J. Anim. Sci. 2006. 84:635-640
© 2006 American Society of Animal Science
Influence of cobalt concentration on vitamin B12 production and fermentation of mixed ruminal microorganisms grown in continuous culture flow-through fermentors1
M. E. Tiffany2,
V. Fellner and
J. W. Spears3
Department of Animal Science and Interdepartmental Nutrition Program, North Carolina State University, Raleigh 27695-7621
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Abstract
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An experiment was conducted to determine the effects of dietary concentrations of Co on vitamin B12 production and fermentation of mixed ruminal microbes grown in continuous culture fermentors. Four fermentors were fed 14 g of DM/d. The DM consisted of a corn and cottonseed hull-based diet with Co supplemented as CoCO3. Dietary treatments were 1) control (containing 0.05 mg of Co/kg of DM), 2) 0.05 mg of supplemental Co/kg of DM, 3) 0.10 mg of supplemental Co/kg of DM, and 4) 1.0 mg of supplemental Co/kg of DM. After a 3-d adjustment period, fermentors were sampled over a 3-d sampling period. This process was repeated 2 additional times for a total of 3 runs. Ruminal fluid vitamin B12 concentrations were affected by Co supplementation (P < 0.01), and there was a treatment x day interaction (P < 0.01). By sampling d 3, cultures fed the basal diet supplemented with 0.10 mg of Co/kg had greater (P < 0.05) vitamin B12 concentrations than those supplemented with 0.05 mg of Co/kg of DM, and increasing supplemental Co from 0.10 to 1.0 mg/kg of DM increased (P < 0.01) ruminal fluid vitamin B12 concentration. Ruminal fluid succinate also was affected (P < 0.10) by a treatment x day interaction. Cobalt supplementation to the control diet greatly decreased (P < 0.05) succinate in ruminal cultures on sampling d 3 but not on d 1 or 2. Molar proportions of acetate, propionate, and isobutyrate, and acetate:propionate were not affected by the addition of supplemental Co to the basal diet. However, molar proportions of butyrate, valerate, and isovalerate increased (P < 0.05) in response to supplemental Co. The majority of long-chain fatty acids observed in this study were not affected by Co supplementation. However, percentages of C18:0 fatty acids in ruminal cultures tended (P < 0.10) to be greater for Co-supplemented diets relative to the control. Methane, ammonia, and pH were not greatly affected by Co supplementation. The results indicate that a total (diet plus supplemental) Co concentration of 0.10 to 0.15 mg/kg of dietary DM resulted in adequate vitamin B12 production to meet the requirements of ruminal microorganisms fed a high-concentrate diet in continuous-flow fermentors.
Key Words: cobalt • ruminal culture • succinate • vitamin B12
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INTRODUCTION
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Cobalt is required by ruminal microorganisms for the synthesis of vitamin B12 (McDowell, 2000
). In addition to a mammalian requirement for vitamin B12, several studies have determined that vitamin B12 is an important growth factor for some ruminal microorganisms (Tanner and Wolfe, 1988; Strobel, 1992) and is utilized by others in pathways that produce propionate (Chen and Wolin, 1981). In ruminal microbes, one function of vitamin B12 is as a cofactor for methylmalonyl-CoA mutase, which catalyzes the conversion of succinyl CoA to methylmalonyl-CoA during propionate formation (Nagaraja et al., 1997). Adding supplemental Co to a high-energy diet that was low in Co increased ruminal fluid vitamin B12 concentrations and molar proportions of propionate in finishing steers (Tiffany et al., 2002). Feeding diets severely deficient in Co (0.004 mg of Co/kg) to sheep caused an immediate (within 3 d) and dramatic increase in ruminal fluid succinate and a decline in ruminal propionate concentration (Kennedy et al., 1991). Tiffany and Spears (2005) found that ruminal vitamin B12 concentrations were greater, whereas ruminal succinate concentrations were lower, in steers fed high-concentrate diets containing 0.19 mg of Co/kg compared with those fed diets containing 0.04 or 0.09 mg of Co/kg of diet.
The Co requirement of mixed ruminal microorganisms for optimal vitamin B12 production and ruminal fermentation has not been defined. The current study was conducted to determine the effects of dietary Co on ruminal vitamin B12 production and fermentation of mixed ruminal microorganisms cultured in continuous dual-flow fermentors.
