the following day. Following treatment (24 h), the samples were homogenized for 10 s with a Waring Blender, filtered through two layers of nylon cloth (40-µm pore size), and the filtrate was removed and retained. The residue was resuspended in the corresponding solution and homogenized; the process was repeated five times, pooling the filtrates each time. The filtrate was then centrifuged at 20,000 x g for 25 min at 4°C to obtain a microbial pellet. This was washed by resuspension in saline solution and the centrifugation was repeated. Microbial pellets were lyophilized, ground to a fine powder with a mortar and pestle, and analyzed for N, 15N enrichment, and PB concentration. The final residue from the contents of the nylon bags was weighed, frozen, and lyophilized for determination of DM, non-ammonia N (NAN), and 15N enrichment. Diets were also analyzed for their natural 15N content, and this value was used for background correction before 15N infusion.
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Figure 1. Procedures used to detach and isolate particle-associated ruminal microorganisms (PAM) from nylon bag residues in semi-continuous fermenters.
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On d 12, 13, 14, and 15, the daily liquid effluent was collected and pooled. One sample (about 300 mL) was frozen and lyophilized for determination of DM, NAN, and 15N enrichment. Approximately 500 mL of effluent were used for isolation of LAM by centrifugation following the procedure described above for PAM. Microscopic examination of microbial pellets (PAM and LAM) showed that they were essentially free from feed particles.
Analytical Procedures
Procedures for determination of DM, ash, N, NDF, ADF, acid detergent lignin, VFA and ammonia N, and preparation of samples for 15N analysis of digesta and microbial pellets have been reported by Carro and Miller (1999). Analyses of 15N were performed by isotope ratio mass spectrometry as described by Barrie and Workman (1984). The PB concentration in bacterial pellets was quantified by HPLC after acid hydrolysis with 2 mL of 2 M perchloric acid at 100°C for 1 h (Martín-Orúe et al., 1995).
Calculations and Statistical Analyses
The proportion of detached microbes (percentage detachment) was calculated as [1 - (15N in residues after treatment/15N in residues before treatment) x 100]. The proportion of recovery from detached microbes (percentage recovery) was calculated as [15N in microbial pellet/(15N in residues before treatment - 15 N in residues after treatment) x 100]. The total recovery was calculated as [(percentage detachment x percentage recovery)/100].
Daily PAM synthesis was estimated by multiplying total NAN production in nylon bag residues by the ratio 15N:N in residues/15N:N in PAM. Daily LAM synthesis was estimated by multiplying total NAN production in the effluent by the ratio 15 N:N in effluent/15N:N in LAM. Total daily microbial production was calculated as the sum of the flows of PAM and LAM.
Data relative to detachment procedures, chemical composition of microbial pellets and microbial synthesis were analyzed by ANOVA according to a split-plot arrangement of treatments with diet as the main-plot and detachment procedure as the subplot treatment. Effect of diet on any of the considered variables was tested using the variance between vessels within diet as the error term. Effect of detachment procedure and the interaction of diet x detachment procedure was tested against the residual error. There were four replicates for each detachment treatment. The sums of squares were further partitioned by orthogonal contrasts to analyze differences among detachment procedures. The contrasts were distributed as follows: 1) methylcellulose vs the others; 2) Tween 80 vs methanol and tertiary butanol; and 3) methanol vs tertiary butanol.
The effect of diet on the main fermentation measurements (pH, VFA and ammonia-N production, and disappearance of diet) was tested by ANOVA with three repeated measurements (3 d of sampling). The GLM procedures of SAS (SAS Inst., Inc., Cary, NC) were used for all statistical analyses.
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Results and Discussion
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There were marked differences in the ruminal fermentation between diets. Ruminal pH values before feeding were greater (P < 0.01) for vessels fed the C20 than for those receiving the C80 diet (6.84 vs 6.25, respectively). Daily ammonia production was greater (P < 0.01) in C20-fed vessels (149 and 99 mg NH3-N for the C20 and the C80 diet, respectively), but daily VFA production was lower (P < 0.05) in these vessels (50.5 mmol) than in those fed the C80 diet (56.0 mmol). The apparent disappearance of OM was lower (P < 0.01) for the C20 (61.5%) than for the C80 diet (69.6%).