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MATERIALS AND METHODS
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Conditions
Whole ruminal contents were collected from a nonlactating, ruminally fistulated Holstein cow fed fescue hay ad libitum and 4.5 kg of cracked corn daily. The study was approved by the North Carolina State University Institution of Animal Care and Use Committee. The cow had free-choice access to a white salt block. Ruminal contents (approximately 4.0 L) were placed in preheated vacuum containers, transported to the laboratory, and strained through double-layered cheesecloth. Approximately 700 mL of the filtered ruminal fluid was placed into each of the 4 vessels. This study utilized all glass, closed-system fermentors, which allowed continuous independent flow of liquid and particulate matter (Teather and Sauer, 1988). Anaerobic conditions of the cultures were maintained by sealing the fermentor openings with rubber and providing a continuous flow of CO2 (20.0 mL/min) to maintain a positive internal pressure. Artificial saliva was prepared as described by Slyter et al. (1966) and delivered by precision pump at a flow rate of 0.73 mL/min. Over a 24-h period, 1.1 L of artificial saliva was delivered to each ruminal culture to yield a fractional dilution rate of 6.8%/h. The temperature of the cultures was maintained at 39°C by a circulating water bath, and ruminal contents were continually stirred at 10 rpm as described by Teather and Sauer (1988).
Diets and Treatments
Feed totaling 14.0 g of DM was added to each fermentor daily in equal portions delivered at 0800 and 1500. Dietary treatments were 1) control (Table 1; corn and cottonseed hull-based diet containing 0.05 mg of Co/kg of DM and no supplemental Co), 2) control supplemented with 0.05 mg of Co/kg of DM, 3) control supplemented with 0.10 mg of Co/kg of DM, and 4) control supplemented with 1.0 mg of Co/kg of DM. Supplemental Co was provided as CoCO3. The control diet was formulated to meet or exceed requirements for finishing beef cattle (NRC, 1996), with the exception of Co. Fermentors were allowed a 3-d stabilization period when cultures were fed dietary treatments and pelleted alfalfa at 25:75, 50:50, and 75:25 on d 1, 2, and 3, respectively. Following the stabilization period, ruminal cultures were fed the dietary treatments exclusively for a 3-d period. This procedure was repeated 3 times so that this experiment utilized 4 continuous culture fermentors on 3 separate 6-d runs (3-d adjustment period followed by a 3-d sampling period).
Sample Collection and Analytical Procedures
Feed samples for the determination of Co concentration were prepared using a microwave digestion (Mars 5, CEM Corp., Matthews, NC) procedure described by Gengelbach et al. (1994). Cobalt was determined by flameless atomic absorption spectrophotometry using a graphite furnace (GFA-6500, Shimadzu Scientific Instruments, Kyoto, Japan). Gas samples with 10 µL of headspace were collected before feeding and at 2 h post-feeding from each fermentor using a gas-tight syringe (Hamilton Co., Reno, NV) and were analyzed for methane using gas chromatography (Model CP-3800, Varian, Walnut Creek, CA). The culture pH was also recorded at the same times. Subsequent to thorough mixing of the ruminal cultures, 5 mL were removed at 2 h post afternoon feeding on each sampling day and analyzed for VFA by GLC (Model CP-3380, Varian) and for NH3 N using a colorimetric assay (Beecher and Whitten, 1970). At the same time, two 1.0-mL aliquots of the mixed ruminal fluid were obtained for the determination of succinate and vitamin B12. Ruminal fluid samples for the determination of ruminal succinate were prepared according to the method of McMurray et al. (1986) using a modified GC method (Tiffany and Spears, 2005). Ruminal fluid was prepared for the determination of vitamin B12 as described by Tiffany and Spears (2005), and vitamin B12 was determined using a competitive binding radioimmunoassay kit (ICN, Costa Mesa, CA) in which nonspecific vitamin B12 binding proteins were removed by affinity chromatography. On the final day (d 3), a separate 5-mL sample was collected 2 h after the final afternoon feeding for analysis of long-chain fatty acids. Samples for the determination of long-chain fatty acids were methylated as described by Kramer et al. (1997) and analyzed for fatty-acid composition by GLC.