As stated by Whitehouse et al. (1994), an ideal protocol for dissociating ruminal microbes from particulate matter undoubtedly requires a combination of physical and chemical methods to disrupt the variety of adherence mechanisms used by microorganisms. Based on the results previously reported by others, we chose several combinations of physical treatments (homogenizing, chilling at 4°C, and low pH) with different chemical treatments (methylcellulose, Tween 80, methanol, and tertiary butanol). Methylcellulose has been reported to remove cellulolytic bacteria from cellulose powder (Minato and Suto, 1978), but few studies have tested its efficiency on removing PAM from ruminal digesta. Homogenization, chilling at 4°C, and methylcellulose treatment were included in all treatments, and the objective was to investigate whether other physical or chemical treatments could increase the removal of PAM from ruminal digesta.
As shown in Table 2, there were no differences (P > 0.05) among detachment procedures in percentage of detachment, recovery of detached, or total recovery of PAM for both diets. Values of detachment for methylcellulose treatment in our study (80.5 and 86.0% for C20 and C80 diets, respectively) were greater than the 66.2% value reported by Martín-Orúe et al. (1998) for ruminal digesta of sheep fed two diets differing in forage:concentrate ratio. The increased removal of PAM found in our study might be due to the longer time of incubation of ruminal samples with the methylcellulose solution (15 vs 5 min in the work of Martín-Orúe et al., 1998). Whitehouse et al. (1994) compared different detachment treatments using purine bases as microbial markers, and reported that the incubation of ruminal digesta with methylcellulose solution (0.1%) previous to treatment with Tween 80 and methanol produced an increase in PAM removal from 61.5 to 83.4%. Detachment values in our study were greater than those obtained by Whitehouse et al. (1994) for treatments with solutions (pH = 2) of Tween 80 (62.2%) and Tween 80 plus methanol (66.6%), but similar to the 83.4% obtained by these authors with a combination of methylcellulose (0.1%), Tween 80, and methanol. Whereas in the study of Martín-Orúe et al. (1998) the treatment with methylcellulose produced the greatest detachment values compared with others (saline solution and tertiary butanol), in our study no other treatment (pH = 2, Tween 80, butanol, or methanol) resulted in an increase of PAM removal. According to these results and those reported by Whitehouse et al. (1994), methylcellulose seems to be efficient for removing PAM from ruminal digesta. If these results are confirmed in future studies, the use of irritant substances such as Tween 80, methanol and tertiary butanol for detachment purposes might be discarded.
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Table 2. Percentage of detachment, recovery of detached and total recovery of particle-associated microbes from ruminal digesta in semicontinuous fermenters fed two diets (low concentrate [C20] and high concentrate [C80]) after applying different detachment procedures (see text for calculations),as determined using 15N as microbial marker
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Although we obtained high values of detachment for all treatments, the values of PAM recovery were considerably lower (see Table 2
), ranging from 36.3 to 69.3%. Total recovery of PAM ranged from 54.2 to 55.8% and from 32.2 to 37.3% for diets C20 and C80, respectively, and the differences among treatments were not significant (P > 0.05). Similar values of recovery (from 32.1 to 52.4%) were reported by Craig et al. (1987) after extracting PAM from ruminal digesta of cows fed a 65% alfalfa haylage diet using an extraction solution containing formaldehyde and Tween 80. In contrast, Martín-Orúe et al. (1998) reported lower values (from 19.2 to 22%) using extraction solutions with NaCl, methylcellulose or tertiary butanol. One factor that could explain the greater recovery values in our study is the time of sampling. Craig et al. (1987) observed that the proportion of PAM recovered from ruminal digesta tended to increase with time after feeding, with the lowest recoveries observed 1 to 3 h after feeding. In the Rusitec system, a nylon bag is removed daily after 48 h of incubation, and microbial attachment could be less tenacious at this time than soon after feeding (Leedle et al., 1982).