Statistical Analysis
Data were analyzed as a randomized complete block (run) design using Proc Mixed of SAS (SAS Inst., Inc., Cary, NC). Fermentation variables and vitamin B12 concentrations were analyzed as repeated measures with the model containing treatment, run, day, and all possible interactions. The model for long-chain fatty acids contained treatment, run, and treatment x run interaction. Preplanned orthogonal contrasts were utilized to detect differences among means. Comparisons made were 1) control vs. all Co supplemented treatments, 2) 0.05 mg of Co/kg vs. 0.10 mg of Co/kg, and 3) 0.10 mg of Co/kg vs. 1.0 mg of Co/kg.
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RESULTS AND DISCUSSION
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Ruminal Fluid Vitamin B12
Vitamin B12 concentration in continuous cultures was affected by Co supplementation (P < 0.01) and a treatment x day interaction (P < 0.01; Table 2). Cobalt supplementation to the experimental diet did not affect vitamin B12 concentrations on sampling d 1 or 2. However, on sampling d 3, cultures fed diets supplemented with Co had greater (P < 0.01) ruminal fluid vitamin B12 concentrations. These results indicate that microbial vitamin B12 production responds rapidly to Co supplementation in cultures fed high-energy diets and grown in continuous-flow fermentors.
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Table 2. Effects of Co concentration on vitamin B12 and succinate concentrations in continuous cultures of ruminal microbes1
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Increasing supplemental Co from 0.05 to 0.10 or from 0.10 to 1.0 mg/kg of DM increased ruminal vitamin B12 concentrations (P < 0.05 and 0.01, respectively) on sampling d 3 (Table 2
). Adding supplemental Co to a low-Co diet (control diet containing 0.05 mg of Co/kg of DM) increased vitamin B12 concentration in ruminal fluid obtained from steers on d 84 of the finishing period (Tiffany et al., 2002). In that study, increasing supplemental Co from 0.10 to 1.0 mg/kg greatly increased vitamin B12 concentration of ruminal fluid. Vitamin B12 concentration in ruminal fluid was also greater in steers supplemented with 0.15 mg of Co/kg than for those fed the control diet or the control diet supplemented with 0.05 mg of Co/kg (Tiffany and Spears, 2005). The present work demonstrated that vitamin B12 production of mixed ruminal microbes grown in continuous culture fermentors also increases substantially in response to increasing dietary Co from 0.05 to 1.05 mg of Co/kg of DM (diet plus supplemental). Earlier work with in vitro cultures showed that ruminal fluid vitamin B12 concentrations declined greatly between d 3 and 5 when the cultures were fed a hay diet that was low (0.03 mg/kg of DM) in Co (McDonald and Suttle, 1986). In our study, ruminal vitamin B12 concentration of cultures fed the control diet that was moderately deficient in Co (0.05 mg/kg of DM) did not decline over the 3-d sampling period. Vitamin B12 production in the current study averaged approximately 1,100 pmol/d in the control vessels. The decline in vitamin B12 between d 3 and 5 in the study by McDonald and Suttle (1986) was from approximately 3,100 to 800 pmol/d.
Ruminal Fluid Succinate
Ruminal fluid succinate concentrations (Table 2) were affected by Co supplementation (P < 0.05), day (P< 0.01), and a treatment x day interaction (P < 0.10). Ruminal fluid succinate concentrations increased across all treatments (P < 0.05) from sampling d 1 to 3, but the increase was dramatic for the unsupplemented controls on sampling d 3, which had greater succinate concentrations (P < 0.05) than Co-supplemented treatments. Tiffany et al. (2002) reported that steers fed moderately Co-deficient corn-based diets had plasma succinate concentrations greater than steers consuming Co-supplemented diets, suggesting increased succinate absorption from the rumen. A subsequent study (Tiffany and Spears, 2005) found that ruminal fluid succinate concentrations were greater in steers consuming a low-Co diet (0.04 mg of Co/kg) or the control diet supplemented with 0.05 mg of Co/kg of DM than for those supplemented with 0.15 mg of Co/kg of DM. When Kennedy et al. (1991) fed sheep a Co-sufficient (1.0 mg of Co/kg of DM) barley-based diet for >2 mo and then abruptly switched to a severely Co-deficient (0.004 mg of Co/kg of DM) diet, ruminal fluid succinate concentrations increased greatly within 4 d of consuming the Co-deficient diet. The sharp and immediate increases in ruminal succinate concentrations observed in that study agree with the findings of the current study utilizing continuous culture fermentors. However, much greater ruminal succinate concentrations were observed by Kennedy et al. (1991). This may relate, in part, to the much lower Co concentrations of the basal diet in that study compared with the present work (0.004 vs. 0.05 mg/kg). However, it is difficult to compare in vitro to in vivo studies because of possible differences in the ratio of DM fed to fermentor or ruminal volume. This can affect ruminal concentrations of Co to which bacteria are exposed.