A critical point to use of alcohols as detaching agents is their ability to react with the lipid structure of the cellular membrane, breaking the noncovalent bounds in the hydrophobic regions of lipids (Harold, 1970), which may cause leakage or cell lysis, and hence, losses of cellular content. We analyzed the chemical composition of the pellets to test the possible existence of cellular losses with some detachment procedures. As shown in Table 3, no marked differences in chemical composition were detected between PAM pellets obtained with methylcellulose treatment and those obtained after Tween 80, tertiary butanol or methanol treatments. For both diets, total N content in PAM was not affected (P > 0.05) by the detachment procedure. Whereas no differences among methods for 15N enrichment in PAM were observed for the C20 diet, for the C80 diet, treatment with methylcellulose produced a microbial pellet with greater (P = 0.03) 15N enrichment than that of pellets obtained with the other treatments. 15N enrichment in bacteria depends on their ability to use free ammonia as the N source and on the 15N enrichment of the ammonia fraction. Although PAM are located bound to plant surfaces, where the actual ammonia concentration may be much less than in ruminal fluid and could fluctuate widely, the lack of differences for the C20 diet in 15N enrichment in PAM detached by different procedures suggests that the bacteria had similar access to ammonia N and incorporation rates. A higher proportion of species that preferentially incorporate ammonia N in the PAM pellet obtained with methylcellulose treatment for the C80 diet might explain the greater 15N enrichment compared with those obtained by the other detachment procedures. In fact, ammonia 15N incorporation by PAM obtained with methylcellulose was 12.8%, whereas the mean value for the other three pellets was 10.1% (Table 3). Despite the greater 15N enrichment observed in PAM pellets obtained with the methylcellulose treatment for the C80 diet, no differences due to the detachment procedure were detected either in the daily PAM growth or in the daily total microbial growth (Table 3).
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Table 3. Microbial growth (particle-associated [PAM] and total microbes [TM]) and ammonia 15N incorporation by PAM in semicontinuous fermenters fed two diets (low concentrate [C20] and high concentrate [C80]) estimated using microbial pellets as reference obtained after applying different detachment procedures
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If any treatment caused microbial lysis, then concentration of PB in the microbial pellet would be lower than the values obtained with other treatments since PB are located exclusively in the microbial cytoplasm. In contrast, N compounds are also located in the cell wall and, therefore, the 15N enrichment could not be as strongly affected by cellular lysis. With both diets, detachment procedure did not affect (P > 0.05) PB concentration, PB:N ratio or guanine:adenine ratio in PAM pellets (Table 4). These results seem to indicate that the different detachment procedures either did not have an adverse effect on microbial cell integrity or they produced it with the same intensity. Moreover, the N content of PAM was in the range of the values previously reported in the literature (Clark et al., 1992), and their PB concentration was only slightly lower than the values reported by Rodríguez et al. (2000) and Carro and Miller (2002) for microorganisms detached from ruminal digesta in sheep and in semi-continuous fermenters, respectively.
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Table 4. Chemical composition of microbial pellets obtained from semicontinuous fermenters fed two diets (low concentrate [C20] and high concentrate [C80]) after applying different detachment procedures (see text for details)
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Whereas Martín-Orúe et al. (1998) did not find differences between diets in the percentage of detachment of PAM from ruminal digesta in sheep fed two diets differing in their forage:concentrate ratio (20:80 and 80:20), in our study the level of detachment was greater (P < 0.01) for the C80 diet compared to the C20 diet for all detachment procedures (mean values of 88.1 and 79.7%, respectively; see Table 2). These results would indicate that PAM population with the C80 diet showed a lower resistance to detachment. However, the lower (P < 0.01) recovery observed with the C80 diet compared to the C20 diet (39.8 and 69.0%, respectively) indicates a greater proportion of microbial losses for the C80 diet during the process of isolation. As a consequence, total recovery of PAM was lower (P < 0.01) for the C80 diet (mean value of 34.9%) compared with the C20 diet (54.9%).
In agreement with the results of Martin et al. (1994), diet did not affect the N content of LAM (62.5 and 63.9 mg/g of DM for the C20 diet and the C80 diet, respectively). In contrast, the N content of PAM was greater (P < 0.01) when vessels received the C20 diet compared with the C80 diet for all detachment treatments (Table 4). The influence of diet on the N content of ruminal microbes is unresolved in the literature. In some studies (Arambel et al., 1982; Yang et al., 2001), forage:concentrate ratio in the diet did not affect the N content of microbes, but in others, significant differences were observed (Chiquette and Benchaar, 1998; Martín-Orúe et al., 1998). In agreement with our results for PAM pellets, McAllan and Smith (1977) reported that in mixed ruminal bacteria the N content declined, whereas carbohydrates rose as concentrate in the diet increased.
Similar to the results of Martín-Orúe et al. (1998), we observed a greater (P < 0.05) PB concentration for the C80 diet compared to the C20 diet in both PAM (26.0 vs 22.8 µmol/mg DM, respectively; Table 4) and LAM (80.6 vs 72.6 µmol/mg DM, respectively). Changes in purine concentration are associated with different bacterial species (Obispo and Dehority, 1999), and with the growth rate of bacteria (Legay-Carmier and Bauchart, 1989; Obispo and Dehority, 1999). Thus, the lower PB concentration found in PAM and LAM with the C20 diet might stem from a lower growth rate of these populations (Cecava et al., 1990), as energy availability is a major factor influencing compositional changes in ruminal microorganisms (Craig et al., 1987).