Increased ruminal succinate concentrations in control cultures may be explained by reduced activity of the vitamin B12-dependent enzyme, methylmalonyl-CoA mutase. This enzyme is responsible in certain bacteria for converting succinyl CoA to methylmalonyl CoA (Banerjee and Chowdhury, 1999), which is subsequently converted to propionate. Although several bacterial species produce succinate; under normal conditions, succinate does not accumulate in the rumen because of the rapid conversion to propionate (Scheifinger and Wolin, 1983). Among microbes that produce propionate, Prevotella ruminicola (Strobel, 1992) and Bacteroides fragilis (Chen and Wolin, 1981) have been shown to require vitamin B12. When vitamin B12 is limiting, both of these organisms produce succinate as a major end product instead of propionate.
Ruminal Fluid VFA and Long-Chain Fatty Acids
Total VFA concentrations and molar proportions of individual VFA were not affected by a treatment x day interaction. Cultures fed the control diet supplemented with 0.10 mg of Co/kg of DM had lower (P < 0.05) total VFA concentrations than those supplemented with 0.05 or 1.0 mg of Co/kg of DM (Table 3). It is unclear why total VFA concentrations were lower in cultures supplemented with 0.10 compared with the lower or greater Co concentration. Molar proportions of acetate tended (P < 0.12) to be lower in cultures fed diets supplemented with Co (Table 3). However, increasing supplemental Co from 0.05 to 0.10 or from 0.10 to 1.0 mg/kg of DM did not affect acetate molar proportions. The molar proportions of propionate and acetate:propionate were not affected by Co addition to the control diet. In contrast, Kennedy et al. (1991) found that propionate concentrations in ovine ruminal fluid decreased substantially within 4 d of consuming a low-Co diet. Previous studies with steers (Tiffany et al., 2002, 2003) fed a low-Co (approximately 0.05 mg/kg), high-concentrate finishing diets reported a decrease in the molar proportions of ruminal propionate relative to those consuming Co-supplemented diets at 84 d. Results of those studies suggest that long-term consumption of a high-concentrate diet that is moderately deficient in Co may decrease microbial methylmalonyl-CoA mutase activity, reducing the conversion of succinate to propionate. The current study might have been of too short in duration for the elevated ruminal succinate observed on sampling d 3 to affect propionate molar proportion. In addition to the succinate pathway, propionate can also be produced by some ruminal microorganisms from lactate via the acrylate pathway (Baldwin and Allison, 1983). The acrylate pathway involves the conversion of lactate to acrylyl-CoA via phospholactyl-CoA and reduction of acrylyl-CoA to propionyl-CoA (Baldwin and Allison, 1983), thus eliminating the need for a vitamin B12-dependent enzyme-catalyzed reaction.
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Table 3. Effects of Co concentration on VFA concentrations and molar proportions in continuous cultures of ruminal microbes1
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Addition of supplemental Co to the control diets increased (P < 0.05) molar proportions of butyrate in continuous cultures (Table 3). In contrast, steers fed high-concentrate diets supplemented with Co had lower ruminal molar proportions of butyrate than unsupplemented controls (Tiffany et al., 2003). Isobutyrate molar proportions were not affected by addition of supplemental Co to the control diet. Molar proportions of valerate and isovalerate in ruminal cultures increased (P < 0.05) in response to Co supplementation of the control diet. Increasing supplemental Co from 0.05 to 0.10 mg/kg of DM increased (P < 0.05) the molar proportion of isovalerate in ruminal cultures.