The mean proportion of bacterial DM in the particulate fraction of the fermenters (nylon bag residues) was 13.2 and 21.6% for the C20 diet and the C80 diet, respectively. Whereas the value observed for the C20 diet is lower than values reported in vivo for diets containing high proportions of forages (Craig et al., 1987), the value observed for the C80 diet is in the range of in vivo values reported in the literature for diets containing high proportions of concentrates (Legay-Carmier and Bauchart, 1989; Yang et al., 2001). The proportion of PAM in the total microbial pool estimated with the different detachment procedures ranged from 66.4 to 67.1% for the C20 diet, and from 53.1 to 56.9% for the C80 diet (Table 3). Increasing the forage:concentrate ratio is usually beneficial for the pool of PAM because more fibrolytic bacteria attach to forage particles (Yang et al., 2001). The observed values are in the range of those found by Merry and McAllan (1983) for steers receiving diets of equal proportions of forage and concentrate; however, values are lower than the 70 to 80% reported by Craig et al. (1987) for cows fed a 65% alfalfa haylage diet, and the 90% reported by Faichney (1980) in sheep fed all-forage diets. These lower values could be due to the different experimental conditions. Ruminal DM content can vary with several factors, ranging from 10 to 25%, whereas in our experiment with semicontinuous fermenters the DM content of the digesta represented only about 2 to 3% of the total volume.
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Implications
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Results suggest that the treatment of ruminal digesta from Rusitec fermenters with a saline solution with 0.1% methylcellulose at 38°C for 15 min combined with homogenizing and chilling at 4°C for 24 h removed more than 80% of particle-associated microorganisms from solid ruminal digesta. However, total recovery of particle-associated microorganisms was only 55.6 and 35.1% for 20 and 80% concentrate diets, respectively. Although these recoveries are higher than some others reported previously, further research is needed to reduce microbial losses during the isolation process. The particle-associated microorganisms constituted more than 50% of the ruminal microbial population in the fermenters with both diets, and differed in chemical composition from liquid-associated microorganisms. Therefore, contribution of particle- and liquid-associated microorganisms should be taken into account to estimate total microbial growth.
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Footnotes
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1 This research was supported by MCYT of Spain (Project AGL2001-0130) and Junta de Castilla y León (Projects LE 29/98 and LE 34/01). M. J. Ranilla gratefully acknowledges receipt of a research contract from MCYT (Programa Ramón y Cajal). The authors thank M. J. Arín and M. T. Díez for analyses of purine bases. Analyses of 15N isotope ratios were performed at the Servicio Interdepartamental de Investigación of the Universidad Autónoma de Madrid (Spain). 
Received for publication July 17, 2002.
Accepted for publication October 14, 2002.
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Literature Cited
|
|---|
Arambel, M. J., E. E. Bartley, G. S. Dufva, T. G. Nagaraja, and A. D. Dayton. 1982. Effect of diet on amino and nucleic acids of rumen bacteria and protozoa. J. Dairy Sci. 65:2095–2101.
Barrie, S., and C. T. Workman. 1984. An automated analytical system for nutritional investigations using N-15 tracers. Spect. Int. J. 3:439–447.
Carro, M. D., and E. L. Miller. 1999. Effect of supplementing a fibre basal diet with different nitrogen forms on ruminal fermentation and microbial growth in an in vitro semi-continuous culture system (RUSITEC). Br. J. Nutr. 82:149–157.[Medline]
Carro, M. D., and E. L. Miller. 2002. Comparison of microbial markers (15N and purine bases) and bacterial isolates for the estimation of rumen microbial protein synthesis. Anim. Sci. 75:315–321.
Cecava, M. J., N. R. Merchen, L. C. Gay, and L. L. Berger. 1990. Composition of ruminal bacteria harvested from steers as influenced by dietary energy level, feeding frequency, and isolation techniques. J. Dairy Sci. 73:2480–2488.[Abstract]
Chiquette, J., and C. Benchaar. 1998. Effect of diet and probiotic addition on chemical composition of free or particle-associated bacterial populations of the rumen. Can. J. Anim. Sci. 78:115–120.
Clark, J. H., T. H. Klusmeyer, and M. R. Cameron. 1992. Microbial protein synthesis and flows of nitrogen fractions to the duodenum of dairy cows. J. Dairy Sci. 75:2304–2323.[Abstract]
Craig, W. M., G. A. Broderick, and D. B. Ricker. 1987. Quantitation of microorganisms associated with the particulate phase of ruminal ingesta. J. Nutr. 117:56–62.
Czerkawski, J. W., and G. Breckenridge. 1977. Design and development of a long-term rumen simulation technique (Rusitec). Br. J. Nutr. 38:371–384.[Medline]
Faichney, G. J. 1980. Measurement in sheep of the quantity and composition of rumen digesta and of the fractional outflow rates of digesta constituents. Austr. J. Agric. Res. 31:1129–1137.
Harold, F. M. 1970. Antimicrobial agents and membrane function. Page 54 in Advances in Microbial Physiology. Vol. 4. A. H. Rose and J. F. Wilkinson, ed. Academic Press, London.
Leedle, J. A. Z., M. P. Bryant, and R. B. Hespell. 1982. Diurnal variations in bacterial numbers and fluid parameters in ruminal contents of animals fed low- or high-forage diets. Appl. Environ. Microbiol. 44:402–412.[Abstract/Free Full Text]
Legay-Carmier, F., and D. Bauchart. 1989. Distribution of bacteria in the rumen contents of dairy cows given a diet supplemented with soya-bean oil. Br. J. Nutr. 61:725–740.[Medline]
Martín-Orúe, S. M., J. Balcells, J. A. Guada, and C. Castrillo. 1995. Endogenous purine and pyrimidine derivative excretion in pregnant sows. Br. J. Nutr. 73:375–385.[Medline]
Martín-Orúe, S. M., J. Balcells, F. Zakraoui, and C. Castrillo. 1998. Quantification and chemical composition of mixed bacteria harvested from solid fractions of rumen digesta: Effect of detachment procedure. Anim. Feed Sci. Technol. 71:269–282.
Martin, C., A. G. Williams, and B. Michalet-Doreau. 1994. Isolation and characteristics of the protozoal and bacterial fractions from bovine ruminal contents. J. Anim. Sci. 72:2962–2968.[Abstract]
McAllan, A. B., and R. H. Smith. 1977. Some effects of variation in carbohydrate and nitrogen intakes on the chemical composition of mixed rumen bacteria from young steers. Br. J. Nutr. 37:55–61.[Medline]
Merry, R. J., and A. B. McAllan. 1983. A comparison of the chemical composition of mixed bacteria harvested from the liquid and solid fractions of rumen digesta. Br. J. Nutr. 50:701–709.[Medline]
Minato, H., and T. Suto. 1978. Technique for fractionation of bacteria in rumen microbial ecosystem. II. Attachment of bacteria isolated from bovine rumen to cellulose powder in vitro and elution of bacteria attached therefrom. J. Gen. Appl. Microb. 24:1–16.
Obispo, N. E., and B. A. Dehority. 1999. Feasibility of using total purines as a marker for ruminal bacteria. J. Anim. Sci. 77:3084–3095.[Abstract/Free Full Text]
Rodríguez, C. A., J. González, M. R. Alvir, J. L. Repetto, C. Centeno, and F. Lamrani. 2000. Composition of bacteria harvested from the liquid and solid fractions of the rumen of sheep as influenced by feed intake. Br. J. Nutr. 84:369–376.[Medline]
Whitehouse, N. L., V. M. Olson, C. G. Schwab, W. R. Chesbro, K. D. Cunningham, and T. Lykos. 1994. Improved techniques for dissociating particle-associated mixed ruminal microorganisms from ruminal digesta solids. J. Anim. Sci. 72:1335–1343.[Abstract]
Yang, W. Z., K. A. Beauchemin, and L. M. Rode. 2001. Effect of dietary factors on distribution and chemical composition of liquid- or solid-associated bacterial populations in the rumen of dairy cows. J. Anim. Sci. 79:2736–2746.[Abstract/Free Full Text]
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L. A. Giraldo, M. L. Tejido, M. J. Ranilla, and M. D. Carro
Effects of exogenous cellulase supplementation on microbial growth and ruminal fermentation of a high-forage diet in Rusitec fermenters
J Anim Sci,
August 1, 2007;
85(8):
1962 - 1970.
[Abstract]
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J. Boguhn, H. Kluth, and M. Rodehutscord
Effect of Total Mixed Ration Composition on Amino Acid Profiles of Different Fractions of Ruminal Microbes In Vitro
J Dairy Sci,
May 1, 2006;
89(5):
1592 - 1603.
[Abstract]
[Full Text]
[PDF]
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Abstract
Introduction