There were no major effects of Co supplementation on the percentages of long-chain fatty acids in continuous ruminal fluid cultures (Table 4). However, cultures fed the control diet supplemented with Co tended (P < 0.10) to have a greater percentage of C18:0 fatty acids than those fed the control diet.
Methane, Ammonia, and pH
Methane production and ammonia concentration in continuous ruminal cultures were not affected by dietary Co or a treatment x day interaction (Table 5). Ruminal fluid pH was affected by Co supplementation (P < 0.10); however, the effect was not consistent with increasing dietary additions of Co to the control diet (Table 5). Ruminal fluid pH was similar between cultures supplemented with 0.05 or 1.0 mg of Co/kg of DM but was greater (P < 0.05) in cultures supplemented with 0.10 mg of Co/kg of DM. The greater ruminal pH in cultures supplemented with 0.10 vs. 0.05 or 1.0 mg of Co/kg of DM can be explained by the lower total VFA concentrations observed for the 0.10-mg of Co/kg of DM treatment. Previous research by Jenkins et al. (2003) utilizing continuous flow fermentors determined that a diet with a 70:30 concentrate to forage ratio (concentrate portion containing 70% corn and 27% soybean meal, DM basis) resulted in a ruminal fluid pH of 6.14. Other research with continuous ruminal cultures found that when dietary corn was increased from 12.6 to 54.6% DM in gammagrass silage and corn-based diets that the pH dropped from 6.1 to 5.9 (Eun et al., 2004). As expected, the range of pH in the present work (5.43 to 5.59) was lower than that observed in these studies, reflecting the greater percentage of concentrate that provides a rapidly fermentable substrate.
A symbiotic relationship seems to exist among ruminal microorganisms that synthesize vitamin B12 from inorganic Co and those that require vitamin B12 as a growth factor and/or in the production of propionate. Dryden et al. (1962) tested a number of strains of ruminal microorganisms for their ability to produce vitamin B12. Among the strains tested, Selenomonas ruminantium and Peptostreptococcus (Megasphaera) elsdenii were found to be the predominant vitamin B12-producing species in rumen medium. Vitamin B12 is a growth factor for other ruminal bacteria such as Prevotella ruminicola (Strobel, 1992) and Methanomicrobium mobile (Tanner and Wolfe, 1988), and when vitamin B12 is limiting, Prevotella ruminicola has an inability to convert succinate to propionate (Strobel, 1992).
In the current study, a high-concentrate diet containing 0.05 mg of Co/kg of DM seemed to be inadequate to meet the vitamin B12 requirements of mixed ruminal microorganisms grown in continuous culture flow-through fermentors. By sampling d 3, ruminal succinate concentrations were greatly elevated in cultures fed the low-Co, control diet, suggesting that the vitamin B12-dependent enzyme, methylmalonyl CoA mutase, limited succinate conversion to methylmalonyl CoA. Recent in vivo studies (Tiffany et al., 2003; Tiffany and Spears, 2005) confirm that high-concentrate diets containing 0.04 to 0.05 mg of Co/kg of DM alter ruminal fermentation, resulting in elevated ruminal succinate and reduced molar proportions of propionate. A total (diet plus supplemental) dietary Co concentration of 0.10 to 0.15 mg/kg of DM increased vitamin B12 production by sampling d 3 and prevented the rise in ruminal succinate in the current study. Although increasing supplemental Co from 0.10 to 1.0 mg/kg of DM increased vitamin B12 production on sampling d 3 by 57%, the current study suggests that 0.10 to 0.15 mg of Co/kg of DM resulted in adequate vitamin B12 synthesis to meet microbial requirements in our continuous-flow fermentor system.
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Footnotes
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1 Use of trade names in this publication does not imply endorsement by the North Carolina Agric. Res. Serv. or criticism of similar products not mentioned. 
2 Present address: Burkmann Feeds, Greeneville, TN 37744.
3 Corresponding author: Jerry_Spears{at}ncsu.edu
Received for publication January 28, 2005.
Accepted for publication October 24, 2005.
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Abstract
INTRODUCTION
